HK1182745B

HK1182745B - Methods and systems for generating, validating and using monoclonal antibodies

Info

Publication number: HK1182745B
Application number: HK13110091.2A
Authority: HK
Inventors: 杰夫．D．伯克; 恒．朱; 塞斯．布莱克肖; 丹尼尔．J．艾兴格; 伊格纳西奥．皮诺
Original assignee: 约翰.霍普金斯大学; Cdi实验室公司
Priority date: 2010-06-16
Filing date: 2011-06-16
Publication date: 2017-08-11

Abstract

Provided herein is a library of antibodies, wherein the library of antibodies can comprise a plurality of monoclonal, monospecific, or immunoprecipitating antibodies. Also provided herein is a method for producing and using the library of antibodies.

Description

Methods and systems for generating, validating and using monoclonal antibodies

Cross-referencing

This application claims benefit of U.S. provisional application No. 61/355,329 filed on 16/6/2010, which is incorporated herein by reference in its entirety.

Statement regarding federally sponsored research

The invention was made with U.S. government support based on RR020839 and GM076102 sponsored by the national institutes of health. The government may have certain rights in the invention.

Background

There is a significant need in the biomedical world for reliable techniques that can produce reproducible antibody reagents with the highest quality possible. Continued improvements in this technological route would directly benefit the health research community and the larger biomedical community.

The detection or binding of epitopes, antigens or proteins is a large part of the research products industry and the diagnostic and therapeutic industry that serves academia and the pharmaceutical industry. The use of antibodies in the detection of epitopes, antigens or proteins can be used to identify new biomarkers and to perform a number of assays. For example, antibodies are also widely used in diagnostic applications, such as clinical medicine (e.g., ELISA and radioimmunoassay systems). Analysis of cells and tissues in pathology laboratories includes the use of antibodies in tissue sections and flow cytometry analysis. Antibodies may also be used as therapeutic agents.

The production of antibodies can be expensive and time consuming, and therefore a more cost effective and less time consuming method for the production of high throughput antibodies, especially highly specific antibodies, is desired. The present disclosure satisfies these needs and provides related advantages.

Disclosure of Invention

The present invention relates generally to antibody libraries, including methods and systems for producing, generating, characterizing, and utilizing antibodies. The antibody may be highly specific. In some embodiments, the antibody is a monoclonal antibody. The library may comprise a plurality of different antibodies, wherein the antibodies are produced by the same platform. The library may comprise a plurality of different antibodies, wherein in the plurality or a subset of the plurality, each antibody is a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is similar to the binding affinity of another antibody of the plurality of antibodies (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,),Within 19% or 20%); has at least 10 to its target^-7M(K_D) Binding affinity (e.g., such as at least 10)^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16M); or any combination thereof.

The antibody library can be produced with high reproducibility. For example, in a first antibody library and a second antibody library, the first and second libraries comprise the same set of different antibodies, and each antibody of the first library has a binding affinity that is within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the binding affinity of the same antibody of the second library.

One aspect of the invention is an antibody library comprising a plurality of different antibodies, wherein at least 10% of the plurality of antibodies are produced by the same platform. In one embodiment, the library comprises a plurality of different antibodies, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies are antibodies produced by the same platform.

Each of the plurality of antibodies produced by the same platform may be a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding of MAffinity; or any combination thereof.

Antibodies produced by the same platform can comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibodies or a subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M (KD), such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

In yet another embodiment, antibodies produced by the same platform bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein the antibodies or a subset of the antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^- ⁷M, such asAt least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^- ¹⁴M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Antibodies produced by the same platform can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibodies or a subset of the antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Another aspect of the invention is an antibody library comprising a plurality of different antibodies, wherein at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the plurality of antibodies are monospecific antibodies. Each monospecific antibody of the plurality of antibodies may bind to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20% of the binding affinity of another antibody of the plurality of antibodies, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

A monospecific antibody comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the library may comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900 or 1000 different antibodies, wherein the antibody or subset of antibodies may bind to the native form of its target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody in at least 10% of the plurality of monospecific antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^- ¹⁵M or 10^-16Binding affinity of M; or any combination thereof.

A monospecific antibody comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of different antibodies can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% of the human proteome90%, or 100%, wherein the antibody or subset of antibodies can bind to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody in at least 10% of the plurality of monospecific antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

A monospecific antibody comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of different antibodies can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibody or subset of antibodies can bind to the native form of their target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Also provided herein are antibody libraries comprising a plurality of different antibodies, wherein at least 10% of the plurality of antibodies have at least 10% to their target, to their target protein^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M. Has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Each antibody of the plurality of antibodies to the binding affinity of M may be a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; or any combination thereof.

Has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16The antibody of binding affinity for M can comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20% of the binding affinity of another antibody of the plurality of antibodies, such as at least 1%, 2%, 3%, 4Percent, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19%; or any combination thereof.

Has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16An antibody of binding affinity for M can bind at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; or any combination thereof.

Has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16An antibody of binding affinity for M can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibodyOr an antibody of the IgG isotype; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; or any combination thereof.

Another aspect of the invention is an antibody library comprising a plurality of different antibodies, wherein at least 10% of the plurality of antibodies bind to a native form of their target protein. In one embodiment, the library comprises a plurality of different antibodies, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies bind to a native form of its target protein. Each antibody of the plurality of antibodies that binds to its native form of the target protein may be a monospecific antibody; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The antibody that binds to its native form of the target protein may comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibody or a subset of antibodies may be monospecific antibodies; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target in another of the plurality of antibodiesWithin at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of the antibody; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^- ¹³M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

In yet another embodiment, an antibody that binds to its native form of a target protein can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein the antibody or subset of antibodies can be monospecific antibodies; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

An antibody that binds to its native form of the target protein can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibody binds to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%Or a subset of the antibodies may be monospecific antibodies; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Another aspect of the invention is an antibody library comprising at least 50 different monoclonal antibodies, such as at least 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900 or 1000 different monoclonal antibodies. Each monoclonal antibody of the library may be monospecific; binds to its native form of the target protein; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^- ¹⁵M or 10^-16Binding affinity of M; or any combination thereof.

In yet another embodiment, at least 50 different monoclonal antibodies are included, such as at least 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425. An antibody library of 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different monoclonal antibodies that binds to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein a monoclonal antibody or a subset of monoclonal antibodies can be monospecific antibodies; binds to its native form of the target protein; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

An antibody library comprising at least 50 different monoclonal antibodies, such as at least 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different monoclonal antibodies, can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein a monoclonal antibody or a subset of monoclonal antibodies can be monospecific; binds to its native form of the target protein; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; to its target toolHas a binding affinity that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Another aspect of the invention is an antibody library comprising a plurality of different antibodies that bind at least 0.5% of the human proteome, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome. Each antibody of the plurality of antibodies can be a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Comprises binding to at least 0.5% of the human proteome, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% of the human proteome,An antibody library of 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of a plurality of different antibodies can comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

An antibody library comprising a plurality of different antibodies that bind at least 0.5% of the human proteome, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the human proteome, can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has binding affinity for its targetA force within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%, of the binding affinity of another antibody of the plurality of antibodies; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Another aspect of the invention is an antibody library comprising a plurality of different antibodies, wherein at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the plurality of antibodies have a binding affinity for their target that is within at least 20% of the binding affinity of another antibody of the plurality of antibodies, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19%. Each antibody of the plurality of antibodies can be a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Comprising a plurality of different antibodies (wherein at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the plurality of antibodies have a binding affinity for their target that is within at least 20% of the binding affinity of another antibody of the plurality of antibodies, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%18%, or 19%) of a library of antibodies may comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibodies or a subset of antibodies may be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

In yet another embodiment, an antibody library comprising a plurality of different antibodies (wherein at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies have a binding affinity for their target that is within at least 20%, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies) binds at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% of the human proteome, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%, wherein the antibody or subset of antibodies may be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

An antibody library comprising a plurality of different antibodies (wherein at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies have a binding affinity for their target that is within at least 20%, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies) can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% of the human proteins listed in table 5, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%, wherein the antibody or subset of antibodies may be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Also provided herein are antibody libraries comprising a plurality of different antibodies, wherein at least 10% of the plurality of antibodies are IgG antibodies or IgG isotype antibodies. In one embodiment, the library comprises a plurality of different antibodies, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies are IgG antibodies (e.g., IgG isotype antibodies) produced by the same platform. Each IgG antibody (e.g., an IgG isotype antibody) can be a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; has a binding affinity for its target that is within at least 20% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform, such as toLess than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

An IgG antibody (e.g., an IgG isotype antibody) can comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

In yet another embodiment, an IgG antibody (e.g., an IgG isotype antibody) binds to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody(ii) a Is an immunoprecipitated antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

An IgG antibody (e.g., an IgG isotype antibody) can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibody or subset of antibodies can be monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^- ¹⁰M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Another aspect of the invention is an array comprising a library of antibodies and a substrate, wherein the antibodies or a subset, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the antibodies, are immobilized on the substrate. The substrate may be planar or particulate, comprising solid or porous material. Immobilization may be reversible or irreversible.

The array may comprise an antibody library comprising a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies can be produced by the same platform, are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The array can comprise an antibody library comprising at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibodies or a subset of the antibodies can be produced by the same platform, are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The array may comprise a library of antibodies that bind at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein the antibodies or a subset of the antibodies may be produced by the same platform and are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The array may comprise a library of antibodies that bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibodies or a subset of the antibodies may be produced by the same platform and are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; having binding affinity for its target at the binding affinity of another antibody in a plurality of antibodies produced by the same platformWithin at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^- ¹³M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Also provided herein is a method of generating a library of antibodies, the method comprising: (a) immunizing an animal with a plurality of antigens; (b) isolating antibody-producing cells from said animal; (c) isolating a plurality of antibodies from the antibody-producing cells; (d) screening the plurality of antibodies in step c) using a human proteome array, wherein the human proteome array may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the human proteome, or at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the human proteins listed in table 5; and (e) selecting an antibody that is monospecific for the proteomic array. In some embodiments, the antibody selected in step (e) is added to a library, wherein the library may comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the plurality of different antibodies may be produced by the same platform and are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; having binding affinity for its target to that of another antibody in a plurality of antibodies produced by the same platformWithin 20% less, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^- ¹¹M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The method can produce an antibody library that can comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibodies or a subset of the antibodies can be produced by the same platform, are monospecific antibodies; binds to its native form of the target protein; does not bind to a denatured form of its target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^- ¹¹M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The method can produce an antibody library that can bind at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, wherein the antibody or antibody thereofSubsets can be generated from the same platform, are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The method can produce a library of antibodies that can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibodies or a subset of the antibodies can be produced by the same platform and are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

The method of generating an antibody library may further comprise pre-screening a plurality of antibodies from antibody-producing cells prior to step c), such as by performing immunocytochemistry or determining binding of antibodies from said antibody-producing cells to a mixture comprising one or more target antigens, such as native proteins. The mixture may comprise crude lysate, one or more cells, one or more proteins, one or more peptides, or one or more nucleic acids or a biological sample, wherein the biological sample may be, but is not limited to, a mixture of cells, tissue, blood, serum, plasma, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper fluid, pre-ejaculatory fluid, female ejaculate (femalejaculate), sweat, tear fluid, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretions, mucosal secretion, fecal water (stoolwater), pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary aspirates, blastocyst fluid (blastocyst fluid), or umbilical cord blood.

A method of generating an antibody library can comprise immunizing an animal with a plurality of antigens, wherein the plurality of antigens can comprise a crude lysate, one or more cells, one or more proteins, one or more peptides, or one or more nucleic acids. The plurality of antigens may also comprise a biological sample, wherein the biological sample may be, but is not limited to, a mixture of cells, tissue, blood, serum, plasma, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper fluid, pre-ejaculatory fluid, female ejaculate, sweat, tear, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretions, mucosal secretion, fecal fluid, pancreatic juice, lavage fluid from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In some embodiments, the plurality of antigens comprises at least 11,000 different antigens, such as at least 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 different antigens. In some embodiments, the plurality of antigens comprises at least 0.5% of the human proteome, such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome.

In generating an antibody library, the antibody-producing cells may be B cells. The method of generating an antibody library may further comprise immobilizing the antibodies on a substrate, wherein the substrate may be planar or particulate, comprise a solid or porous material, or any combination thereof. The antibody may be reversibly or irreversibly immobilized on the substrate.

Also provided herein is a method of identifying an antibody that is monospecific for a human protein, the method comprising: (a) contacting a plurality of antibodies with a human proteome array; (b) determining binding between the plurality of antibodies and a target present on a human proteomic array; and (c) identifying the antibody as monospecific when the antibody is monospecific for a single target on the proteome array, wherein the human proteome array may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome, or at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% of the human proteins listed in table 5, 50%, 60%, 70%, 80%, 90% or 100%.

Also provided herein is a method of identifying an antibody to a target, the method comprising: (a) contacting a target with an antibody library or an array comprising an antibody library, (b) determining between the target and the plurality of antibodiesCombination of (1); and (c) identifying an antibody to the target when the target binds to the antibody of the library. The library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies can be produced by the same platform, are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

A method of identifying antibodies to a target can include contacting the target with an antibody library or an array comprising an antibody library comprising at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different antibodies, wherein the antibodies or a subset of the antibodies can be produced by the same platform, are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Methods of identifying antibodies to a target can include contacting the target with an antibody library or an array comprising an antibody library that can bind at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of a human proteome, wherein antibodies or a subset of antibodies can be produced by the same platform and are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Methods of identifying antibodies to a target can include contacting the target with an antibody library or an array comprising an antibody library that can bind at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5, wherein the antibodies or a subset of the antibodies can be produced by the same platform, are monospecific antibodies; binds to its native form of the target protein; is monoclonalAn antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10-⁸M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof.

Also provided herein is a method of detecting a target, the method comprising: (a) contacting a target with an antibody library or an array comprising an antibody library, (b) determining the presence or absence of binding between the target and the plurality of antibodies; and (c) detecting the target when the target binds to at least one antibody of the library. The library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies can be produced by the same platform and are monospecific antibodies; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a binding affinity for its target that is within at least 20%, such as within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% of the binding affinity of another antibody of the plurality of antibodies produced by the same platform; has at least 10 to its target^-7M(K_D) Such as at least 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16Binding affinity of M; or any combination thereof. In some embodiments, the method further comprises detecting a plurality of targets. In some embodiments, the method includes detecting 50,75. 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different targets. In some embodiments, the method comprises detecting the level of one or more targets.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 depicts: A. a method of selecting antibodies by immunizing an animal, producing hybridomas from an animal, inoculating hybridomas, probing the produced antibodies against a library, and selecting antibodies with a desired binding profile (profile); methods for the production and validation of super-specific monoclonal antibodies (MAbs) by live cell immunization, hybridoma selection, MAb identification and validation.

FIG. 2 depicts a 3-D pool analysis (pooling) strategy.

FIG. 3 depicts a flow chart of LR recombinant cloning into the target vector using BsrGI digestion of 8,064 clones.

FIG. 4 depicts the preparation of a human proteome chip. A.17,000 human proteins were purified as N-terminal GST-Hisx6 fusion proteins and the quality was monitored by silver staining and immunoblot analysis. B. 17,000 purified human proteins and controls were spotted in duplicate onto one slide. The immobilized proteins were visualized using anti-GST. Higher magnification view of c.b indicates that the array is of good quality.

FIG. 5 depicts the mass spectrometry (profiling) of monoclonal antibodies (mAbs) on the human protein chip. A.1,058 protein GST images of human protein chips. B. Monoclonal antibodies (mabs) raised against pirin were tested against 1,058 protein pairs on the chip, and the mabs only recognized pirin. The purple boxes indicate mouse IgG loci. C. The other four mabs also showed similar specificity as the anti-pirinmAb.

Fig. 6 depicts a pool analysis strategy to facilitate mAb-specific protein microarray analysis. Hybridoma supernatants (A-L) were pooled in "vertical" and "horizontal" pools before their binding specificity was detected using human protein microarrays. The specificity of a single mAb was identified by determining which proteins on the microarray were bound by which combination of vertical and horizontal pools.

In the examples shown here, the pool of mabs that recognize a particular protein is denoted by X. The results show that only the "D" pool containing the mAb recognizes the antigen.

Figure 7 depicts a high throughput protein purification scheme developed for bacteria. A. A flow chart of the scheme. B. Coomassie staining of purified proteins from 1.5mL e.coli (e.coli) cultures. C. Preparation of Escherichia coli proteome chip.

FIG. 8 depicts the subcellular localization of four human proteins using ICC. C11orf68, CNTD1, DYDC2 and TXNDC9, which were not previously known for their subcellular localization, now localized to the mitochondria, cytosol, plasma membrane and endoplasmic reticulum/golgi apparatus, respectively.

Figure 9 depicts the validation and characterization of mmabs (monospecific monoclonal antibodies). Cells were transfected with either the V5-labeled antigen construct (lane 1) or the empty vector (lane 2), or with the antigen constructs co-transfected with their corresponding shRNA constructs (lane 3). At 2 days post-transfection, cells were lysed and immunoblotted with each corresponding mMAb (upper panel). For comparison, the same blot was removed (strip) and re-probed with anti-V5 antibody (lower panel).

Figure 10 shows many mmabs as IP levels. HeLa cells transfected with the V5-labeled antigenic construct were immunoprecipitated with their corresponding mmabs and immunoblotted with anti-V5 antibody. Lane 1: inputting; lane 2: an antigen immunoprecipitated using mMAb; lane 3: IgG negative control; lane 4: anti-V5 positive control.

Figure 11 depicts the characterization of anti-HNRPCmMAb. A. ICC analysis using anti-HNRPC clearly showed different nuclear localisation. B. After immunoprecipitation of transfected HeLa cells with anti-HNRPC and immunoblotting with anti-V5, a single band of V5-labeled HNRPC was visualized. Anti-mouse IgG and anti-V5 antibodies were used as negative and positive controls, respectively. C. Successful use of chromatin immunoprecipitation (ChIP)

Fig. 12 depicts the continuous measurement of mAb affinity using oblique incidence light reflectance difference method (OIRD). Red, blue and black curves represent smoothed and averaged Im { OIRD } values obtained by probing the pilot (pilot) IgG microarray with anti-rabbit, anti-mouse and anti-human secondary antibodies, respectively. The red dashed line shows the start of the estimated saturation phase.

Detailed Description

The present invention relates generally to antibody libraries, including methods and systems for producing, generating, characterizing, and utilizing antibodies. The antibody may be highly specific. The library may comprise a plurality of different antibodies, wherein the antibodies are produced by the same platform. The library may comprise a plurality of different antibodies, wherein within the plurality of antibodies or a subset of the plurality of antibodies, each antibody is a monospecific antibody; binds to its native form of the target protein; is a monoclonal antibody; is an immunoprecipitated antibody; is an IgG antibody or an IgG isotype antibody; has a junction to its target with another antibody of the plurality of antibodies(ii) binding affinities similar to avidity; has at least 10 to its target^-7M(K_D) Binding affinity of (a); or any combination thereof.

Platform

The antibody library may comprise a plurality of different antibodies produced by the same platform. In one embodiment, the antibody library comprises a plurality of different antibodies, wherein at least 10% of the plurality of antibodies are produced by the same platform. For example, the library can comprise a plurality of different antibodies, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies are antibodies produced by the same platform.

When the first and second antibodies are produced by the same protocol, these antibodies are considered to be produced by the same platform. For example, a plurality of antibodies can be produced by a platform as shown in figure 1A and/or figure B. In one embodiment, an animal can be immunized with a plurality of antigens as described in step 102 of fig. 1A. The animal may be a non-human animal, such as a bovine, avian, canine, equine, feline, ovine, porcine, or primate. The animal may be a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, or goat.

The plurality of antigens may comprise purified or non-purified samples, such as purified or non-purified cells, proteins, peptides, or nucleic acids. In another embodiment, the plurality of antigens may comprise a non-purified sample, such as a crude lysate or an unpurified cell, protein, peptide, or nucleic acid sample.

The plurality of antigens may comprise a biological sample. For example, the plurality of antigens may comprise a single cell or a plurality of cells, proteins, representative peptides, or tissues from an organism. The organism may be a human or a non-human. The non-human organism may be a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, or goat. The biological sample can be tissue, blood, serum, plasma, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In one embodiment, the biological sample may be substantially depleted of common serum proteins, such as, but not limited to, albumin or IgG. Exclusion may include filtration, fractionation or affinity purification.

The biological sample may comprise cells, such as stem cells, undifferentiated cells, differentiated cells, or cells from a diseased individual or an individual with a particular condition. The disease or condition may be cancer, an inflammatory disease, an immune disease, an autoimmune disease, a cardiovascular disease, a neurological disease, an infectious disease, a metabolic disease, or a perinatal condition. For example, the cancer may be breast cancer, ovarian cancer, lung cancer, colon cancer, colorectal cancer, prostate cancer, melanoma, pancreatic cancer, brain cancer, hematologic malignancies, hepatocellular cancer, cervical cancer, endometrial cancer, head and neck cancer, esophageal cancer, gastrointestinal stromal tumors (GIST), Renal Cell Carcinoma (RCC), or gastric cancer.

The plurality of antigens may also comprise samples produced in bacterial (such as, but not limited to, e.g., e.coli), yeast, mammalian, or insect cells, such as proteins overexpressed by an organism. Purified or unpurified antigens, such as in the form of over-expressing cells or cell mixtures expressing an antigen of interest, can be used to immunize an animal.

The plurality of antigens may comprise at least 11,000 different antigens, such as at least 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 different antigens.

In step 104, antibody-producing cells, such as lymphoid cells, from the animal can then be isolated for production of a variety of antibodies. Antibody-producing cells can be used to produce hybridomas. The antibody-producing cell may be a B cell. B cells can be fused with myeloma cells to produce hybridomas. Examples of myeloma cells include, but are not limited to, NS-1, P3U1, SP2/0, AP-1, and the like.

In step 106, antibody-producing cells, such as hybridoma cells, may then be used to produce clones. For example, hybridoma cells can be placed on a semi-solid medium to rapidly produce clones. Clones of interest can be isolated using fluorescently labeled antigens or isotype specific probes for characterization and propagation.

In step 108, the clone of interest is probed against the antigen library to detect the antigen recognized by each antibody and at the same time obtain information about which antigens it does not react with (eliminating false positives). The antigen library may represent at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteome of the organism. The antigen library may represent most or the entire proteome of an organism, such as the proteome of a bacterium, virus, fungus. The antigen library may represent most or the entire proteome of an insect or mammal (such as a mouse, rat, rabbit, cat, dog, monkey, goat, or human). For example, an antigen library may comprise at least 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 different antigens. The antigen library may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the human proteins listed in table 5. The antigen library may also comprise a library of fusion proteins that represent the proteome of the organism in unpurified form, such as a collection of over-expressed cells (e.coli, yeast, mammalian, insect, or other organisms).

Detection can be performed by arraying cells overexpressing an antigen such as a fusion protein or cells of a different cell line (e.g., cancer cell line) and detecting hybridoma supernatants against the arrayed cells. The arranged cells can be permeabilized. Detection can also be performed by fluorescence flow cytometry and the target identified by PCR or sequence analysis of the intracellular recombinant DNA.

In step 110, the antibody has a desired binding profile. The selected antibody may be a highly specific monoclonal antibody that recognizes only one target and does not cross-react with other targets in the organism's proteomic library. Cell lines secreting these monoclonal antibodies can be expanded. Antibodies produced by the same platform or protocol can be used to generate or form antibody libraries.

Antibodies can also be produced by the same platform when they are produced by the same method or protocol as described further below in the method of producing the library.

Monospecificity

The antibody library may comprise a plurality of different antibodies, each antibody having a specific binding specificity for its target. For example, an antibody library may comprise a plurality of different antibodies, wherein the antibodies are monospecific. In one embodiment, the antibody library comprises a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies are monospecific.

When incubated on an array comprising the antigens listed in table 5, an antibody is monospecific if it has an a value greater than 6 and an S value greater than 3 for the protein or antigen. For one antibody, the number of standard deviations above the average signal intensity across the array is referred to as the a value. When ranking the signal intensity of the antibody against the entire array, the difference between the highest signal and the second highest signal on the array is the S value. For example, the a and S values may be calculated by: separate supernatants from antibody-producing cells or hybridomas are combined into a 12 x 12 two-dimensional pool and these pools are incubated on an array, such as a human proteome microarray, and the antibodies are labeled (such as by Cy 5-conjugated anti-IgG secondary antibodies or any other method of detecting antibodies). After washing and scanning, the signal intensity of each spot (representing the antibody bound to the protein or antigen on the array) is expressed as the ratio of the foreground signal to the background signal. The number of standard deviations above the average signal strength of the entire array is called the a-value. Repeat spots (for each repeat pair of proteins or antigens) with a > 3 were labeled and the results deconvoluted to identify proteins or antigens that were present at the intersection of a single horizontal pool and a single vertical pool and therefore recognized by a single monoclonal antibody. Each candidate highly specific monoclonal antibody (i.e., a > 3) was tested individually against the entire array and a was measured for each spotted protein. The signal strengths are then rank-ordered and the difference between the highest signal and the second highest signal on the array is calculated to obtain the S value. Monoclonal antibodies with A > 6 and S > 3 were identified as monospecific monoclonal antibodies (mMAbs). Bispecific monoclonal antibodies (dMAbs) bind strongly to two different proteins on the array and have values of A > 6 and S > 3.

In some embodiments, the mMAb may have an a value greater than 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, and/or an S value greater than 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

In one embodiment, the array for determining monospecificity of an antibody comprises a proteome library of an organism, such as at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteome of the organism. The antigen library may represent most or the entire proteome of an organism, such as the proteome of a bacterium, virus, fungus. The antigen library may represent most or the entire proteome of an insect or mammal (such as a mouse, rat, rabbit, cat, dog, monkey, goat, or human). For example, a proteomic library of an organism can comprise at least 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 different antigens. A proteomic library of an organism may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5.

Binding affinity

The antibody library may comprise a plurality of different antibodies having a particular binding affinity for their targets. For example, the library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies have a particular binding affinity. For example, the plurality of antibodies can have a dissociation constant (K) for their target proteins, e.g., by their dissociation constant_D) Is determined to be at least 10^-7M, such as at least 10^- ⁸M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^- ¹⁶Binding affinity of M.

The plurality of antibodies can have an association rate constant (k), e.g., by their binding rate constant_on) An assayed binding affinity, wherein the plurality of antibodies has at least 10⁴M^-1s^-1At least 5 × 10⁴M^-1s^-1At least 10⁵M^-1s^-1At least 5 × 10⁵M^-1s^-1At least 10⁶M^-1s^-1At least 5 × 10⁶M^-1s^-1At least 10⁷M^-1s^-1At least 5 × 10⁷M^-1s^-1Or at least 10⁸M^-1s^-1Binding affinity of (4). The plurality of antibodies can have a structure as defined byConstant of rate of dissociation (k)_off) An assayed binding affinity, wherein the plurality of antibodies has less than 10³M^-1s^-1Less than 5 × 10³M^-1s^-1Less than 10⁴M^-1s^-1Less than 5 × 10⁴M^-1s^-1Less than 10⁵M^-1s^-1Less than 5 × 10⁵M^-1s^-1Less than 10⁶M^-1s^-1Less than 5 × 10⁶M^-1s^-1Less than 10⁷M^-1s^-1Less than 5 × 10⁷M^-1s^-1Or less than 10⁸M^- ¹s^-1Less than 5 × 10⁷M^-1s^-1Less than 10⁸M^-1s^-1Less than 5 × 10⁸M^-1s^-1Less than 10⁹M^-1s^-1Less than 5 × 10⁹M^-1s^-1Or less than 10¹⁰M^-1s^-1Binding affinity of (4).

Binding affinity can be determined by surface plasmon resonance, chromatography, or any other method known in the art. Binding affinity can also be determined optically, such as by using real-time methods for detecting biomolecular interactions and/or label-free methods. In one embodiment, oblique incident light reflection difference method (OIRD) is used.

Antibody libraries may comprise a plurality of different antibodies in their native form that bind their target proteins. For example, the library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies bind to the native form of its target protein. In one embodiment, the plurality of antibodies that bind to native forms of their target proteins do not bind to denatured forms of their target proteins under the same binding conditions.

The antibody library may comprise a plurality of different antibodies, wherein one or more antibodies of the library have a binding affinity for its target that is similar to the binding affinity of another antibody of the plurality of antibodies. For example, an antibody library may comprise a first antibody and a second antibody, wherein the binding affinity of the first antibody to a first protein is similar to the binding affinity of the second antibody to a second protein. An antibody of the library can have a binding affinity for its target that is within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the binding affinity of one or more other antibodies of the library. The first antibody of the library may have a binding affinity for its target that is within at least 20% of the binding affinity of the one or more other antibodies of the library. In one embodiment, an antibody library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the different antibodies have a binding affinity for their target protein that is within at least 20% of the binding affinity of the remaining antibodies in the plurality of different antibodies for their respective target proteins.

Proteome

Also provided herein are antibody libraries that can comprise a plurality of different antibodies that bind to a portion of a proteome of an organism. The plurality of different antibodies can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of a proteome of an organism. For example, the plurality of different antibodies can bind to at least 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 proteins of an organism. The proteome can be a proteome of a bacterium, a virus, a fungus. The proteome can be that of an insect or mammal such as a mouse, rat, rabbit, cat, dog, monkey, goat, or human. In some embodiments, the proteome is a human proteome.

For example, the plurality of different antibodies can bind at least 0.5% of the human proteome. In one embodiment, the plurality of different antibodies can bind to at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome. The plurality of different antibodies can bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5.

Other antibody characteristics

The antibody library can also comprise a plurality of different antibodies, wherein the antibodies are IgG antibodies (e.g., IgG isotype antibodies). For example, an antibody library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the antibodies are IgG antibodies or IgG isotype antibodies.

The antibody library may further comprise a plurality of different antibodies, wherein the antibodies are immunoprecipitated antibodies. For example, an antibody library can comprise a plurality of different antibodies, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the antibodies are immunoprecipitating antibodies. Immunoprecipitating antibodies are antibodies that are capable of immunoprecipitating the target protein from cell homogenates as compared to the no antibody negative control and the anti-V5 antibody positive control. Detection of immunoprecipitated targets can be performed by Western blotting. Immunoprecipitation can be performed with clear cell lysate and approximately 2 μ g of antibody, followed by incubation at 4 ℃ for 2 hours, followed by addition of a substrate to bind any antibody-protein complex (such as protein-gdynabare), followed by incubation at 4 ℃ for an additional 2 hours. After incubation, the substrate is washed twice, for example with ice-cold TBST, transferred to a new reaction vessel, washed again with ice-cold TBST, followed by SDS-PAGE and Western blot analysis.

Biological pathways

In one aspect, the invention relates to an antibody library comprising a plurality of antibodies specific for antigens of a common pathway. Antigens belong to a common pathway when they have one or more common attributes in the gene ontology (assigning a defined characteristic to a set of genes and their products). This ontology, governed by the gene ontology ("GO") consortium, is particularly useful in this regard. Antigens belonging to a common pathway can be identified by searching for gene ontologies (such as GO) of genes sharing one or more attributes. A common attribute may be, for example, a common structural feature, a common location, a common biological process, or a common molecular function.

The wealth of information present in published, peer-reviewed literature about the function of human genes and proteins has been organized and managed using a collaborative system of controlled vocabularies managed by the Gene Ontology (GO) Association (http:// www.geneontology.org /). Of the approximately 40,000 transcribed units in the human genome, approximately 20,000 of these encode annotated proteins, and approximately 14,000 of these proteins have functional annotations in the GO database. The functional annotations contained in the GO database are organized in a hierarchical manner, and this information can be accessed from the GO database and searched for all genes in the human genome that are annotated as being involved in the same biological process, present in the same cellular components, or performing the same molecular function.

In some embodiments, the antigens in the common pathway are expression products of genes involved in the same biological process or molecular function as annotated by the gene ontology. An example of this is a gene involved in response to DNA damage. Another example is the gene product of a transcription factor such as a particular tissue, cell type or organ. One example is the gene product of a transcription factor of the brain.

In some embodiments, the antigens in the common pathway are gene products of genes that are all bound by the same transcription factor protein, complex of transcription factor proteins, other nucleic acids or other molecules that bind the protein. These interactions can occur in living cells (in vivo) or in solution of purified molecules (in vitro). For example, all gene products of genes for which transcription factor protein binding is induced by hypoxia.

In some embodiments, the antigen in the common pathway is the gene product of a gene whose transcription level or protein level is altered and thus synergistically regulated when treated or exposed to the same stimulus. For example, all antigens that are induced or inhibited when treated with UV radiation.

In some embodiments, the antigens in the common pathway are antigens comprising similar sequence features. These features may be enrichment of DNA sequence motifs, collections of DNA sequence motifs, or higher order sequence features distinguishable from a background model of random genomic sequence. A sequence motif as used herein is a strand of two or more nucleobases (A, T, C or G). DNA sequence motifs can be determined by consensus sequences or probability matrices, in which the identity of each base at each position of the motif is defined as a probability.

In some embodiments, the antigen in the common pathway may be the gene product of a gene whose sequence, transcript or protein is linked by metabolic transformation and/or physical protein-protein, protein-DNA and protein-compound interactions. These reactions are catalyzed by enzymes, and often require dietary minerals, vitamins and other cofactors for proper functioning. The route can be quite complex, as many chemicals may be involved.

In some embodiments, members of a pathway share a common structural or functional attribute. For example, proteins may share the same sequence motifs, such as zinc fingers or transmembrane regions.

In some embodiments, the antigens in the common pathway belong to the same signal transduction pathway. Generally, in biology, signal transduction refers to any process by which a cell converts one signal or stimulus to another, most commonly involving an ordered series of biochemical reactions within the cell, which are carried out by enzymes, activated by second messengers, resulting in pathways that are considered signal transduction pathways. Generally, signal transduction involves the binding of an extracellular signaling molecule (or ligand) to a cell surface receptor that faces outward from the plasma membrane and triggers an intracellular event. In addition, intracellular signaling cascades can be triggered by cell-substrate interactions, as is the case for integrins that bind ligands found in the extracellular matrix. Steroids represent another example of extracellular signaling molecules that can cross the plasma membrane due to their lipophilic or hydrophobic properties. Many, but not all steroids, possess receptors in the cytoplasm and typically act by stimulating the binding of their receptors to the promoter regions of steroid-responsive genes. Within a multicellular organism, there are a number of different small molecules and polypeptides that are used as a whole to coordinate the individual biological activities of cells in the environment of the organism. Examples of such molecules include hormones (e.g., melatonin), growth factors (e.g., epidermal growth factor), extracellular matrix components (e.g., fibronectin), cytokines (e.g., gamma-interferon), chemokines (e.g., RANTES), neurotransmitters (e.g., acetylcholine), and neurotrophins (e.g., nerve growth factor).

In addition to many of the conventional signal transduction stimuli listed above, there are also examples of other environmental stimuli that trigger signal transduction processes in complex organisms. The environmental stimulus may also be a molecule in nature or more a physical stimulus such as light illuminating cells on the retina of the eye, a flavoring agent that binds to olfactory receptors in the nasal epithelium, bitter and sweet tastes stimulating taste receptors in the taste buds, ultraviolet light that alters DNA in cells, and hypoxia that activates a series of events in cells. Certain microbial molecules, such as viral nucleotides, bacterial lipopolysaccharides, or protein antigens, are capable of eliciting responses of the immune system against invading pathogens that are mediated through signal transduction processes.

Activation of genes, alterations in metabolism, sustained proliferation and death of cells, and stimulation or inhibition of motility are some of the cellular responses to extracellular stimuli that require signal transduction. Gene activation leads to further cellular effects, as many protein products in response to genes include enzymes and transcription factors themselves. Transcription factors produced as a result of the signal transduction cascade may in turn activate more genes. Thus, an initial stimulus can trigger the expression of an entire set of genes, and in turn can lead to the activation of many complex physiological events. These events include, for example, increased glucose uptake from the blood stream stimulated by insulin, and neutrophil migration to the site of infection stimulated by bacterial products.

Most mammalian cells require stimulation to control not only cell division, but also survival. In the absence of growth factor stimulation, programmed cell death will ensue in most cells. Such a need for extracellular stimulation is essential for controlling cellular behavior in both unicellular and multicellular organisms. It is not surprising that signal transduction pathways are so important for biological processes that a large number of diseases are attributed to their dysregulation.

In some embodiments, the invention provides methods and compositions comprising libraries comprising a plurality of antibodies specific for an antigen that is part of an oncology pathway. Antigens in oncological pathways are the gene products of those genes involved in hyperplasia, tumor formation and/or the development of cancer. Examples of oncology pathways include, but are not limited to, hypoxia, DNA damage, apoptosis, cell cycle, and the p53 pathway.

In some embodiments, the invention provides methods and compositions comprising a library comprising a plurality of antibodies specific for an antigen that is part of the membrane pathway. Examples of membrane pathways include, but are not limited to, transporter proteins, G-coupled receptors, ion channels, cell adhesion proteins, and receptor pathways.

In some embodiments, the invention provides methods and compositions comprising libraries comprising a plurality of antibodies specific for an antigen that is part of the nuclear receptor pathway. Examples of antigens in the nuclear receptor pathway include, but are not limited to, gene products regulated by glucocorticoid receptor proteins, estrogen receptor proteins, peroxisome proliferator activated receptor proteins, androgen receptor proteins, and transporter pathways including ABC and SLC transporters.

In some embodiments, the invention provides methods and compositions comprising libraries comprising a plurality of antibodies specific for an antigen that is part of a neuronal pathway. Examples of antigens in neuronal pathways include, but are not limited to, gene products of genes expressed in neurons, such as neurotransmitters and cell adhesion proteins.

In some embodiments, the invention provides methods and compositions comprising a library comprising a plurality of antibodies specific for an antigen that is part of a vascular pathway. Examples of antigens in the vascular pathway include, but are not limited to, antigens involved in angiogenesis, lipid metabolism, and inflammation.

In some embodiments, the invention provides methods and compositions comprising libraries comprising a plurality of antibodies specific for an antigen that is part of a signaling pathway. Examples of antigens in signaling pathways include, but are not limited to, gene products involved in intercellular signaling, hormones, hormone receptors, cAMP responses, and cytokines.

In some embodiments, the invention provides methods and compositions comprising libraries comprising a plurality of antibodies specific for an antigen that is part of an enzymatic pathway. Examples of antigens in the enzymatic pathway include, but are not limited to, gene products of genes involved in glycolysis, anaerobic respiration, tricarboxylic/citric acid cycle, oxidative phosphorylation, fatty acid oxidation (beta-oxidation), gluconeogenesis, HMG-CoA reductase pathway, pentose phosphate pathway, porphyrin synthesis (or heme synthesis) pathway, urea cycle, photosynthesis (plants, algae, cyanobacteria), and chemical synthesis (some bacteria).

The invention also provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of which are part of a common pathway.

In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of an oncology pathway. In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a hypoxia pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a DNA damage pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of an apoptotic pathway. In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a cell cycle pathway. In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the p53 pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are differentially selected from the hypoxic pathway, the DNA damage pathway, the apoptotic pathway, the cell cycle pathway, and the p53 pathway.

In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a membrane binding pathway.

In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the nuclear receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the glucocorticoid receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the peroxisome proliferator activated receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the estrogen receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the androgen receptor pathway. In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of the cytochrome P450 receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a transporter receptor pathway. In some embodiments, the present invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are differentially selected from the glucocorticoid receptor pathway, the peroxisome proliferator activated receptor pathway, the estrogen receptor pathway, the androgen receptor pathway, the cytochrome P450 pathway, and the transporter pathway.

In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a vascular pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a neuronal pathway. In some embodiments, the invention provides libraries comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a transcription factor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of which are part of a signaling pathway.

The invention also provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in a genome that are part of a common pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of an oncology pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a hypoxia pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a DNA damage pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in a genome that are part of an apoptotic pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a cell cycle pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a p53 pathway.

In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a membrane binding pathway.

In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a nuclear receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in the genome that are part of the glucocorticoid receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a peroxisome proliferator activated receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of an estrogen receptor pathway. In some embodiments, the present invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of an androgen receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a cytochrome P450 receptor pathway. In some embodiments, the invention provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of all antigens in a genome that are part of a transporter receptor pathway.

The invention also provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in a genome that are part of a neuronal pathway. The invention also provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in a genome that are part of a signaling pathway. The invention also provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in a genome that are part of a vascular pathway. The invention also provides a library comprising a plurality of antibodies specific for a plurality of antigens, wherein the library represents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of all antigens in a genome that are part of a transcription factor pathway.

Method for generating antibody library

Also provided herein are methods of generating antibody libraries. In one embodiment, the method comprises (a) immunizing an animal with a plurality of antigens; (b) isolating antibody-producing cells from the animal; (c) isolating a plurality of antibodies from the antibody-producing cells; (d) screening the plurality of antibodies of step c) using a protein array (such as a human protein array); and (d) selecting antibodies that are monospecific for a single target on the proteomic array. In one embodiment, the method may further comprise pre-screening for a plurality of antibodies from the antibody-producing cells prior to step c).

The animal can be a non-human animal, such as a bovine, avian, canine, equine, feline, ovine, porcine, or primate. The animal may be a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, or goat.

Animals may be immunized with a variety of antigens that may comprise purified or non-purified samples, such as purified or non-purified cells, proteins, peptides, or nucleic acids. In another embodiment, the plurality of antigens may comprise a non-purified sample, such as a crude lysate or an unpurified cell sample, protein sample, peptide sample, or nucleic acid sample. The plurality of antigens may comprise a biological sample. For example, the plurality of antigens may comprise a single cell or a plurality of cells, proteins, representative peptides or tissues from an organism such as bovine, avian, canine, equine, feline, ovine, porcine, or primate. The organism may be a human or non-human. The non-human organism may be a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, or goat.

The biological sample can be tissue, blood, serum, plasma, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In one embodiment, the biological sample may be substantially depleted of common serum proteins, such as, but not limited to, albumin or IgG. Exclusion may include filtration, fractionation or affinity purification.

The plurality of antigens may also comprise a sample produced in a bacterial (such as, but not limited to, e.coli), yeast, mammalian or insect cell, such as a protein overexpressed by an organism. Purified or unpurified antigens, such as in the form of over-expressing cells or cell mixtures expressing an antigen of interest, can be used to immunize an animal.

The plurality of antigens may comprise at least 11,000 different antigens, such as at least 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 different antigens. In some embodiments, the plurality of antigens comprises at least 0.5% of the human proteome. The plurality of antigens may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteome of the organism. The plurality of antigens may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome. The plurality of antigens may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteins listed in table 5. The antigen library may also comprise a library of fusion proteins that represent the proteome of an organism in unpurified form, such as a collection of over-expressed cells (e.coli, yeast, mammalian, insect, or other organisms).

The antibody-producing cells from the animal can be lymphoid cells, such as B cells. Antibody-producing cells can be used to produce hybridomas, for example, by fusion with immortalized cells such as myeloma cells to produce hybridomas.

In one embodiment, a pre-screening step is performed in which a plurality of antibodies from antibody-producing cells are screened prior to isolating the plurality of antibodies from the antibody-producing cells. Pre-screening can be performed by using serum or supernatant of antibody-producing cells to determine binding of antibodies from the antibody-producing cells to a mixture comprising one or more target antigens, such as native antigens or proteins. The pre-screening may be performed using immunohistochemistry, immunocytochemistry, ELISA, chromatography, or any other suitable method known in the art for determining binding. Pre-screening can be used to select antibody-producing cells (which can include antibody-secreting cells that have been fused to immortal cells, such as hybridomas) for further screening, such as by a protein array as described herein.

The mixture may comprise crude lysate, cells, proteins, peptides, or nucleic acids. The mixture may comprise a biological sample such as tissue, blood, serum, plasma, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper fluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretions, mucosal secretion, fecal water, pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In one embodiment, the biological sample may be substantially depleted of common serum proteins, such as, but not limited to, albumin or IgG. Exclusion may include filtration, fractionation or affinity purification. The biological sample may comprise cells, such as stem cells, undifferentiated cells, differentiated cells, or cells from a diseased individual or an individual with a particular condition.

Multiple antibodies from antibody-producing cells can be isolated with or without a prior pre-screening step, and then screened using the whole or a portion of the organism's proteome. For example, an isolated antibody can be screened using at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteome of the organism. For example, isolated antibodies can be screened using at least 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 organism proteins. The proteome can be a proteome of a bacterium, a virus, a fungus. The proteome can be that of an insect or mammal such as a mouse, rat, rabbit, cat, dog, monkey, goat, or human. In some embodiments, the proteome is a human proteome.

For example, at least 0.5% of the human proteome can be used to screen for isolated antibodies. In one embodiment, the isolated antibody is screened using at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome. Isolated antibodies can be screened using at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5. The proteome or portion thereof used to screen the antibodies can be present on an array.

After screening, antibodies can be selected based on their binding profiles. For example, if an antibody binds to a single target of a proteome, e.g., binds to a single target of a human proteome array, the antibody can be selected for the library described herein.

The method can be used to perform high throughput production of antibodies. For example, antigens for immunizing mice can be produced in a high throughput manner. This can be done as follows: 1) recombinant proteins are produced from gene expression libraries in a targeted manner, wherein the protein of interest can be expressed as desired or by using whole heterologous cells, tissues or crude or enriched cell extracts from the target species (non-directed methods) or whole mammalian cells designed for expression of the heterologous protein (directed methods). Extracts for injection may include subcellular components such as cell membranes and intracellular membranes, organelles, particles, or protein complexes, and may be prepared using differential centrifugation or other well-known methods. These immunizations with complex mixtures of proteins eventually produce antibodies against the most immunogenic proteins; in this event, the antigen source may be immunodepleted using previously isolated antibodies to remove any particular antigenic protein. In addition, soluble proteins and membrane proteins can be chromatographed in their native or denatured state by size exclusion, ion exchange, hydrophobic chromatography, or affinity chromatography, or by using small fractions collected separately to immunize with tens to hundreds, rather than thousands, of proteins. Likewise, simple ammonium sulfate fractionation of the extract will provide a variety of antigen subsets. Specific techniques for targeting specific classes of proteins can be used. For example, proteins involved in SNO signaling can be purified by Immobilized Metal Affinity Chromatography (IMAC), after which eluted metal-free proteins can be used as antigens for immunization, or phosphoproteins can be bound to TiO₂Thereby being greatly concentrated on the affinity matrix. Native proteins isolated or obtained directly from living or fixed cells may be a preferred source of immunogen.

High throughput methods may also include short-time immunizations to produce antibody-secreting lymphoid cells. For example, animals can be immunized in a footpad fashion with a variety of antigen and adjuvant formulations. The popliteal lymph nodes were collected from 7 to 21 days after immunization. Lymph nodes in immunized animals were visualized directly by injection of evans blue at the footpad.

High throughput methods may also include the production of antibodies in GST-tolerant mice. Since many antigens are expressed as GST fusions, a strain of mice expressing GST (mice actively expressing GST from schistosoma japonicum (s.) japonicum) can be used to increase the yield of specific antibodies against the non-GST component of the fusion protein and eliminate the production of anti-GST antibodies.

High throughput methods for producing antibodies may also include the construction of cell fusions/hybridomas for antibody production. The antigen-contacted lymphoid cells and myeloma cells are fused, and the fusion product is seeded on a semi-solid medium (methylcellulose) containing a fluorescently labeled antibody that recognizes the desired subclass of immunoglobulin molecules, or a semi-solid medium containing a fluorescently labeled antigen that will label colonies that secrete the desired antibody. Combinations of the above methods may also be used. Fluorescently labeled colonies were transferred from the semi-solid medium to a liquid medium using an inverted fluorescence microscope in combination with manual picking using a Drummond microtube and Drummond Wiretrol device, and individual clones were then grown.

For monoclonal antibodies produced in high throughput methods using recombinant or native proteins, the corresponding antigen can be identified by deconvolution of protein microarrays. A single step deconvolution can be performed.

For example, when whole cells or cell lysates are used to generate hybridomas, a two-dimensional pool analysis strategy is applied to reveal the identity of the antigen that each monoclonal antibody will recognize. Supernatants from hybridomas were arranged in a two-dimensional grid and pool analyses were performed horizontally and vertically at pool sizes of 3 × 3 to 100 × 100. Three-dimensional pool analysis strategies can also be performed in which hybridomas are arranged in flat panel sets to produce flat-panel pools in a3 x 3 to 100 x 100 format as well as horizontal and vertical pools. The resulting pools were individually hybridized to protein microarrays and scored using microarray analysis software. In two-and three-dimensional designs, respectively, the positives (hits) shared by each horizontal and vertical pair or at each 3-way intersection of the plate, horizontal and vertical pools (called triple recombination) were identified as antigens recognized by monoclonal antibodies located at the same pair or triple intersection. If necessary, the identified antigen is confirmed by probing the microarray with the corresponding antibody. Pool analysis strategies can reduce the cost of characterizing or producing antibodies because the cost of the array is high, and the use of, for example, 10 x 10 pools reduces the number of arrays required to analyze 100 clones from 100 to 20; for a 20 x 20 pool, it can reduce the number of arrays required from 400 to 40. For example, a three-dimensional pool can screen a batch of 4096 hybridomas, whereas a 16 x 16 3D strategy requires only 48 arrays. Using the 10 x 10 strategy, 820 arrays would be available. The 96-well plate based 3D pool assay strategy (12 × 8 × 12) is depicted in fig. 2.

Characterization of monoclonal antibodies produced in high throughput methods can be performed using complete proteome microarrays. The microarray can be used to determine the specific antigen to which a given monoclonal antibody binds. Key quality information about monoclonal antibody affinity, potential cross-reactivity with other antigens, or lack of cross-reactivity is provided by array analysis.

Production of monoclonal antibodies in high throughput methods can be from fusion clones. Monoclonal antibodies are produced in desired amounts in vitro or in vivo. These antibodies can be purified using various established methods.

Based on the characterization of monoclonal antibodies using protein microarrays, monoclonal antibodies of high quality (e.g., high affinity and low cross-reactivity) can be selected and used to generate antibody arrays. Antibodies were selected and their concentrations were normalized to similar titers. They can be aligned in a multi-well format (e.g., 96-well, 384-well, or 1562-well) with appropriate positive (e.g., diluted human IgG) and negative (e.g., human IgM and BSA) controls to prepare antibody microarrays using a microarray robot (e.g., Nanoprint, ArrayIt, Inc.). The arrangement of monoclonal antibodies in different microarray configurations can be tailored to facilitate a series of whole proteomic studies.

Methods of using antibody libraries

Also provided herein are methods of identifying an antibody to a target, the method comprising contacting the target with a library of antibodies, determining binding between the target and a plurality of antibodies; and identifying an antibody to the target when the target binds to the antibody of the library. Also provided herein are methods of identifying a target, the method comprising contacting the target with a library of antibodies, determining binding between the target and a plurality of antibodies; and identifying an antibody to the target when the target binds to the antibody of the library. The antibody library can be attached to a substrate such that the target is in contact with an array comprising the antibody library.

The library may comprise a plurality of different antibodies such as those described above. For example, an antibody library may comprise antibodies produced by the same platform. The library may comprise a plurality of different antibodies, wherein in the plurality of antibodies or a subset of the plurality of antibodies (such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the plurality of antibodies), the antibodies are produced by the same platform, are monospecific, bind to the native form of their target protein; is monoclonal; to immunoprecipitate antibodies; is an IgG antibody (e.g., an IgG isotype antibody); has a binding affinity for its target that is similar to (e.g., within at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) of the binding affinity of another antibody of the plurality of antibodies; each antibody has at least 10 for its target^-7M(K_D) (e.g., such as at least 10)^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M、10^-14M、10^-15M or 10^-16M) binding affinity; or any combination thereof. The libraryCan comprise at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 1000 different monoclonal antibodies that bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of a biological proteome (e.g., a human proteome), bind to at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 100% of human proteins listed in Table 5, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%, or any combination thereof.

Also provided herein are methods of identifying an antibody that is monospecific for a protein (such as a human protein), the method comprising: contacting a plurality of antibodies with a proteome array (such as a human proteome array); determining binding between the plurality of antibodies and a target present on the proteomic array; and identifying the antibody as monospecific. The array can comprise a plurality of antigens or proteins comprising at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteome of the organism. For example, a proteomic array can comprise at least 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 proteins of an organism. The organism may be a bacterium, virus or fungus. The organism may be an insect or a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, goat or human. For example, the proteome may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteome. The proteome array may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5.

Binding can be detected by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay (e.g., using colloidal gold, enzyme, or radioisotope labels), Western blot, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, immunofluorescence assay, protein a assay, and immunoelectrophoresis assay).

Binding of the antibody can be detected by detecting a label on the primary antibody. Alternatively, the primary antibody is detected by detecting the binding of a secondary antibody or reagent to the primary antibody. For example, the secondary antibody may be labeled. In some embodiments, an automated detection assay or high throughput system is employed. For example, in a capture micro-enzyme-linked immunosorbent assay (ELISA), the antibody/antigen reaction is made measurable by immobilization of the antibody and subsequent direct or indirect colorimetric, fluorescent, luminescent or radioactive detection of the bound labeled antigen. For example, the antigen may be labeled with biotin or other marker that will allow downstream detection.

The immobilized antibody will typically bind to a single epitope present. The antigenic determinants can be labeled by, for example, a label comprising a biomarker for the antigenic determinant. The specificity of this reaction will allow quantification in an ELISA assay. All hybridoma supernatants can be screened via the following steps using ELISA reactions in a high throughput format. Screening assays based on other principles than ELISA can be used (e.g., antibody microarray, high throughput screening based on MALDI/MS and/or multi-channel capillary electrophoresis). ELISA or microarray data can be evaluated by, for example, published methods. The objective of the data analysis process is to select hybridoma supernatants that show the best set that has important clinical parameters and is specific for one analyte group.

Microarray substrate

In some embodiments, the invention provides an array (or microarray) comprising a library of antibodies and a substrate. In some embodiments, each antibody is immobilized on a substrate. The antibody may be reversibly or irreversibly immobilized on the substrate. The substrate may be planar or particulate and may comprise a solid or porous material. The substrate may be organic or inorganic, biological or non-biological, or any combination of these materials.

In one embodiment, the substrate is transparent or translucent. The surface portion of the substrate on which the patches (patches) are located is preferably flat and strong or semi-strong. Many materials are suitable for use as the substrate. The substrate may comprise silicon, silicon dioxide, glass, or a polymer. For example, the substrate may comprise a material selected from the group consisting of silicon, silicon dioxide, quartz, glass, controlled pore glass, carbon, alumina, titanium dioxide, germanium, silicon nitride, zeolites, and gallium arsenide. Many metals, such as gold, platinum, aluminum, copper, titanium, and alloys thereof, are also options available for the array substrate. In addition, many ceramics and polymers may also be used as substrates. Polymers that can be used as substrates include, but are not limited to, the following materials: polystyrene; polytetrafluoroethylene; polyvinylidene fluoride; a polycarbonate; polymethyl methacrylate; a polyvinyl ethylene; a polyethyleneimine; polyether ether ketone; polyoxymethylene (POM); polyvinyl phenol; polylactic acid; polymethacrylimide (PMI); polyalkene sulfone (PAS); polyhydroxyethyl methacrylate; polydimethylsiloxane; polyacrylamide; a polyimide; a block copolymer; andphotoresistEtching agents, polymeric L-B films, and LIGA structures may also be used as substrates in the present invention.

In one embodiment, a microarray of different antibodies is bound to a slide coated with a polycationic polymer. According to another aspect of the invention, a substrate is formed and is intended for detecting binding of a target molecule to one or more different antibodies. In one embodiment, the substrate comprises a glass substrate having a coating of a polycationic polymer (preferably, a cationic polypeptide such as polylysine or polyarginine) formed on a surface thereof. Microarrays of different biopolymers are formed on the polycationic coating, each localized to a known selected area of the array, such as a region.

The glass slide can be coated by placing a uniform thickness film of a polycationic polymer, e.g., poly-L-lysine, on the surface of the glass slide and drying the film to form a dried coating. The amount of polycationic polymer added is sufficient to form at least a monolayer of polymer on the glass surface. The polymer film is bound to the surface by electrostatic binding between negatively charged silyl-OH groups on the surface and charged amine groups in the polymer. Poly L-lysine coated glass slides are commercially available from, for example, sigma chemical co (st.

Suitable microarray substrates are also prepared by chemical derivatization of glass. A silane compound having a suitable leaving group on the terminal Si is covalently bound to the glass surface. The derivatized molecules may be designed to impart desired chemical properties to the surface of the glass substrate. An example of such a bifunctional agent is amino-propyl-tris (ethoxy) silane, which reacts with the glass surface at the tris (ethoxy) silane portion of the molecule while leaving the amino portion of the molecule free. The surface with terminal amino groups is suitable for adsorbing biopolymers in the same way as polylysine coated slides. The identity of the terminal surface groups may be modified by further chemical reactions. For example, the reaction of a terminal amine with glutaraldehyde in the above examples produces a terminal aldehyde group. Further modification layers may be applied prior to spotting the microarray to obtain the desired reactivity, such as by applying a protein a or protein G solution to a silylated (silylated) glass. Other binding polypeptides have surfaces that are nitrocellulose-coated glass slides and protein-bound plastics such as polystyrene, commercially available from Schleicher and Schuell.

The spotted antibodies can be attached by adsorption or covalent binding. Adsorption occurs through electrostatic interactions, hydrophobic interactions, van der waals interactions, or hydrogen bonding interactions between the spotted polypeptide and the array substrate. Simple application of a polypeptide solution to a surface in an aqueous environment is sufficient to adsorb the polypeptide. Covalent attachment is achieved by reaction of functional groups on the polypeptide with a chemically activated surface. For example, if the surface has been activated with a highly reactive electrophilic group such as an aldehyde or succinimide group, the unmodified polypeptide reacts at the amine group, such as at a lysine residue or a terminal amine, to form a covalent bond.

To form a microarray, defined volumes of different biopolymers are deposited on polymer-coated slides using any suitable method known in the art. According to an important feature of the substrate, when an aqueous sample is applied to the substrate under conditions that allow binding of the labeled ligand in the sample to the cognate binding pair in the substrate array, the deposited antibody remains non-covalently bound to the coated slide surface.

In some embodiments, each microarray is less than about 1cm each²Comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 different antibodies on the surface area of (a). In one embodiment, at about 16mm²The microarray comprises 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or 400 regions, or 2.5 × 10³Area/cm². In addition, in a preferred embodiment, the antibodies in each microarray region are present in a defined amount between about 0.1 femtomoles and 100 nanomolar.

In addition, in a preferred embodiment, the biopolymers have a length of at least about 50 units, e.g., amino acids, nucleotides, etc., i.e., are significantly longer than polymers that can be formed in high density arrays by various in situ synthesis schemes.

Use of microarrays

The microarrays of the invention can be used for medical diagnostics, drug discovery, molecular biology, immunology and toxicology.

Microarrays of immobilized antibodies prepared according to the invention are useful in large scale binding assays in a number of diagnostic and screening applications. Multiplex measurements of quantitative variation at the level of a large number of targets (e.g., proteins) allow for the identification of patterns defined by several to many different targets (e.g., proteins). Many physiological parameters and disease-specific patterns can be evaluated simultaneously.

One embodiment relates to the isolation, identification and characterization of proteins present in a biological sample. For example, by comparing a disease sample to a control sample, a "disease-specific protein" can be identified. These proteins can be used as targets for drug development or as molecular markers for disease.

Antibody arrays can be used to monitor the expression levels of proteins in samples, where such samples can include biopsies of tissue of interest, cultured cells, microbial cell populations, biological fluids including blood, plasma, lymph, synovial fluid, cerebrospinal fluid, cell lysates, culture supernatants, amniotic fluid, and the like, and derivatives thereof. Of particular interest are clinical samples of biological fluids, including blood and its derivatives, cerebrospinal fluid, urine, saliva, lymph, synovial fluid, and the like. Such assays may be quantitative, semi-quantitative or qualitative. Where the assay is quantitative or semi-quantitative, it preferably comprises a competitive format, for example between labelled and unlabelled samples, or between differentially labelled samples.

The assay for detecting the presence of the target molecule of the immobilized polypeptide can be performed as follows, but the method is not necessarily limited to those methods described herein and includes any suitable method known in the art.

The sample, fraction or aliquot thereof is added to a microarray comprising antibodies. The sample may comprise a plurality of biological fluids or extracts as described above. Preferably, a series of standards containing known concentrations of control ligands are assayed as controls in parallel with the sample or fraction thereof. The incubation time should be sufficient for the target molecule to bind to the polypeptide. Typically about 0.1-3 hours, usually 1 hour, but may be as long as 1 day or longer.

After incubation, the non-binding components on the insoluble support are typically washed away. Typically, a dilute non-ionic detergent medium of suitable pH (typically 7-8) is used as the wash medium. 1-6 washes can be employed, using a volume sufficient to thoroughly wash away non-specifically bound proteins present in the sample.

To detect the presence of bound target, a number of methods can be used. These are divided into three general groups. The target itself can be labeled with a detectable label and the amount of bound label determined directly. Alternatively, a labeled sample may be mixed with a differentially labeled or unlabeled sample in a competition assay. In yet another embodiment, the sample itself is not labeled, but a second stage labeling reagent is added to quantify the amount of ligand present.

Examples of labels that allow direct measurement of ligand binding include radioisotope labels (such as³H or¹²⁵I) Fluorescent agents, dyes, beads, chemiluminescent agents, colloidal particles, and the like. Suitable fluorescent dyes are known in the art and include Fluorescein Isothiocyanate (FITC); rhodamine and rhodamine derivatives; texas Red; phycoerythrin; allophycocyanin; 6-carboxyfluorescein (6-FAM); 2 ', 7' -dimethoxy-4 ', 5' -dichloro-6-carboxyfluorescein (JOE); 6-carboxy-X-Rhodamine (ROX); 6-carboxy-2 ', 4 ', 7 ', 4, 7-Hexachlorofluorescein (HEX); 5-carboxyfluorescein (5-FAM); n, N' -tetramethyl-6-carboxyrhodamine (TAMRA); sulfonated rhodamine; cy 3; cy5, and the like. Preferably, the compound to be labelled is bound to an activated dye which binds to a group present on the ligand (e.g.amino, thiol, or the like),Aldehyde group, etc.) to react.

In particular, where the second stage detection is carried out, for example by addition of a labelled antibody which recognises the target, the label may be a covalently bound enzyme which is capable of providing a detectable product signal following addition of a suitable substrate. Examples of suitable enzymes for use in the conjugate include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, and the like. Such antibody-enzyme conjugates can be readily produced by techniques known to those skilled in the art when not commercially available. The second stage binding reagent may be any compound that specifically binds the target molecule sufficiently such that it is distinguishable from the other components present. In a preferred embodiment, the second stage binding reagent is a ligand-specific antibody that is a monoclonal or polyclonal serum, e.g., a mouse anti-human antibody or the like.

For amplification of the signal, the ligand may be labelled with a reagent such as biotin, digoxigenin, etc., where the second stage reagent will comprise avidin, streptavidin, an anti-digoxigenin antibody, etc., suitable for the label.

Binding of the ligand can be detected by scanning the microarray, for example, using a scanning laser microscope, a fluorescence assay, a modified ELISA plate reader, and the like. For example, a scanning laser microscope may scan individually for each fluorophore used, using a suitable excitation light line. The digital images produced by the scan are then combined for subsequent analysis. For any particular array element, the ratio of the fluorescence signal from one label is compared to the fluorescence signal from another labeled DNA, and the relative abundance is determined.

Microarrays and methods for detecting target molecules can be used in a variety of screening, research, and diagnostic assays. In one application, the antibody array binds to total protein from an organism, thereby monitoring protein expression for research or diagnostic purposes. Labeling total proteins from normal cells with one color of fluorophore and from diseased cells with another color of fluorophore, and simultaneously combining both samples with the same array, allows determination of different protein expression as a ratio of the intensities of the two fluorophores. The two-color experiment can be used to monitor expression in different tissue types, disease states, response to drugs, or response to environmental factors.

In screening assays, for example, to determine whether a protein or proteins are associated with a disease pathway or are associated with a disease-specific phenotype, the assay can be performed on cultured cells. These cells can be manipulated experimentally by adding pharmacologically active agents that act on the target or pathway of interest. This application is critical to elucidating biological functions or the discovery of therapeutic targets.

For many diagnostic and research purposes, it is useful to determine the level of a target molecule, such as a protein, in blood or serum. This application is crucial for the discovery and diagnosis of clinically useful markers relevant to a particular diagnosis or prognosis. For example, by monitoring a series of antibody or T cell receptor specificities in parallel, the level and kinetics of antibodies in the course of autoimmune disease, during infection, in graft rejection, etc. can be determined. Alternatively, novel protein markers associated with a disease of interest can be developed by comparison of normal and diseased blood samples or by comparison of clinical samples at different stages of the disease.

Information about protein expression in the genome of an organism can have many applications, including but not limited to the diagnosis and treatment of disease in a personalized manner by combining phenotypes such as pathogenesis, disease development, disease resistance, disease susceptibility or drug response (also referred to as "personalized medicine"). The identification and characterization of proteins associated with biological pathways in the genome of an organism in a cell or tissue specific sense may also help to design transgene expression constructs for therapies with enhanced therapeutic efficacy and reduced side effects. The identification and characterization of protein expression in terms of cell or tissue specificity may also aid in the development of functional markers for diagnosis, prevention and treatment of disease. "disease" includes, but is not limited to, any condition, trait, or characteristic of an organism for which an alteration is desired. For example, a condition can be physical, physiological, or psychological, and can be symptomatic or asymptomatic.

In another embodiment of the invention, the antibody array is used to detect post-translational modifications in proteins that are important in studying signaling pathways and cellular regulation. Post-translational modifications can be detected with antibodies specific for a particular state of the protein, such as phosphorylation, glycosylation, farnesylation (farnesylated), and the like.

Detection of these interactions between ligands and polypeptides can lead to medical diagnosis. For example, the identity of a pathogenic microorganism can be unambiguously determined by binding a sample of an unknown pathogen to an array comprising many types of antibodies specific for known pathogenic antigens.

Reagent kit

In one embodiment, a kit comprising a library of antibodies is provided. In some embodiments, the antibody library is arrayed in a support such as 96 or 384 well. In one embodiment, the kit comprises an antibody microarray. The kit may further comprise: reporting the assay substrate; agents for inducing or inhibiting a particular biological pathway (cytokines or other purified proteins, small molecules, cdnas, sirnas, etc.), and/or data analysis software.

In addition, kits are provided that comprise reagents and instructions for performing the methods of the invention or performing a test or assay using any of the compositions, libraries, arrays, or combinations of articles of the invention. The kit may further comprise buffers, enzymes, adaptors, labels, secondary antibodies and instructions necessary for using the kit, optionally including trouble-shooting information.

In yet another embodiment, a kit can comprise an antibody library such as described herein and an antigen library such as a proteome of an organism. A kit can comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the proteome of an organism. The antigen library may represent most or the entire proteome of an organism, such as the proteome of a bacterium, virus, fungus. The antigen library may represent most or the entire proteome of an insect or mammal (such as a mouse, rat, rabbit, cat, dog, monkey, goat, or human). For example, a proteomic library of an organism can comprise at least 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 different antigens. A proteome library of an organism can comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the human proteins listed in table 5.

Examples

Example 1: subcloning of approximately 17,000 full-length human ORFs into an expression vector。

Obtaining about 16,000 unique ORFsThe ORF pool (Invitrogen, CA) was simultaneously subcloned with an additional 1,000 full-length human ORFs. Constructed for yeastA compatible expression vector (pEGH-A) that produces an N-terminal 6XHis6:: glutathione-S-transferase (GST) fusion protein in yeast after galactose induction (FIG. 3). Subsequently, all about 17,000 human ORFs were subcloned with a 99% success rate as determined by restriction digestion. In particular, three replicates of this pool were prepared, and the incoming DNA (EntryDNA) was extracted from one of them and agaroseThe quality of the plasmid DNA was checked on the gel. The resulting recombinants were then transformed into bacteria and single colonies were selected on Amp-containing plates. For each LR recombinant, four single colonies were picked to generate glycerol stocks, two of which were further processed to extract plasmid DNA in a 96-well format. The extracted plasmid DNA was digested with restriction enzymes to release the insert and electrophoresed on an agarose gel to check the vector and insert size as an indication of successful subcloning (fig. 3). Each restriction event was scored based on the expected insert size and the success rate was determined to be 99.3%.

Sequencing of more than 200 randomly expressed clones revealed that 100% of the sequences were correct. The confirmed expression constructs were rearranged to generate a master set of expression clones (masterset) for yeast. Similar large-scale cloning was done in bacterial expression vectors.

Example 2: design and construction of microarrays of 5000 human antigens.

Preliminary experiments were performed to test the ability to rapidly purify correctly folded recombinant proteins for microarray production. These proteins are divided into 5 different functional classes: transcription factors and transcriptional accessory regulators, RNA binding proteins, protein kinases, chromatin-associated and chromatin-modifying proteins, and mitochondrial proteins. Proteins were placed into these categories according to primary sequence, literature, and gene ontology annotation. The expressed ORF represents up to 85% (in terms of transcription factors) of all human proteins in the relevant functional class. More than 90% of the expressed protein was purified at a level sufficient for array construction (Ho, s.w., et al, LinkingDNA-binding protein sequencing sequences by using protein microarrays. proc. natl acads sciusa, 2006.103 (26): p.9940-5).

Several functional assays were performed to confirm that the expressed protein was functional after immobilization on a solid surface. Autophosphorylation assays were performed on kinase chips containing 119 independently purified protein kinases from yeast and it was observed that approximately 85% of the kinases exhibited detectable kinase activity (Zhu, h., et al, analytes of protein kinase using protein chips. natgenet, 2000.26(3) p.283-9.). Although covalently attached to a solid surface, most of these protein kinases apparently retain their enzymatic activity. Specific interactions between various DNA motifs and transcription factors were identified and characterized using a protein chip consisting of approximately 300 yeast transcription factors (Ho, S.W., et al, Linking DNA-binding protein sequencing sequences by using protein microarray, Proc Natl Acad Sci USA, 2006.103 (26): p.9940-5.). The interaction between DNA motifs and human transcription factors was also studied, wherein a leader protein chip consisting of approximately 1,000 human transcription factors was used to demonstrate that known DNA motifs specifically bind to their documented transcription factors; in contrast, point mutations in these motifs significantly reduced their binding affinity (Hu, S., et al, Profile the humanin protein-DNAinteractive molecular ERK2 asatranscriptionaltemporal compression of the interaction of cells, 2009.139 (3): p.610-22.).

Example 3: preparation of human antigen protein chip

To purify a full panel of 17,000 human protein antigens from yeast cells, the entire human ORF master was cloned into yeast in pEGH-a, single colonies were picked and glycerol stocks were prepared. Yeast cells were induced for recombinant protein production and stored at-80 ℃. To monitor the quality of the induced cultures, 24 random strains were inoculated in duplicate for each batch of culture preparations and first subjected to a protein purification step. Using immunoblotting and silver staining techniques, the success rate of culture induction was estimated (fig. 4A). Success was only indicated when at least 85% of the purified protein showed a major band in the expected molecular weight range in the immunoblot and silver stain analysis. Otherwise, culture preparation is repeated for the failed batch. Using this standard, all 17,000 antigenic proteins were purified with 85% success. Purified human antigen proteins were spotted onto various glass surfaces (e.g., FAST, Ni-NTA, and fullmon) using a microarray spotter to produce human antigen chips. The quality of the chip was monitored by probing the slide with anti-GST antibody and Cy 3-labeled secondary antibody. The chip was scanned and signals were obtained and analyzed using GenePix software. The results show that the chip is of high quality and > 90% of the spotted proteins produce signals significantly above background (fig. 4B, fig. 4C).

Example 4: antibody mass spectrometry on antigen microarrays

Monoclonal antibodies against proteins expressed in human liver were generated by conventional methods (Hu, S., et al, aproteinproppaproachforhigh-throughput differentiation. proteomics, 2007.7 (13): p.2151-61). Large amounts of monoclonal antibodies were purified from ascites fluid and diluted 10,000-fold in PBS containing 1% BSA. The quality (e.g., affinity and specificity) of mabs was assessed using a human liver protein chip. From literature/database searches, 1058 full-length cdnas known to be expressed in human liver were cloned into expression vectors expressing these proteins as N-terminal GST fusion proteins. To prepare the human liver protein chip, these genes were expressed and purified from yeast cells using glutathione affinity chromatography and spotted onto a glass slide. Mouse IgG was simultaneously spotted onto the chip for use as a site marker. The amount of immobilized human liver protein on the antigen microarray was visualized and quantified with anti-GST antibody (fig. 5A). The conclusion was that 98.5% (1,042/1,058) of the protein showed significantly higher signal than background, and the protein chip was of high quality.

To assess the specificity of several monoclonal antibodies raised against different human liver proteins, the chips were blocked with 1% BSA in PBS buffer for at least 1h at Room Temperature (RT) in a home-made wet chamber with gentle shaking. Meanwhile, the monoclonal antibody purified from ascites fluid was diluted 20,000 times in Phosphate Buffered Saline (PBS). After blocking, diluted mAb was added to different chips and incubated at room temperature with gentle shaking for 30 min. The chip was then washed 3 times in PBST (1% Tween 20) buffer for 10min with shaking at 42 ℃. An anti-mouse IgG antibody labeled with Cy3 (Jackson laboratories, USA, diluted 11,000-fold in PBS) was added to the chip and incubated for 1h at room temperature in the dark. The chips were then washed 3 times with pre-warmed PBS + 0.1% Triton at 42 ℃ for 10min each time with gentle shaking. After rinsing twice in filter sterilized di water, the chip was spun dry. To visualize the binding spectra, we scanned the chip with a microarray scanner and further analyzed the binding signals with GenePix software. As shown in fig. 5B, the specificity of the mAb raised against pirin was detected against 1,058 proteins spotted on the slide, and the mAb recognized only pirin. This result indicates that the antibodies are highly specific, as the amount of each spotted protein does not vary significantly. Similar results were obtained with mabs raised against the other four human proteins (fig. 5C).

Example 5: production and characterization of monospecific antibodies.

Immunization in response to recombinant human proteins and live human cancer cell lines has produced over 2,000 strong IgG-secreting monoclonal antibodies. Protein microarrays containing 17,000 human proteins have been used to analyze the binding specificity of 88 of these monoclonal antibodies.

To reduce the number of microarrays used for this analysis, an equal mixture of supernatants from up to 7 different hybridomas was used in the cross-over analysis illustrated in fig. 6 to check the specificity of these pooled antibodies. 11 of the 88 monoclonal antibodies were truly monospecific because only one protein on the microarray was recognized by these monoclonal antibodies. The other 7 monoclonal antibodies bound only 3 different proteins on the array, showing highly restricted specificity. For 4 of these monospecific monoclonal antibodies, no antibodies of any kind (polyclonal or monoclonal) can be purchased, while it is also unknown whether any of the commercially available antibodies for the other 7 species are monospecific. A list of these monospecific monoclonal antibodies and the commercially available antibodies that recognize them is shown in table 1. The antigens recognized by the 11 monospecific monoclonal antibodies are listed according to the gene symbol ID. The number of all commercially available antibodies (monoclonal and polyclonal) to the protein is also listed, and the number of monoclonal antibodies to the antigen is listed in parentheses.

Table 1: the antigen recognized by the generated monospecific mAb.

Example 6: monoclonal antibodies directed against transcription factors expressed by the human brain.

Antigen production

Human proteins were expressed from proprietary expression vectors in E.coli. As previously described, the human protein was expressed and purified as N-terminally labeled GST: his6 fusion protein. Because some human proteins are more easily expressed and purified in large quantities than other proteins from bacterial hosts, large-scale screening is performed to determine which human chromosomal proteins fall within this class. A very high throughput protein purification protocol that allows purification of more than 4,000 individual proteins from E.coli in 10 hours can be performed as described in FIG. 7 (Chen, C.S., et al, aprotomepaproaches, science, DNA and engineering bacteria purification methods, 2008.5 (1): p.69-74.). Using this protocol, approximately 200 candidate proteins that are more easily purified will be identified. Approximately 30% of the strains should produce 2mg protein/250 mL of culture, 60% should produce 0.5mg protein/250 mL of culture, and 40% should produce < 0.1mg protein/250 mL of culture.

The "easy" higher yield of protein from more bacterial cultures was purified. Briefly, the strains were activated in fewer overnight cultures and inoculated250mL of medium to reach a final OD of about 0.1 the next morning₆₀₀. When the cells were grown to OD at 37 ℃ with shaking at 250rpm₆₀₀After 0.7-0.9, protein expression was induced for about 3.5h with isopropyl β -D-thiogalactoside (IPTG) at a final concentration of 1mM cells were harvested by centrifugation and incubated at 4 ℃ in the presence of 50mM NaH₂PO₄(pH8) and 300mM NaCl, 20mM imidazole, CelLytic B (Sigma), 1mg/mL lysozyme, 50 units/mL benzonase, protease inhibitor cocktail (Sigma) and 1mM PMSF lysis buffer. After incubation on ice for 30min, cell debris was removed by centrifugation and pre-washed Ni-ntacuperflow (qiagen) was added to the cell lysate. After 40min incubation on ice, the nickel resin was collected and washed with wash buffer I (50mM NaH) in the cold chamber₂PO₄And 300mM NaCl, 10% glycerol, 20mM imidazole and 0.01% Triton X-100, pH8) three times, washing with washing buffer II (50mM NaH)₂PO₄And 150mM NaCl, 25% glycerol, 20mM imidazole and 0.01% TritonX-100, pH8) were washed three times. The purified protein was eluted by cleavage with 3C protease to remove GST and His6 tag. Combining the eluted proteins and dividing into fractions; one aliquot was used to determine the mass and quantity of protein on a PAGE gel using Coomassie staining.

For proteins falling in the medium yield category, the culture volume was increased to 1L to achieve a yield of 2 mg. For those proteins that are poorly expressed in bacteria, the protein was purified from yeast culture using a GST affinity tag. As previously described, the expression of human proteins can be induced in yeast by the addition of galactose. To purify these difficult to obtain proteins, the proteins are synthesized using a commercially available in vitro transcription/translation system (e.g., the Roche rabbit reticulocyte system). Since the T7 promoter is already built into the vector system used, no additional subcloning is required. In summary, at least 85% of human protein is expected to yield about 2 mg. This amount of antigen is more than adequate for immunization, monoclonal antibody production and screening purposes.

Antibody production

Animals were immunized using multiple site tachyphylaxis (RIMMS) using up to 4 dorsal subcutaneous antigen injections in a single round, Intraperitoneal (IP) boosting after 14 days, and splenocytes were harvested after 3-4 days (Bynum, j., et al, development of spleen-switch, after fine-matrix monoclonal antibody injection 7-synthetic drug. hybrid, 1999.18 (5): p.407-11) or single round of antigen/adjuvant injection to the hind footpad, after which draining (popliteal and inguinal) lymph nodes were harvested after 7-14 days without boosting injection (Mirza, i.h., et al, Acomprision paragonon. degree. f. infiltration of lung tissue fusion, et al, mutation of lung tissue antigen injection 63235.52).

Hybridomas are produced by fusing two cell types (immune B cells and culturing stable myeloma cell lines). This is done in the presence of a fusogenic compound such as PEG. The desired hybrid cell product can be selected from non-fused cells by exploiting the presence of two metabolic pathways for pyrimidine/purine synthesis (de novo and clearance pathways). Commonly used myeloma cell lines lack a salvage pathway because they have been selected for 8-azaguanine or 6-thioguanine resistance and thus lack hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The fusion reaction was therefore inoculated in HAT medium to eliminate unfused immune cells and myeloma cells, but allow the hybridomas to grow out. The myeloma cell lines used are X63-Ag8.653, NSO/1 or Sp 2/0-Ag-14.

Screening was performed by analyzing each microtiter well culture supernatant aliquot from the dispersed cell fusion reactions to identify the desired hybridomas. ELISA-type assays were designed for each individual antigen, where the antigen was immobilized on a 96-well polyethylene plate and then incubated with supernatants from all wells containing colonies. Cells in culture wells producing antigen binding antibody were harvested and reseeded in diluted form for isolation of clonal colonies. This limiting dilution cloning step requires an incubation time of up to about 2 weeks to allow single cells to grow into detectable colonies. Usually, ELISA assays incorporate a second screen that indicates the type of antibody secreted by the hybridoma.

The two final steps in the isolation of monoclonal antibodies include establishment of clonality of hybridoma lines, and then propagation of these lines to produce useful amounts of antibody. When hybridomas have been produced using standard methods, cell lines are cloned to eliminate the presence of contaminating hybridomas that produce irrelevant antibodies. This is usually done by plating the cells in microtiter wells at limiting dilution, followed by a second screening of the supernatant with an antigen-specific ELISA. If the original fusion reaction is seeded in semi-solid medium (methylcellulose) in a petri dish, limiting dilution cloning (Davis, J.M., et al, Asimple, single-steptechniqueform for selecting and cloning of bacterial clones. jimmunol methods, 1982.50 (2): p.161-71) can be bypassed because the cells grow to established clones from the beginning. Clones expressing antibodies against the antigen can be identified by including the antigen in a semi-solid medium, in effect performing an Ouchterlony reaction that will precipitate the antigen-antibody complex in the vicinity of the clone. Clones were then picked into 12-well plates using capillary tubes as the first step of propagation. The small scale cultures from either type of cloning procedure were then expanded using increasingly larger culture flasks. The hybridomas are maintained in a large volume culture system (e.g., large resting flasks, spinner flasks, hollow fiber reactors) to harvest useful amounts of antibody, which typically yields 20-100mg/L, or by injecting cells into mice for ascites production, which can yield 5-10mg/mL, but the total volume is quite small.

Preparation of human antigen microarray

To provide an adequate human antigen chip, approximately 17,000 human antigens as N-terminal GST-His6 fusion proteins were first purified from yeast cells using an established high throughput protocol (discussed above). First, yeast strains on SC-Ura agar plates were cultured overnight in 800. mu.l of SC-Ura medium in 96 wells. Each saturated culture was then inoculated into 12mL of SC-Ura and induced with 2% galactose for 4 hours after the culture reached an o.d. of 0.7. Induced yeast cells were collected and stored at-80 ℃. To monitor the quality of the induced cultures, each batch of culture preparations was double inoculated with 24 random strains and first passed through a protein purification step to determine sufficient induction levels. Using immunoblotting and silver staining techniques, the success rate of culture induction was estimated and only those batches that showed > 85% success rate were subjected to large scale purification. To produce human antigen chips, purified human antigen proteins and control proteins including a dilution series of BSA and GST-His6 as negative controls and a dilution series of histone, human IgG and IgM, and EBNA1 as positive controls were spotted in duplicate on a fullmonon slide (fullmonon biosystems, usa) at a throughput of 150 chips/day using two microarray spotter instruments, ChipWriterPro (Bio-Rad) and NanoPrint (TeleChem, usa). The quality of each batch of chips and the amount of immobilized protein on the surface was monitored by probing the chips with anti-GST antibody followed by Cy 3-labeled secondary antibody. It is expected that the success rate of dot printing (printing) will reach 90% or more.

Antibodies against chromosomal proteins were confirmed using human antigen microarrays.

Determination of mAb specificity: to assess the specificity of mabs produced against human chromosomal proteins, mabs purified from ascites fluid were diluted 1000-fold in PBS as working stock. The chip (microarray) was blocked with 1% BSA in PBS buffer for at least 1h at Room Temperature (RT) in a home-made wet chamber with gentle shaking. mAb diluted appropriately in PBS buffer was incubated on the chip for 30min at room temperature with gentle shaking. The chip was then washed in PBST (1% Tween 20) buffer for 3-10min with shaking at 42 ℃. An anti-mouse IgG antibody labeled with Cy-5 (Jackson laboratories, USA, 1: 1,000 dilution in PBS) was added to the chip and incubated for 1h in the dark at room temperature. After the same washing steps, the chip was briefly rinsed in filter-sterilized di water and spun dry. To visualize the binding spectra, the chip was scanned with a microarray scanner and the binding signals were analyzed with GenePix software. As a negative control experiment, the chip was probed with Cy 5-labeled secondary antibody to identify non-specific binding activity. When a microarray of 17,000 proteins was tested, about 150 or so proteins (e.g., MGMT and PCBP1) showed binding activity to a secondary antibody of Cy 5-labeled anti-mouse IgG. Non-specific interactors were excluded from further analysis.

Pool analysis: based on preliminary data, mabs that showed monospecificity were rapidly identified using pool analysis methods. Supernatants from hybridomas secreting high levels of IgG positive mabs in the "horizontal" and "vertical" pools are schematically shown in fig. 6. Antibodies are classified as monospecific if one protein on the microarray is recognized by only one horizontal and vertical pool, both containing supernatant from the hybridoma in question. It is expected that approximately 10% of all screened hybridomas will exhibit a single specificity, and that a large number of such mabs can be isolated. A total of 14 microarrays were available to analyze the specificity of 49 different hybridomas, greatly increasing the throughput of the analysis.

Secondary screening: antibodies determined to be monospecific by analysis of pooled supernatants were then subjected to a round of secondary screening using a protein microarray to further characterize their affinity and specificity. Antibodies recognizing no more than three different proteins on the array will also be picked for further analysis in this manner, although they will be given lower priority than monospecific mabs, since mabs with highly selective (although not necessarily monospecific) protein recognition properties can also be used in a range of applications. For these experiments, one human proteome chip was probed with each mAb at 10,000 fold dilution during the secondary screening. If the mAb can specifically recognize its corresponding target protein, meaning that no significant binding signal was observed for any other proteins on the chip (other than those non-specific proteins described above), the chip was again probed with a 50,000 fold dilution of mAb to help determine the optimal dilution. If no binding signal was observed, the titer was increased to 100-fold and the chip was probed again. If binding signal occurs, 1000-fold dilution is increased. Those that did not show a binding signal at 100-fold dilution were considered to be failed. Thus, for a typical antigen, 6 chips per antigen (3 mabs per antigen, 2 chips per mAb) were used for this second round of microarray-based screening.

The main tool for specificity evaluation was the reaction with a single protein on a microarray of 17,000 antigens as outlined above. Antibodies that show high specificity on antigen microarray-based screening are further tested by a series of tests that are combined into a test workflow called an application testing route. The immunoblot assay examined the specificity of each antibody against the cognate antigen and against at least three human cell extracts from different tissue culture cell types. These were tested with and without the use of mixed homologous antigens (since the antigens were GST-labelled, which can be distinguished from the native antigens).

Several different neuroblastoma cell lines were used to conduct these additional studies of antibody specificity. A large amount of gene expression data is publicly available in the gene expression integrated database (GEO) website, which allows for easy determination of whether the transcription factor in question and its putative target gene are both expressed in a human cell line that can be readily cultured and analyzed. As shown in table 2, previously published microarray data has been analyzed to determine which readily available cell lines exhibit strong mRNA expression levels for each antigen used to produce monoclonal antibodies.

Table 2: an antigen for the production of monoclonal antibodies.

Abbreviations: BFLS-Forssman-Lehmann syndrome, MD affective disorder, NI unidentified, NSMR-non-syndromic retardation, PWS/AS-pra-Williams syndrome/anglman syndrome, RS-rett syndrome, SZ-schizophrenia, WBS-Williams-beeren syndrome.

Indicates that ChIP-grade antibodies are not commercially available.

For most antigens of interest, a well characterized SH-SY5Y neuroblastoma cell line was used. However, in other cases, for example, in different readily culturable neuroblastoma or non-neuronal cell lines, mRNA corresponding to the antigen of interest is expressed significantly higher. These are shown in table 3.

Table 3: cell lines for immunohistochemistry-based and/or chromatin immunoprecipitation (ChIP) -based analysis of the produced antibodies. Target genes for ChIP analysis are also listed when known.

Indicates the target gene inferred from the loss-of-function assay and not directly confirmed by ChIP, luciferase assay, etc.

If this is the case, these other cell lines are used for immunocytochemical analysis and for chromatin immunoprecipitation as described later. To determine whether the monoclonal antibodies produced during the screening process can detect endogenous proteins by immunoblotting, the following test groups were used: antigen alone, extracts from rows 1-3 + antigen mixed at 1 μ Gm/mL. For the Immunoprecipitation (IP) assay, the same starting material was used, except that the final read was to detect an Immunoblot (IB) of antigen formed with anti-GST. To examine the expression of endogenous proteins by Immunohistochemistry (IHC), an indirect immunofluorescence-based assay was performed to screen neuroblastoma cells grown on poly D-lysine coated coverslips. As a negative control, a mouse fibroblast cell line (3T3) was screened.

To this end, quantitative ChIP protocols (Onishi, A., et al, Pias3-dependent fashion directive phosphor analyzer maintenance. neuron, 2009.61 (2): p.234-46; Peng, G.H. and S.Chen, Chromatinmumbranched transfection efficiency and transfection efficiency, Newfindingding and mining, Vis Neurosis, 2005.22 (5): p.575-86. briefly), immunoprecipitated with 1 microgram of each antibody in the same amount of cross-linked formaldehyde, and sonicated from cultured cells (355) 5 × 10. the same amount of cross-linked formaldehyde was immunoprecipitated with 1 microgram of each antibody, and sonicated from cultured cells (3510. sup.5. th⁶Individual cells). Real-time qPCR with gene-specific primers was used to detect and quantify specific genomic regions enriched in IP samples versus input controls. Non-specific mouse IgG will be used as a negative control. The ratio of IP/import in each genomic region of a gene will be used to compare the specificity and effectiveness of different antibodies made against the same protein. Cell lines expressing the target antigen at high levels were used for this assay, as shown in table 3.

When selecting genomic regions for amplification and analysis, several criteria are considered. In the case of monoclonal antibodies against an antigen in which ChIP has been made available (as shown in Table 2), polyclonal antibodies to that antigen are selected in which the protein in question is expressed and in which the target gene for the transcription factor is a known genomic region and cell type, so that the effectiveness of the novel agent can be determined. For many of these antigens, ChIP analysis of SH-SY5Y cells has been performed using polyclonal antibodies, and in these cases, genomic regions that have been shown to be bound by these factors in this cell type have been analyzed. For other proteins, ChIP assays have been performed in other cell or tissue types, although generally, commercially available antibodies are used. Table 3 lists the known and proposed direct target genes for the antibodies to be produced. In the case where ChIP has been performed against the target genes of these factors, the previously tested primer sets were used. In other cases, the consensus binding sites for these factors are determined using bioinformatic tools such as TRANSFACDb, and then primer pairs are designed that evolutionarily flank conserved sequences corresponding to sites found in putative regulatory regions of these transcripts, or have been characterized using protein microarray-based high throughput analysis of protein-DNA interactions. By combining these bioinformatic data with previously published gene expression data, a series of high probability direct targets for the transcription factors under study were developed and primer sets were used for ChIP analysis on antibodies.

Example 7: characterization of monospecific monoclonal antibodies (mMAb):

and (3) single specificity MAbICC verification.To further characterize mmabs generated using the shotgun method, high-resolution Immunocytochemistry (ICC) was performed to assess the ability of mmabs to bind to proteins in fixed cells. Permeabilized HeLa, HepG2 or HCT-116 cells were fixed in paraformaldehyde and blocked in 3% BSA + 3% FBS. Each mMAb was diluted to 10. mu.g/mL and was at 4^1/4Incubate overnight at C. Mixing DyLight^TMConjugated goat anti-mouse IgG antibody (Rockland) was used as the secondary antibody. The stained cells were observed and a photograph was taken with a fluorescence microscope. Benefit toThe subcellular localization of a given antigen detected by mMAb was classified by 5 simple categories including nucleus, mitochondria, endoplasmic reticulum/golgi apparatus, cytosol, and plasma membrane.

As expected, 52 of the 59 mmabs tested produced a discernible pattern in HeLa cells whereas 6 and 1 mmabs correctly recognized their corresponding antigens in HCT-116 and HepG2 cells, respectively, given that HeLa cells were the source antigen. The distribution of the subcellular localization of the 59 human proteins is summarized in table 4.

TABLE 4 summary of subcellular localization of antigens in cells

By comparing ICC results to literature and databases (e.g., NCBI), 39 of these match known patterns, while 16 do not. This difference is likely to reflect differences in the cell lines/tissues tested and/or differences in basic biology. In addition, 4 antigens whose subcellular localization was unknown, namely C11orf68, CNTD1, DYDC2 and TXNDC9, have now been localized to the mitochondria, cytosol, plasma membrane and endoplasmic reticulum/golgi apparatus, respectively (fig. 8). Thus, the resulting mmabs not only show 100% ICC success, but also help to determine the localization of proteins for which information was not previously known.

mMAb pairs using immunoblotting and shRNA-based Gene knockdown (knockdown) Confirmation/characterization of (a).We constructed human tet-regulated plasmid expression vectors. Has passed throughLR recombination, subcloning of 42 human ORFs into the commercial mammalian expression vector pcDNA3.1-V5(Invitrogen) in order to overexpress them in HeLa as V5-tagged fusion proteins briefly, all plasmids were amplified in E.coli DH5 α and isolated by alkaline lysisFor transient transfection of human cells, transfection with the corresponding ORF expression construct was performed at about 0.6 × 10⁴Individual cells/well HeLa cells seeded on 6-well tissue culture plates. Two days after transfection, cells were lysed, after which a portion of each lysate was subjected to immunoblot analysis using mMAb (1: 1000 dilution).

Cells transfected with empty vector DNA as a negative control were also included in the immunoblot analysis. In some cases, a given mMAb detects two bands in transfected cells. For example, α -EFHD2 recognized two bands in cells transfected with V5 labeled EFHD2, but only one band in vector transfected cells (fig. 9, lane 1, lane 2 in the upper left panel). When the same blot was taken and probed with anti-V5 antibody, the faster migration band disappeared in both cells, indicating that α -EFHD2 faithfully detected V5-labeled and native EFHD2 in the cells. Of the 42 mmabs tested, 30 (71%) were confirmed to correctly detect the corresponding proteins by comparing the mmabs with the V5 blot (fig. 9, lanes 1, 2 in the top and bottom panels).

To further confirm that these mmabs are highly specific, gene knock-down assays were performed using shRNA. Mu.g of the expression construct was co-transfected with 3. mu.g of the shRNA expression construct in HeLa cells. Cells were harvested 24-36h after transfection, lysed and subjected to immunoblot analysis with the corresponding mMAb and anti-V5 antibody (fig. 9, lane 3 in upper and lower panels). In all 6 cases tested (4 of which are shown in figure 9), the corresponding shrnas effectively inhibited antigen expression as determined by mMAb and anti-V5 immunoblot analysis. These results directly confirm a very stringent test, i.e. the mmabs generated in the process route are likely to be highly specific and therefore very useful for immunoblot analysis. Furthermore, they provide such final verification: throughout the process route, the correct identity of the proteins was still maintained from preparation of the array to interpretation of the array images, since the shRNA knockdown reagent was from a completely different set (Sigma) than the set of ORFs used to express proteins placed on the array (Invitrogen).

Characterization of mmabs using immunoprecipitation

Immunoprecipitation (IP) validation protocols for human cells were performed. HeLa cells were transfected with a plasmid expressing the antigen of interest in the form of an N-terminal V5-labeled protein and lysed. The antigen was immunoprecipitated with the corresponding mAAB and analyzed by immunoblot analysis using anti-V5 antibody. Anti-mouse IgG and anti-V5 antibodies were used in IP as negative and positive controls, respectively. 16 of the 27 mmabs tested showed efficient immunoprecipitation of their corresponding antigens in HeLa cells with an overall power of 59% (example shown in fig. 10). Thus, a high proportion of mmabs are IP levels.

Characterization of mmabs using chromatin immunoprecipitation.

Many non-conventional DNA binding proteins (uDBP) exhibit sequence-specific binding properties, including the RNA binding protein HNRPC, a kind of hnRNP (nuclear heterogeneous ribonucleoprotein) (Hu et al, Cell 2009). It is known that hnRNP complexes with nuclear heterogeneous RNA (hnRNA). They are also associated with pre-mRNA in the nucleus and appear to affect pre-mRNA processing as well as other aspects of mRNA metabolism and transport. Although hnRNP protein has unique nucleic acid binding properties, such as the formation of 40ShnRNP particles, recent studies using Protein Binding Microarrays (PBMs) have shown that it also binds to double-stranded DNA and single-stranded DNA in a sequence-dependent manner. Since a highly specific mMAb against HNRPC (IgG2b isotype) was generated in the CDI route, we decided to fully characterize the antibody with various standard assays.

As shown in fig. 11A, ICC staining with this antibody in HeLa cells showed clear nuclear localization of HNRPC, consistent with its known subcellular localization (i.e., nucleoplasm and pronuclei). Since the mMAb can detect native HNRPC in HeLa cells, before testing whether it can specifically immunoprecipitate DNA-crosslinked HNRPC, it is tested to determine whether it is useful for immunoprecipitation analysis.

Two million HeLa cells overexpressing V5-labeled HNRPC were lysed and 20. mu.g of anti-HNRPCmMAb was added to the lysate and allowed to incubate overnight. Protein G-coupled beads were added to capture the antibody, followed by extensive washing. Anti-mouse IgG and anti-V5 antibodies were also used in parallel IP experiments as negative and positive controls, respectively. The supernatant of the boiled beads was then immunoblotted with anti-V5 antibody. As shown in figure 11B, one band of V5-labeled HNRPC was detected at the same molecular weight as that observed in the input lane and anti-V5 lane, indicating that the HNRPC immunoprecipitation using this mMAb was successful. As expected, the IgG negative control did not show any signal.

Next we performed chromatin immunoprecipitation (ChIP) against endogenously expressed HNRPC to determine if the mMAb is capable of performing the ChIP of HNRPC and, if successful, which specific DNA sequences the HNRPC protein is associated with in vivo⁷Individual HeLa cells were cross-linked, quenched, harvested and washed. After lysis of the cells under mild conditions, the nuclear pellet is collected, washed and sheared by sonication. For ChIP of HNRPC, 10. mu.g of anti-HNRPC antibody (or 10. mu.g of mouse IgG as a negative control) was added and incubated overnight at 4 ℃. Protein G-bound Dynabeads were added to capture the DNA-HNRPC complex, followed by elution. About 10% of the eluate was used for immunoblot analysis. As shown in fig. 11C, endogenously expressed HNRPC could immunoprecipitate chromatin as observed in the first and second elutions, whereas the IgG control showed no detectable signal.

To determine if any DNA fragments were chromatin immunoprecipitated by the antibody, the chromatin immunoprecipitated complexes were reverse crosslinked overnight at 65 ℃. Since HNRPC is a known RNA binding protein, the eluate is treated with ribonuclease, followed by deproteinization. The mass and concentration of the concentrated DNA sample was determined using an Agilent2100 bioanalyzer. As shown in FIG. 11D, high-yield and high-quality DNA was obtained.

Continuous measurement of MAb affinity

As shown in fig. 12, the kinetics of 3 antigen-antibody interactions were monitored simultaneously in real time using unlabeled antibodies. The 3 antibodies were injected sequentially into the flow cell covered IgG microarray and the binding signal appeared to saturate around 20min as shown by the red line in figure 12. This data demonstrates the feasibility of this method of continuously measuring MAb affinity in a high throughput system.

Example 8: for the production and characterization of monospecific monoclonal antibodies (mMAbs) Platform:

protein microarrays consisting of 17,263 unique full-length human proteins that account for over 70% of the annotated human proteome were constructed. For expression and purification of these proteins, the invitrogen ultimate ORF pool together with 400 additional ORFs was subcloned into a yeast expression vector that allowed galactose-dependent overexpression of N-terminal GST and 6x-His tagged recombinant proteins. The quality of the purified protein was determined by GST immunoblot analysis and silver staining of random subsets of recombinant proteins. Based on these results, approximately 85% of all proteins can be purified with high yield to a purity of at least 80%. Microarrays consisting of these recombinant proteins were prepared and their quality was tested by probing with anti-GST antibodies. More than 85% of the spotted proteins produced a signal significantly above background.

Highly complex mixtures of monoclonal antibody (mAb) producing antigens, such as intact cells or tissues, are effective for Immunocytochemistry (ICC). The production of mabs in this "shotgun" manner increases the likelihood that the mAb recognizes epitopes of the native protein, thus increasing the range of potential applications. However, the main limitation of this approach is the difficulty in identifying antigens that are preferentially recognized by a given mAb, which identification is usually only possible by affinity purification in combination with mass spectrometry. By screening mabs directly against a human proteome microarray, it is possible to directly identify the corresponding antigen recognized by a given mAb produced by immunization with a complex biological sample.

Using this shotgun method, mice were immunized with a variety of different cancer cell lines. Viable human cells were injected directly into the footpad and the popliteal lymph node was collected three weeks later and hybridomas were generated. To enrich for useful mabs, a high-throughput ICC prescreening step was performed against one or more cell lines using supernatants from 12200 hybridomas. Thereafter ICC positive hybridomas were further grown and isotype-typed, and IgG isotype positive supernatants were selected for microarray analysis.

The individual supernatants were pooled into 12 x 12 two-dimensional pools before these pools were incubated on a human proteome microarray and incubated with Cy 5-coupled anti-IgG secondary antibodies. After washing and scanning, the signal intensity of each spot is expressed as the ratio of the foreground signal to the background signal. The number of standard deviations above the mean signal intensity of the entire array was then measured for each replicate pair of proteins, which we call the value a. The duplicate spots for a > 3 were then labeled and the results deconvoluted to identify proteins that were present at the intersection of a single horizontal and a single vertical pool and thus recognized by a single mAb. To confirm the specificity of these mabs, each candidate highly specific mAb was then tested individually against the entire array and the a value of each spotted protein was measured. The signal strengths are then rank ordered and the difference between the highest signal and the second highest signal on the array is calculated, which we denote as S. A number of mAbs were obtained which identified A > 6 and S > 3 and we called monospecific mAbs (mMAbs). In many cases, mmabs show very high selectivity (i.e., S > 100). Many mAbs bind strongly to two different proteins on the array (A > 6, S < 3), and they are called non-specific mAbs (dMAbs).

To further characterize the utility and specificity of mmabs and dmabs, a number of different assays were performed. First, ICC analysis on human cancer cell lines was repeated and images were collected to assess the subcellular expression pattern of the target antigen. Confirmation of the observed specificity of individual mabs comes from the following findings: 85% of all ICC profiles obtained in this study matched that previously reported for their target proteins (when this data was available). The subcellular distribution of proteins that yielded high quality mabs did not show a clear preference for any particular cellular compartment, confirming that shotgun immunization can produce mabs against a wide range of cellular proteins.

Next, the ability to detect its highest ranked target protein using immunoblot analysis was analyzed. In HeLa cells, a single target protein was overexpressed as an N-terminal V5-labeled fusion protein, and expression was confirmed by immunoblot analysis of V5. Of the 56 mabs tested, 31 (55%) recognized their target protein, while 18 (33%) recognized the endogenous, unlabeled protein in these cells. To further confirm the accuracy of these results, the V5-labeled ORF was co-transfected with a single shRNA targeting this gene. In all 9 cases tested, a large reduction in immunoblot signal was observed, but the control shRNA did not show a corresponding reduction in signal.

These mabs were then tested to determine if they could be used effectively for immunoprecipitation to recognize native proteins in cell homogenates. Of the 51 mabs tested, 28 (55%) efficiently precipitated the transfected protein, which was then detected by immunoblot analysis of V5. A substantial portion of the mabs were effective for immunoblot analysis and immunoprecipitation applications. In summary, the protein microarray-based shotgun method is a rapid and efficient method to generate highly specific mabs that can be used in many applications.

TABLE 5

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. An antibody library comprising: a plurality of different monospecific monoclonal antibodies, wherein each antibody of the plurality of antibodies is

(i) Produced by antibody-producing cells, and wherein said antibody-producing cells are isolated from an animal immunized with a plurality of natural antigens;

(ii) has an A-value of greater than 6 and an S-value of greater than 3, wherein the A-value is the number of standard deviations above the mean signal intensity for a protein or antigen when incubated on an array comprising the antigens listed in Table 5, wherein the S-value is the difference between the highest signal and the second highest signal for a protein or antigen when incubated on an array comprising the antigens listed in Table 5, and

(iii) (a) has at least 10 for its target protein^-7M(K_D) (ii) is an immunoprecipitating antibody, or (c) binds to its native form of the target protein.

2. The library of claim 1, wherein the S and a values are determined by incubating antibodies from a repertoire of antibody-producing cells isolated from an immunized animal on a human proteome microarray.

3. The library of claim 1, wherein each antibody of the plurality of antibodies has at least 10 for its target protein^-7M(K_D) Binding affinity of (4).

4. The library of claim 3, wherein said binding affinity is at least 10^- ¹³M。

5. The library of claim 1, wherein each antibody of the plurality binds to a native form of its target protein.

6. The library of claim 1, wherein said plurality of antibodies comprises at least 50 different antibodies.

7. The library of claim 6, wherein said plurality of antibodies comprises at least 125 different antibodies.

8. The library of claim 1, wherein the plurality of antibodies bind at least 0.5% of the human proteome.

9. The library of claim 8, wherein the plurality of antibodies bind at least 5% of the human proteome.

10. The library of claim 1, wherein the plurality of antibodies bind at least 0.5% of the human proteins listed in Table 5.

11. The library of claim 1, wherein at least 10% of the plurality of antibodies have a binding affinity for their target that is within at least 20% of the binding affinity of another antibody of the plurality of antibodies.

12. The library of claim 10, wherein the plurality of antibodies bind at least 1% of the human proteins listed in table 5.

13. The library of claim 7, wherein said plurality of antibodies comprises at least 500 different antibodies.

14. The library of claim 1, wherein each antibody of the plurality of antibodies is an immunoprecipitating antibody.

15. The library of claim 1, wherein each antibody of the plurality of antibodies is an IgG antibody.

16. An array, comprising: the antibody library of claim 1, wherein each antibody of the plurality of antibodies is immobilized on a substrate.

17. The array of claim 16, wherein the substrate is planar.

18. The array of claim 16, wherein the substrate is a particle.

19. The array of claim 16, wherein the substrate comprises a solid material.

20. The array of claim 16, wherein the substrate comprises a porous material.

21. The array of claim 16, wherein the immobilization is reversible.

22. The array of claim 16, wherein the fixation is irreversible.

23. A method of generating a library of a plurality of different monoclonal antibodies, the method comprising:

a) immunizing an animal with a plurality of antigens;

b) isolating antibody-producing cells from the animal;

c) isolating a plurality of antibodies from the antibody-producing cells;

d) screening the plurality of antibodies of step c) of the panel;

e) selecting antibodies to the plurality of antibodies of c) based on the screening of d);

f) screening the selected antibodies using a human proteome array; and

g) selecting antibodies for the library that are monospecific for a single target on the proteomic array and have an a value of greater than 6 and an S value of greater than 3, wherein the a value is the number of standard deviations above the mean signal intensity for a protein or antigen when incubated on an array comprising the antigens listed in table 5, and wherein the S value is the difference between the highest signal and the second highest signal for a protein or antigen when incubated on an array comprising the antigens listed in table 5.

24. The method of claim 23, further comprising pre-screening said plurality of antibodies from said antibody-producing cells prior to c).

25. The method of claim 24, wherein the prescreening is by performing immunocytochemistry or by determining binding of antibodies from the antibody-producing cells to a mixture comprising one or more target antigens.

26. The method of claim 23, wherein the plurality of antigens comprises a cell lysate, a cell, a protein, a peptide, or a nucleic acid.

27. The method of claim 23, wherein the plurality of antigens comprises at least 11,000 different antigens.

28. The method of claim 23, wherein the plurality of antigens comprises at least 0.5% of the human proteome.

29. The method of claim 23, further comprising immobilizing the selected antibody on a substrate.