HK1181112B - Single b-cell cultivation method - Google Patents

Description

Single B cell culture method

Herein is reported a method for obtaining the amino acid sequence of at least the variable domain of a monoclonal antibody secreted by a single B cell obtained from a population of B cells from an experimental animal by single cell deposition and co-culture with feeder cells in the presence of a feeder mix.

Background

In order to obtain cells that secrete monoclonal antibodies, the hybridoma technology developed by Koehler and Milstein is widely used. However, in the hybridoma technique, only a part of the B cells obtained from the immunized experimental animal is fused and proliferated. The source of the B cells is typically an organ of the immunized experimental animal, such as the spleen.

Zubler et al developed different methods for obtaining monoclonal antibody secreting cells since 1984 (see, e.g., Eur. J. Immunol.14(1984)357-63, J. exp. Med.160(1984) 1170-1183). Wherein B cells are obtained from the blood of an immunized experimental animal and co-cultured with murine EL-4B5 feeder cells in the presence of a feeder mix comprising cytokines. Using this method, up to 50ng/ml of antibody was obtained after 10-12 days of co-culture.

Weitkamp, J-h et al, (J. immunological. meth.275(2003) 223-. Methods of producing a variety of isolated antibodies against a variety of homologous antigens are reported in US 2006/0051348. Antibodies against IL-6 and uses thereof, and culture methods to obtain clonal populations of antigen-specific B cells are reported in WO2008/144763 and WO2008/045140, respectively. In US2007/0269868 a culture method is reported to obtain a clonal population of antigen-specific B cells. Masri et al (in mol. Immunol.44(2007) 2101-2106) reported the cloning and expression of functional Fab fragments in E.coli (E.coli) obtained from single human lymphocytes against anthrax toxin. A method for preparing immunoglobulin libraries is reported in WO 2007/031550.

Summary of The Invention

Herein is reported a method for isolating B cells from a B cell population with specific properties. First, induced antibody-producing cells can be isolated already within 4 weeks after the first immunization of the experimental animal, and the binding specificity of the antibody can be determined. Second, the number and/or quality of antibody-producing cells (e.g., antibody production/secretion capacity) can be increased by any of the following steps: i) a pre-incubation step, and/or ii) a centrifugation step, and/or iii) an elutriation step. Third, the feeder mix for co-culture of B cells and feeder cells can be improved by adding IL-21, or IL-6, or SAC, or BAFF.

Thus, in one aspect herein is reported a method of selecting B cells, the method comprising the steps of:

a) optionally labeling B cells of the B cell population,

b) separately co-culturing each B cell of the B cell population that has been deposited as a single cell with a feeder cell,

c) selecting the B cells that proliferate and secrete the antibody in step B).

In one aspect, herein is also reported a method of obtaining a B cell clone, the method comprising the steps of:

a) b cells were obtained from the experimental animals,

b) the labeling of the B cells is carried out,

c) the labeled B cells were deposited as single cells,

d) separately co-culturing the single cell deposited B cells and feeder cells,

e) selecting the B cells that proliferate and secrete the antibody in step d) and thereby obtaining a B cell clone.

In another aspect, herein is reported a method of producing an antibody that specifically binds to a target antigen, the method comprising the steps of:

a) optionally labeling cells of the B cell population with at least one fluorescent dye,

b) culturing each B cell of the B cell population that has been deposited as a single cell in a separate container in the presence of a feeder cell and feeder mix to obtain separate B cell clones and culture supernatant,

c) selecting a B cell clone that produces an antibody that specifically binds to the target antigen,

d) culturing cells containing a nucleic acid encoding an antibody that specifically binds to the target antigen, which antibody is produced by the B cell clone selected in step c), or is a humanized variant thereof, and recovering the antibody from the cells or culture supernatant, thereby producing the antibody.

In one embodiment, the method comprises one or more of the following steps:

after step c): c1) determining the nucleic acid sequences encoding the variable light and heavy domains of the antibody by reverse transcriptase PCR,

after step c 1): c2) cells are transfected with a nucleic acid comprising nucleic acid sequences encoding the variable light and variable heavy domains of an antibody.

In one aspect, herein is also reported a method of producing an antibody, comprising the steps of:

a) providing a population of (mature) B cells (obtained from the blood of experimental animals),

b) labeling cells of the B cell population with at least one fluorescent dye (in one embodiment with 1 to 3, or 2 to 3 fluorescent dyes),

c) depositing individual cells of the labeled B cell population in a separate container (in one embodiment, the container is a well of a multi-well plate),

d) culturing the deposited individual B cells in the presence of feeder cells and a feeder mix (in one embodiment, the feeder cells are EL-4B5 cells, in one embodiment, the feeder mix is natural TSN, in one embodiment, the feeder mix is a synthetic feeder mix),

e) determining the binding specificity of the antibody secreted in the culture medium of the respective B-cell,

f) determining the amino acid sequences of the variable light and heavy domains of the specific binding antibody by reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining nucleic acids encoding the variable light and heavy domains of the monoclonal antibody,

g) for expression of the antibody, monoclonal antibody variable light and heavy chain variable domain-encoding nucleic acids are introduced into an expression cassette,

h) introducing a nucleic acid into a cell, introducing the nucleic acid into the cell,

i) culturing the cells and recovering the antibody from the cells or cell culture supernatant, and thereby producing the antibody.

In one embodiment of all aspects reported herein, the method comprises the step of incubating the B cell population in a co-culture medium prior to single cell deposition. In one embodiment, the incubation is performed at about 37 ℃. In one embodiment, the incubation is performed for 0.5 to 2 hours. In a particular embodiment, the incubation is performed for about 1 hour. In one embodiment, the incubation is performed at about 37 ℃ for about 1 hour.

In one embodiment of all aspects reported herein, the method comprises the step of centrifuging single cell deposited B cells prior to co-cultivation. In one embodiment, centrifugation is performed for about 1 minute to about 30 minutes. In a particular embodiment, centrifugation is performed for about 5 minutes. In one embodiment, the centrifugation is performed at about 100x g to about 1,000x g. In a particular embodiment, centrifugation is performed at about 300x g. In one embodiment, centrifugation is at about 300x g for about 5 minutes.

In one embodiment of all aspects reported herein, the method comprises the following step immediately preceding the labeling step: panning B cells with immobilized antigen.

In one embodiment of all aspects reported herein, the B cell population is obtained from animal blood by density gradient centrifugation.

In one embodiment of all aspects reported herein, the B cell population is obtained from the blood of the experimental animal 4 days after immunization. In another embodiment, the B cell population is obtained from the blood of the experimental animal from 4 days to at least 9 days after immunization. In a further embodiment, the B cell population is obtained from the blood of the experimental animal from 4 to 9 days after immunization.

In one embodiment of all aspects reported herein, the B cell population is separated by density gradient centrifugation.

In one embodiment of all aspects reported herein, the B cell is a mature B cell.

In one embodiment of all aspects reported herein, the labeling is performed with 1 to 3 fluorescent dyes. In particular embodiments, the labeling is with 2 to 3 fluorescent dyes.

In one embodiment of all aspects reported herein, the labeling of B cells results in labeling of 0.1% to 2.5% of cells of the total B cell population.

In one embodiment of all aspects reported herein, the B cell is a mouse B cell, or a hamster B cell, or a rabbit B cell.

In one embodiment of all aspects reported herein, single cell deposition is performed in the wells of a multiwell plate.

In one embodiment of all aspects reported herein, the feeder cells are murine EL-4B5 cells.

In one embodiment of all aspects reported herein, the antibody is a monoclonal antibody.

In one embodiment of all aspects reported herein, the label is an IgG⁺CD19⁺-B cells, IgG⁺CD38⁺-B cells, IgG⁺CD268⁺-B cells, IgG^-CD138⁺B cells, CD27⁺CD138⁺B cells, or CD3-CD27⁺-B cells.

In one embodiment of all aspects reported herein, the B cell is of mouse origin and the label is IgG⁺CD19⁺-B cells, and/or IgG^-CD138⁺-B cells.

In one embodiment of all aspects reported herein, the B cell is hamster derived and the marker is IgG⁺IgM^--B cells.

In one embodiment of all aspects reported herein, the B cell is rabbit derived and the label is IgG⁺B cells and/or CD138⁺-B cells, or CD138⁺IgG⁺B cells and/or IgG⁺IgM^--B cells.

In one embodiment of all aspects reported herein, the co-cultivation is in RPMI1640 medium supplemented with 10% (v/v) FCS,1% (w/v) 200mM glutamine solution comprising penicillin and streptomycin, 2% (v/v) 100mM sodium pyruvate solution, and 1% (v/v) 1M2- (4- (2-hydroxyethyl) -1-piperazine) -ethanesulfonic acid (HEPES) buffer. In another embodiment, the co-cultivation medium further comprises 0.05mM β -mercaptoethanol.

In one embodiment of all aspects reported herein, B cells are co-cultured with feeder cells and a feeder mix. In one embodiment, the feeding mix is a natural thymocyte culture supernatant (TSN) or a synthetic feeding mix.

In a particular embodiment, the feeding mix is a synthetic feeding mix. In one embodiment, the synthetic feeding mix comprises interleukin-1 β and tumor necrosis factor α. In one embodiment, the synthetic feeding mix comprises interleukin-2 (IL-2) and/or interleukin-10 (IL-10). In one embodiment, the synthetic feeding mix further comprises Staphylococcus aureus (Staphylococcus aureus) Cowans strain cells (SAC). In one embodiment, the synthetic feeding mix comprises interleukin-21 (IL-21). In one embodiment, the synthetic feeder mix comprises B cell activating factor of the tumor necrosis factor family (BAFF). In one embodiment, the synthetic feeding mix comprises interleukin-6 (IL-6). In one embodiment, the synthetic feeding mix comprises interleukin-4 (IL-4).

In one embodiment, the co-culturing is performed in the presence of thymocyte culture supernatant as a feeding mixture. In a particular embodiment, the thymocyte culture supernatant is obtained from thymocytes of the thymus of a young animal.

In one embodiment, the method of obtaining a B cell clone further comprises the steps of:

determining the amino acid sequences of the variable light and heavy chain domains of the antibody produced by the selected B cell clone of step e) by reverse transcriptase PCR and nucleotide sequencing and thereby obtaining monoclonal antibody amino acid variable domain sequences.

In one embodiment, the experimental animal is selected from the group consisting of a mouse, a hamster, and a rabbit.

Detailed Description

The method reported herein allows for a rapid characterization of the binding specificity of monoclonal antibodies obtained from individual B cell clones, i.e. the induced antibody producing cells can be isolated within 4 weeks of the first immunization of the experimental animal and the binding specificity of the antibodies produced therefrom can be determined, wherein at least 4 different experiments can be performed depending on the amount/concentration of antibodies in the B cell co-culture supernatant.

Immunization:

non-human animals, such as mice, rabbits, hamsters, and rats are commonly used as animal models for evaluating antibody-based therapies. Thus, it is often desirable to provide cross-reactive antibodies that bind to non-human animal antigens as well as human antigens. The methods reported herein can be used to provide cross-reactive antibodies. In the methods reported herein, B cells obtained from, for example, mice, hamsters and rabbits may be used. In one embodiment, the mouse is an NMRI-mouse or a balb/c-mouse. In another embodiment, the hamster is selected from the hamsters of the sub-meiya (Cricetulus migtorius), chinese (Cricetulus griseus) and syrian hamster (moso Cricetulus auratus). In a particular embodiment, the hamster is a hamsters of methylene. In one embodiment, the rabbit is selected from the group consisting of New Zealand White (NZW) rabbits, zeeman rabbits (ZIKA), Alicia mutant rabbits, basilea mutant rabbits, transgenic rabbits with human immunoglobulin loci, rbIgM knockout rabbits, and hybrids thereof.

In one embodiment, the experimental animal selected for immunization, e.g., mouse, hamster, and rabbit, is no more than 12 weeks.

Source and isolation of B cells:

the blood of the experimental animals provided a high diversity of antibody-producing B cells. The B cell-secreted antibodies derived therefrom have little identical or overlapping amino acid sequences within the CDRs and thus show high diversity.

In one embodiment, B cells, e.g., B cells from blood, are obtained from the experimental animal from 4 days post immunization until at least 9 days post immunization or recent boosting. This time span allows for high flexibility in the methods reported herein. During this time span, B cells that provide most of the familial (affine) antibodies may migrate from the spleen to the blood (see, e.g., Paus, D., et al, JEM203(2006) 1081-.

The B cells in the blood of the experimental animal can be obtained using any method known to those skilled in the art. For example, Density Gradient Centrifugation (DGC) or red blood cell lysis (lysis) may be used. Density gradient centrifugation provides a higher overall yield, i.e. B cell clone number, than hypotonic lysis. In addition, among the cells obtained by density gradient centrifugation, more cells were divided and grown in the co-culture step. The secreted antibody concentration was also higher than in cells obtained by different methods. Thus, in one embodiment, the B cell population is provided by density gradient centrifugation.

Table 1: number of IgG production wells/cell clones when cells were obtained by Density Gradient Centrifugation (DGC) or hypotonic lysis of erythrocytes

Selection step before co-cultivation:

b cells producing antibodies that specifically bind to an antigen can be enriched from Peripheral Blood Mononuclear Cells (PBMCs). Thus, in one embodiment of all methods reported herein, the B cell population is enriched from Peripheral Blood Mononuclear Cells (PBMCs).

The term "specifically binds" and grammatical equivalents thereof means that the antibody is represented by 10^-7M or less, in one embodiment at 10^-8M to 10^-13M, in another embodiment, is at 10^-9M to 10^-13The dissociation constant (Kd) of M binds to its target. This term is also used to indicate that the antibody does not specifically bind other biomolecules present, i.e. it is present at 10^-6M or greater, in one embodiment at 10^-6The dissociation constant (Kd) of M to 1M binds to other biomolecules.

In one embodiment of all methods reported herein, macrophages are removed from PBMCs. This is advantageous, for example, in one embodiment of rabbit-derived B cells, for use in the co-culture step, as described below.

Macrophages can be removed by adhesion to the surface of the cell culture plate (see preculture step).

In one embodiment of the method reported herein, the cells are from animals immunized with the protein and macrophages are removed before labeling.

It has been found that incubating the B cell population in a co-culture medium prior to single cell deposition increases the total number of antibody secreting cells obtained after single cell deposition compared to direct single cell deposition after isolation and optional enrichment of the B cell population from the blood of experimental animals (see tables 2a and 2B for rabbit as an example). In particular, incubation in EL-4B5 medium at about 37 ℃ for about 1 hour, for example using a cell culture incubator.

Table 2 a: IgG positive wells/cell clones were incubated or not incubated in EL-4B5 medium for 1 hour (rb ═ rabbit) before single cell deposition of all cells.

Removing macrophage cells

Cells and monocytes

Table 2 b: IgG-positive well/cell clones incubated or not incubated in EL-4B5 medium for 1 hour before B-cell single-cell deposition

In one embodiment of the method reported herein, cells are obtained from animals immunized with the protein and macrophages are removed.

By using a panning approach, cells that do not produce antibody bound to the antigen can be reduced or similarly enriched for cells that produce antibody bound to the antigen, respectively. Wherein the binding partner bound to the surface is present, and the cell population is selectively enriched for cells bound thereto if the bound cells are further processed, or depleted for cells bound thereto if the remaining cells in the solution are further processed.

Table 3: enrichment of B cells secreting antigen-specific antibodies by panning with the respective antigen

In one embodiment the method as reported herein comprises a selection step prior to single cell deposition, wherein B cells producing specific and/or non-cross-reactive antibodies are selected based on cell surface markers and fluorescence activated cell sorting/gating. In one embodiment, mature B cells are sorted/enriched/selected. To select B cells from different experimental animal species, different cell surface markers can be used. It has been found that many of the available cell surface markers do not provide suitable markers, either alone or in combination.

By labeling non-target cell populations and non-specifically bound lymphocytes, these cells can be selectively removed. In this removal step, only incomplete removal can be achieved. Although removal is not quantitative, it provides an advantage for subsequent fluorescent labeling of the remaining cells, as the number of interfering cells can be reduced or even minimized. By using the markers outlined below, single cell deposition of mature B cells (memory B cells, affinity matured plasmablasts and plasma cells) by fluorescence activated cell sorting, higher numbers of IgG can be obtained in the co-culture step⁺Well/cell cloning.

The term "label" denotes the presence or absence of a surface marker, which can be determined by the addition of an anti-surface marker antibody that specifically binds and labels. Thus, after incubation with the respective specifically binding and labeled anti-surface marker antibody, the presence of the surface marker is determined by the presence of fluorescence, e.g. in the case of a fluorescent marker, and the absence of the surface marker is determined by the absence of fluorescence.

The different cell populations may be labeled by using different surface markers, such as CD3⁺-cells (T cells), CD19⁺-cells (B cells), IgM⁺Cells (mature naive B cells), IgG⁺Cells (mature B cells), CD38⁺-cells (e.g. plasmablasts), and IgG⁺CD38⁺-cells (pre-plasma cells).

As reported herein, IgG's have been developed for selection of mature⁺B cells (B cells)E.g., memory B cells, plasmablasts, and plasma cells). To select or enrich for B cells, cells are singly labeled, or doubly labeled, or triply labeled. Labeling is also required to result in labeled cells in about 0.1% to 2.5% of the total cell population. In one embodiment, the B cells deposited as single cells are selected by labeling of surface molecules present on 0.1% to 2.5% of the B cells in the cell population, in another embodiment, on 0.3% to 1.5% of the B cells in the cell population, and in another embodiment, on 0.5% to 1% of the B cells in the cell population.

0.5% -1% IgG in PBMC population⁺B cells can be double-labeled IgG⁺CD19⁺-cells, IgG⁺CD38⁺-cells and IgG⁺CD268⁺-a cell. Thus, in one embodiment of all methods reported herein, IgG⁺CD19⁺-B cells, IgG⁺CD38⁺-B cells or IgG⁺CD268⁺B cells are deposited as single cells.

0.5% -1% IgG in PBMC population^-B cells can be double-labeled IgG^-CD138⁺-a cell. Thus, in one embodiment of all methods reported herein, IgG^-CD138⁺B cells are deposited as single cells.

CD27⁺CD138⁺-cells or CD3^-CD27⁺The labeling of the cells results in about 1.5% of the cells in the cell population being individually labeled. Thus, in one embodiment of all methods reported herein, CD27⁺CD138⁺B cells or CD3^-CD27⁺B cells are deposited as single cells.

0.6% + -0.1% IgG in PBMC population⁺Hamster B cells can be double-labeled IgG⁺IgM^--hamster B cells. Thus, in one embodiment of all methods reported herein, IgG⁺IgM^-Hamster B cells are deposited as single cells.

At one endIn one embodiment, the IgG is derived from B cells obtained from an immunized animal^-CD138⁺B cells are deposited as single cells. In one embodiment of all methods reported herein, IgG is isolated from B cells obtained from an unimmunized animal⁺CD19⁺B cells are deposited as single cells. In another embodiment of all methods reported herein, IgG is isolated from B cells obtained from an unimmunized or an immunized animal⁺IgM^-B cells are deposited as single cells. In one embodiment of all methods reported herein, the IgG is⁺CD19⁺Murine B cells are deposited as single cells. This selection step results in an increased or even highest yield of IgG in the subsequent co-cultivation step⁺-a well. In another embodiment of all methods reported herein, the IgG is^-CD138⁺Murine B cells are deposited as single cells. The cells producing the highest amount of B cell clones were selected first and the cells producing the highest IgG concentration were selected second (see table 5). In another embodiment of all methods reported herein, the IgG is⁺CD19⁺Murine B cells and IgG^-CD138⁺Murine B cells are deposited as single cells. In a particular embodiment, the method is provided with the proviso that if the cell is of rabbit origin, the label is not IgG⁺B cells and/or CD138⁺-of B cells.

IgG⁺Murine B cells can be labeled with an anti-mouse-IgG-antibody 227(Ab227), IgG⁺Hamster B cells can be labeled with anti-hamster IgG-antibody 213(AB213) and/or anti-hamster IgG-antibody 225(AB225), rabbit B cells can be labeled with anti-IgG-antibody 184 (see Table 4).

Table 4: immunofluorescent labelling of B cells-the Table shows the average labelled ratio of the populations of murine B cells (A-E), hamster B cells (F-H) and rabbit B cells (I-J)

AB 120-goat anti-rabbit IgG-antibody Southern Biotech4030-09

AB 184-goat anti-rabbit IgG Fc-antibody AbDSerotech STAR121F

AB 185-goat anti-mouse IgG-antibody Caltag M35004-3

AB 200-goat anti-mouse IgM-antibody Invitrogen M31504

AB 212-goat anti-hamster IgG-antibody AbDSerotech STAR79F

AB 213-mouse anti-hamster IgG-antibody Becton Dickinson554010

AB 215-goat anti-mouse IgG-antibody Sigma B0529

AB 217-goat anti-mouse IgG-antibody AbDSerotech STAR120F

AB 218-rat anti-mouse CD 19-antibody Abcam AB22480

AB 219-goat anti-mouse IgM-antibody Rockland710-1607

AB 222-goat anti-mouse IgG-antibody Abcam AB7064

AB 223-mouse anti-hamster IgM-antibody Becton Dickinson554035

AB 224-mouse anti-hamster IgM-antibody Becton Dickinson554033

AB 225-mouse anti-hamster IgG-antibody Becton Dickinson554056

AB 227-goat anti-mouse IgG-antibody Sigma F8264

PE: phycoerythrin

APC: allophycocyanin

FITC: fluorescein isothiocyanate

It must be noted that not all commercially available antibodies can be used for labeling because of their low or non-existent specificity.

Murine B cells can be labeled with anti-IgG-antibody 227 and hamster B cells can be labeled with anti-IgG-antibody 213.

IgG⁺CD19⁺Murine B cells can be labeled with antibody 227 and antibody 218,

IgG⁺IgM^-murine B cells can be labeled with antibody 227 and antibody 219,

IgG⁺IgM^-hamster B cells can be labeled with antibodies 213 and 224,

IgG⁺rabbit B cells can be labeled with antibody 184,

IgG⁺IgM^-rabbit B cells can be labeled with antibody 184 and antibody 254 and SA263,

IgG⁺CD138⁺rabbit B cells can be labeled with antibody 259 and antibody 256.

Murine B cells can be labeled with anti-CD 27 antibody 235 or 236(AB235, AB236), anti-CD 38 antibody 192(AB192), anti-CD 138 antibody 233(AB233), and anti-CD 268 antibody 246(AB 246).

Table 5: immunofluorescent markers for determination of mature mouse (A-J), hamster (K) and rabbit (L-N) B cells

AB 184-goat anti-rabbit IgG-antibody AbD Serotec STAR121F

AB 189-hamster anti-mouse CD 3-antibody Becton Dickinson553062

AB 192-rat anti-mouse CD 38-antibody Becton Dickinson553764

AB 213-mouse anti-hamster IgG-antibody Becton Dickinson554010

AB 218-rat anti-mouse CD 19-antibody Abcam AB22480

AB 224-mouse anti-hamster IgM-antibody Becton Dickinson554033

AB 227-goat anti-mouse IgG-antibody Sigma F8264

AB 233-rat anti-mouse CD 138-antibody Becton Dickinson553714

AB 235-hamster anti-mouse CD 27-antibody Becton Dickinson558754

AB 236-hamster anti-mouse CD 27-antibody Becton Dickinson558753

AB 241-hamster anti-mouse CD 3-antibody Becton Dickinson553060

AB 246-rat anti-mouse BAFF-R-antibody eBioscience51-5943

AB 254-mouse anti-Rabbit IgM-antibody Becton Dickinson customization

AB 256-goat anti-rat IgG-antibody Southern Biotech3030-09

AB 259-rat anti-rabbit CD 138-antibody Roche Glycart AG

SA 263-streptavidin Invitrogen S866

A647：

FITC: fluorescein isothiocyanate

In one embodiment, the method comprises the steps of depleting macrophages from the B cell population and enriching the B cells in the B cell population for B cells that secrete antibodies that specifically bind to the target antigen.

Single cell deposition:

the method as reported herein comprises the step of depositing B cells of a B cell population as single cells. In one embodiment of all methods reported herein, the deposition into single cells is by Fluorescence Activated Cell Sorting (FACS). The labeling required for FACS single cell deposition can be performed as reported in the previous section.

In one embodiment of all methods reported herein, specifically labeled B cells are deposited as single cells. In another embodiment of all methods reported herein, the label is a cell surface marker labeled with a fluorescently labeled antibody. In another embodiment, the methods reported herein provide monoclonal antibodies. In one embodiment of all methods reported herein, mature B cells are deposited as single cells.

It has also been found that an additional centrifugation step after single cell deposition and before co-cultivation provides an increased number of antibody secreting cells and an increased amount of secreted IgG (see table 6 for experimental animals with human immunoglobulin loci).

Table 6: IgG positive wells/cell clones with or without centrifugation step after single cell deposition

In one embodiment of all methods reported herein, the method comprises the step of centrifuging the single sedimented cells prior to co-cultivation. In a particular embodiment, centrifugation is performed at 300x g for 5 minutes.

Co-culturing:

there may be a number of additional steps before and after the co-cultivation step with feeder cells.

In one embodiment of all methods reported herein, the individual deposited B cells and feeder cells are co-cultured in the presence of a feeder mix. In a particular embodiment, B cells are co-cultured with murine EL-4B5 feeder cells. The increase in yield during the co-cultivation step (IgG) can be achieved by suitable immunofluorescent labelling as described above⁺Well/cell clone number and IgG concentration), and from PBEnrichment or isolation of mature IgG in MC⁺-B cells.

By IgG from freshly isolated PBMC⁺CD19⁺-and/or IgG⁺CD38⁺Single cell deposition of B cells, giving the highest amounts of IgG⁺Well/cell cloning. By IgG after removal of macrophages or KLH-specific cells (keyhole limpet hemocyanin)⁺CD19⁺，IgG⁺CD38⁺-and/or IgG^-CD138⁺Single cell deposition of B cells, with good results. By IgG after removal of antigen-specific B cells⁺CD19⁺，IgG⁺CD38⁺-and/or IgG^-CD138⁺Single cell deposition of B cells, with improved results. Thus, in one embodiment of all methods reported herein, IgG is used⁺CD19⁺，IgG⁺CD38⁺-and/or IgG^-CD138⁺B cells are deposited as single cells.

It has been found that single cell deposition based on labelling as described above results in the highest proportion of IgG⁺Wells/cell clones and wells/cell clones with the highest IgG concentration in the supernatant. Thus, in one embodiment of all methods reported herein, IgG is used⁺CD19⁺-and/or IgG^-CD138⁺Murine B cells are deposited as single cells. In one embodiment of all methods reported herein, the IgG is⁺IgM^-Hamster B cells are deposited as single cells. In one embodiment of all methods reported herein, the IgG is⁺And/or IgG⁺CD138⁺And/or CD138⁺-and/or IgG⁺IgM^-Rabbit B cells are deposited as single cells.

Table 7: production in Co-culture depending on immunofluorescence labeling

For mouseB cells, by IgG after each enrichment (enr.) and/or depletion (depl.) step⁺CD19⁺Single cell deposition of cells, giving the highest quantities of IgG after co-cultivation⁺Well/cell cloning. Alternatively, by IgG^-CD138⁺Single cell deposition of cell wells/cell clones, optimal IgG concentration in supernatant could be obtained. IgG^-CD138⁺Single cell deposition of cells can be used for B cells from immunized animals. IgG⁺CD19⁺Single cell deposition of cells can be used for B cells from non-immunized animals. IgG⁺IgM^-Single cell deposition of cells can be used for hamster B cells of immunized and non-immunized animals. IgG⁺-, and/or IgG⁺CD138⁺And/or CD138⁺-and/or IgG⁺IgM^-Single cell deposition of B cells can be used for rabbit B cells.

Immunofluorescent labeling of B cells obtained from blood of experimental animals can also be used to label B cells obtained from the spleen and other immune organs of experimental animals (e.g., mice, hamsters, and rabbits). For mouse B cells, IgG from spleen⁺The proportion of B cells is about 0.8%, compared to 0.4% IgG⁺CD19⁺-a cell. For hamster B cells, corresponding values are 1.9% and 0.5% IgG⁺IgM^--a cell. For rabbit blood-derived B cells, 0.2% IgG was found after macrophage depletion⁺-a cell. Peyer' sche plaques from rabbits showed 0.4% IgG after macrophage removal⁺Cells, spleen shows 0.3% IgG⁺-a cell.

Using the methods reported herein, antibody concentrations ranging from about 30ng/ml up to 15 μ g/ml or more (average about 500ng/ml) can be obtained after about 7 days of co-cultivation, i.e. after 5, 6, 7 or 8 days, in particular after 7 or 8 days. Using the amount of antibody thus provided, a number of different assays can be performed to characterize the antibody in more detail, e.g., with respect to binding specificity. By improved characterization of the antibody during the screening/selection process at this early stage, the number of nucleic acid isolation required and sequencing reactions that must be performed can be reduced. Additionally, B cell cloning provides some amount of mRNA encoding monoclonal light and heavy chain variable regions that allows for the use of degenerate PCR primers and avoids the need for highly specific primers. The number of PCR cycles required is also reduced. Thus, in one embodiment, reverse transcriptase PCR uses degenerate PCR primers for the light and heavy chain variable domains.

In one embodiment of all methods reported herein, the feeding mixture is thymocyte culture supernatant. In a particular embodiment, the thymocyte culture supernatant is obtained from thymocytes of the thymus of the corresponding young animal. The thymus of young animals is particularly suitable for use compared to the isolation of thymocytes from the blood of adult animals. The term "young animal" means an animal prior to sexual maturity. For example, young hamsters are less than 6 weeks, particularly less than 4 weeks, of age. For example, the age of young mice is less than 8 weeks, particularly less than 5 weeks.

Since the source of the feeding mixture is the culture-derived thymocyte supernatant (thymocyte culture supernatant: (thymocyte cultivation supernatant) -TSN), there is considerable lot-to-lot variation. To overcome this variability, synthetic feeding mixes consisting of synthetic components have been developed. A feed mixture consisting of IL-1 β (interleukin-1 β), TNF α (tumor necrosis factor α), IL-2 (interleukin-2) and IL-10 (interleukin-10) is known from Tucci, A., et al, J.Immunol.148(1992) 2778-.

Synthetic feeder mixtures for co-culturing of single sedimented B cells and feeder cells are reported herein. Also reported herein are B cell species-specific additives for synthetic feed mixtures that are used to increase the amount of antibodies secreted by the corresponding B cell clones. While highly productive cells contain more mRNA, which in turn facilitates reverse transcription and sequencing of the encoding nucleic acid, e.g., using redundant, non-specific primer sets.

By adding SAC (staphylococcus aureus Cowans strain cells, using a single SAC batch), the number of antibody secreting B cells and the average IgG concentration in the supernatant after co-cultivation can be increased. It has been found that the concentration range of SAC added in co-culture can be limited, since decreasing as well as increasing SAC concentration decreases the amount of secreted antibody.

Table 8 a: results of huCkELISA (huCk ═ human ck) or rbIgG ELISA of cell culture supernatants of B cells obtained from experimental animals with human IgG loci or wild type rabbits (NZW) co-cultured with EL-4B5 feeder cells, using TSN as the feeder mix, with or without SAC.

It can be seen that SAC ratios from 1: 20000 to 1: 150000 provide increased amounts of IgG⁺Well/cell clones, where the ratio from 1: 50000 to 1: 100000 shows the highest numbers. In one embodiment, the amount of SAC added to the medium is determined by providing a dilution series and determining at which dilution SAC added provides the highest number of IgG positive wells/cell clones.

It has been observed that by adding SAC to the feeding mix, co-culture of B cells is surprisingly altered in such a way that only a single deposited B cell has a growth advantage, whereas when a mixture of PBLs (e.g. B cells and endogenous T cells) is used for co-culture, B cell growth is inhibited.

Table 8 b: results of huCk ELISA or rbIgG ELISA of cell culture supernatants of PBL and Single sedimented B cells co-cultured with EL-4B5 feeder cells, using TSN as the feeder mix, with addition of SAC

Removing macrophage

Cells

Additional data obtained with different feeding mixes are shown in tables 9 and 10 below.

In one embodiment of all methods reported herein, the synthetic feeder mix used for B cell co-culture comprises IL-1 β, TNF α, IL-2, IL-10 and IL-21 (interleukin-21). In one embodiment of all methods reported herein, the synthetic feeding mix used for B cell co-culture comprises IL-1 β, TNF α, IL-2, IL-10 and SAC. In a particular embodiment, IL-1 β, TNF α, IL-2, IL-10, and IL-21 are recombinant murine IL-1 β, murine TNF α, murine IL-2, murine IL-10, and murine IL-21.

In one embodiment of all methods reported herein, the synthetic feeding mix used for co-culture of murine B cells comprises IL-1 β, IL-2, IL-10, TNF- α and BAFF. In a particular embodiment, BAFF is added at a concentration of 5 ng/ml.

In one embodiment of all methods reported herein, the synthetic feeding mix for hamster B cell co-culture comprises IL-1 β, IL-2, IL-10, TNF- α, IL-6 and SAC. In a particular embodiment, IL-6 is added at a concentration of 10 ng/ml. In a particular embodiment, SAC is added in a ratio of 1: 75,000.

Table 9: rbIgG ELISA results of cell culture supernatants of rabbit B cells co-cultured with EL-4B5 feeder cells and different synthetic feeder mixes containing different combinations of recombinant murine material

Table 10: IgG of cell culture supernatant of rabbit B cells⁺Wells, said rabbit B cells are incubated with EL-4B5 feeder cells and TSN orCo-culture of a feeding mix comprising recombinant murine material and SAC (rb ═ rabbit, m ═ mouse)

Co-culture of feeder cells and murine B cells without IL-2, without IL-10, and without IL-2 and IL-10 resulted in IgG⁺Increase in well yield but decrease in IgG concentration. Absence of TNF α also reduced IgG concentrations. Without IL-1. beta. no IgG could be found in the supernatant.

Co-culture of hamster B cells without IL-2 or without IL-10, respectively, resulted in IgG having a detectable IgG concentration⁺-a well. In contrast, little B cell growth was detected in co-cultures without IL-2 and IL-10. In the absence of TNF- α or IL-1 β, no IgG secretion was detected.

Co-culture of mouse, hamster, and rabbit B cells requires at least IL-1 β and TNF α in the presence of EL-4B5 feeder cells. For co-culture of murine cells, IL-2 and IL-10 can be omitted. Hamster B cells can be cultured in the absence of IL-2 or IL-10. Rabbit B cells can be cultured in the absence of IL-2 or IL-10 or IL-6.

For murine and hamster B cells, addition of IL-4 to the feeding mix increased IgG⁺Well/cell clone number and IgG concentration in supernatant. Thus, in one embodiment of all methods reported herein, the feeder mix used for co-culture of murine or hamster B cells comprises IL-4.

Addition of IL-6 to a feeding mix for co-cultivation of murine B cells or hamster B cells resulted in increased IgG, respectively⁺Well/cell clone number or increased IgG concentration. Thus, in one embodiment of all methods reported herein, the feeder mix used for co-culture of murine or hamster B cells comprises IL-6. In a particular embodiment, IL-6 is added at a concentration of 50 ng/ml. In a specific embodiment, if high IgG concentrations are required, IL-6 is added at a concentration of 10 ng/ml. In a particularIn embodiments, IL-6 is added after 3 days of co-culture of selected B cells and EL-4B5 cells.

One aspect as reported herein is a synthetic feeder mix for co-culturing B-cells and feeder cells comprising IL-1 β, TNF α, IL-10, and one or more selected from the group consisting of IL-21, SAC, BAFF, IL-2, IL-4, and IL-6.

One aspect as reported herein is a synthetic feeder mix for co-culturing B cells and feeder cells comprising IL-1 β, TNF α, IL-2, IL-10 and SAC.

One aspect as reported herein is a synthetic feeding mix for co-culturing murine B cells and feeder cells consisting of IL-1 β, TNF α and optionally comprising IL-21 and/or SAC and/or BAFF and/or IL-6.

One aspect as reported herein is a synthetic feeder mix for co-culturing murine B cells and feeder cells comprising IL-1 β, IL-2, IL-10, TNF- α and BAFF.

One aspect as reported herein is a synthetic feeding mix for co-culturing murine or hamster B cells and feeder cells comprising IL-1 β, TNF α, IL-2, IL-10 and IL-6.

One aspect reported herein is a synthetic feeding mix for co-culturing hamster B cells and feeder cells, consisting of IL-1 β, TNF α, and IL-2 or IL-10, and optionally comprising IL-21, and/or SAC, and/or BAFF.

One aspect reported herein is a synthetic feeding mix for co-culturing hamster B cells and feeder cells comprising IL-1 β, IL-2, IL-10, TNF- α, IL-6 and SAC.

One aspect as reported herein is a synthetic feeding mix for co-culturing rabbit B cells and feeder cells comprising IL-1 β, TNF α, IL-10 and IL-6.

One aspect as reported herein is a synthetic feeding mix for co-culturing rabbit B-cells and feeder cells comprising IL-1 β, TNF α, IL-10, IL-6 or IL-2, and SAC.

In a particular embodiment, IL-1 β, TNF α, IL-2, IL-10, and IL-21 are recombinant murine IL-1 β, murine TNF α, murine IL-2, murine IL-10, and murine IL-21.

In a particular embodiment, BAFF is added at a concentration of 5 ng/ml.

In a particular embodiment, IL-6 is added at a concentration of 10 ng/ml.

In a particular embodiment, SAC is added in a ratio of 1: 75,000.

In a particular embodiment, the feeder cells are murine EL-4B5 cells.

The addition of some potassium channel inhibitor (═ PAP-1, 5- (4-phenoxybutoxy) psoralen) surprisingly increased B cell rbIgG secretion in a concentration-dependent manner without reducing B cell clone numbers. In general, cytokines that induce rbIgG productivity can be associated with a reduction in the total number of B cell clones. This is not the case with PAP-1.

Table 11: rbIgG ELISA of cell culture supernatants of B cells co-cultured with EL-4B5 feeder cells in the presence of TSN and SAC (═ w/o) and varying concentrations of PAP-1. DMSO, DMSO: PAP-1 solvent (1. mu.M)

The highest IgG concentration in the supernatant was obtained using a TSN concentration of 7.5%.

Table 12: effect of TSN on co-cultivation. TSN concentrations of 7.5% resulted in improved B cell growth and productivity

By using 30,000 feeder cells per well in a 96-well plate, the highest IgG was obtained⁺-the number of wells and the IgG concentration in the supernatant. In one embodiment of all methods reported herein, the number of feeder cells per single deposited B cell is about 30,000.

Table 13: effect of the amount of feeder cells EL-4B5 on Co-culture

In one embodiment of all methods reported herein, the co-culturing is performed in polystyrene multiwell plates with round bottom wells. In one embodiment of all methods reported herein, the working volume of the well is between 50 μ l and 250 μ l. In a particular embodiment, the pores are at least partially coated with a non-fibrous substrate (superstrate) prepared from a blend of a polymeric plastic resin and an amphiphilic molecule, wherein the amphiphilic molecule comprises a hydrophilic portion and a hydrophobic region, wherein the hydrophobic region is anchored within the substrate and the hydrophilic portion is exposed on the substrate. In a particular embodiment, the amphiphilic molecule is selected from ethoxylated alkylamines, poly (ethyleneimine), octyl deluxe base (octyledecamine) or mixtures thereof (see e.g. EP 1860181).

Characterization of the co-cultured cells:

for the (qualitative and quantitative) determination of the IgG secreted after co-cultivation, in general all methods known to the person skilled in the art, such as ELISA, can be used. In one embodiment of all methods reported herein, ELISA is used. In one particular embodiment for the determination of IgG secreted by murine B cells, an ELISA is used in which anti-IgG antibodies AB216 (capture antibody) and AB215 (tracer antibody) are used. In one particular embodiment for the determination of IgG secreted by hamster B cells, an ELISA is used in which monoclonal antibodies AB220 (capture antibody) and AB213 (tracer antibody) are used.

Depending on the characterization results, B cell clones can be obtained, i.e. selected. The term "clone" means a dividing and antibody-secreting B cell population derived from/derived from a single B cell. Thus, the B cell clone produces monoclonal antibodies.

Isolation, cloning and sequencing of mRNA:

total mRNA can be isolated from B cells and transcribed into cDNA. Homologous VH-and VL-region encoding nucleic acids can be amplified using specific primers. By sequencing the nucleic acids thus obtained, it was confirmed that the majority (71-95%) of the antibodies obtained were monoclonal antibodies. From the sequencing of the individual B cells, it can also be seen that almost no identical sequences were obtained. Thus, the methods provide highly diverse antibodies that bind to the same antigen.

Primers for amplifying VH-encoding nucleic acids can be used for cDNA obtained from cells from NMRI-mice, the sub-Mernia hamster, Balb/c-mice, and the Syrian hamsters and rabbits.

In one embodiment of all methods reported herein, the amino acid sequence is derived from an amplified VH-encoding nucleic acid, and the exact start and end points are identified by mapping the amino acid sequence EVQL/QVQL to VSS (VH region) and DIVM/DIQM to KLEIK (VL region).

The term "antibody" refers to a protein consisting of one or more polypeptide chains substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include various constant region genes as well as numerous immunoglobulin variable region genes. Immunoglobulins may exist in a variety of forms including, for example, Fv, Fab and F (ab)₂And single chain (scFv), diabodies, monovalent, divalent, trivalent or tetravalent forms, and bispecific, trispecific or tetraspecific forms (e.g., Huston, J.S., et al, Proc. Natl. Acad. Sci. USA85(1988)5879-Immunology, Benjamin n.y., second edition (1984); and Hunkapiller, T. and Hood, L., Nature323(1986) 15-16).

Also reported herein is a method of producing an antibody, comprising the steps of:

a) providing a population of (mature) B cells (obtained from the blood of experimental animals),

b) staining cells of the B cell population with at least one fluorescent dye (in one embodiment with 1 to 3, or 2 to 3 fluorescent dyes),

c) depositing individual cells of the stained B cell population in a separate container (in one embodiment, the container is a well of a multi-well plate),

d) culturing the deposited individual B cells in the presence of feeder cells and a feeder mix (in one embodiment, the feeder cells are EL-4B5 cells, in one embodiment, the feeder mix is natural TSN, in one embodiment, the feeder mix is a synthetic feeder mix),

e) determining the binding specificity of the antibody secreted in each B-cell culture,

f) determining the amino acid sequences of the variable light and heavy chain domains of the antibody specifically bound by reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining nucleic acids encoding the variable light and heavy chain domains of the monoclonal antibody,

g) introducing nucleic acids encoding the light and heavy chain variable domains of a monoclonal antibody into an expression cassette for expression of the antibody,

h) introducing a nucleic acid into a cell, introducing the nucleic acid into the cell,

i) culturing the cells and recovering the antibody from the cells or cell culture supernatant, and thereby producing the antibody.

An "expression cassette" refers to a construct containing the necessary regulatory elements (e.g., promoter and polyadenylation site) for the expression of at least a nucleic acid contained in a cell.

"laboratory animal" means a non-human mammal. In one embodiment, the laboratory animal is selected from the group consisting of rat, mouse, hamster, rabbit, non-human primate, sheep, dog, cow, chicken, amphibian, and reptile.

The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is to be understood that changes may be made in the illustrated methods without departing from the spirit of the invention.

Examples

Example 1

Media and buffers:

blocking buffer used for ELISA contained 1X PBS and 1% BSA.

The coating buffer used for ELISA contained 4.29g Na₂CO₃＊10H₂O and 2.93g NaHCO₃Water was added to a final volume of 1 liter and adjusted to pH9.6 with 2N HCl.

Ethanol solutions used for RNA isolation contained 70% ethanol or 80% ethanol.

FACS buffer for immunofluorescent staining contained 1X PBS and 0.1% BSA.

IMDM buffer used for ELISA contained 1X PBS, 5% IMDM and 0.5% BSA.

Incubation buffer 1 used for ELISA contained 1X PBS, 0.5% CroteinC.

Incubation buffer 2 used for ELISA contained 1X PBS, 0.5% CroteinC, and 0.02% tween 20.

Incubation buffer 3 used for ELISA contained 1X PBS,0.1% BSA.

Incubation buffer 4 for ELISA contains1 XPBS, 0.5% BSA, 0.05% Tween, PBS (10X), 0.01M KH₂PO₄，0.1M Na₂HPO₄，1.37M NaCl，0.027M KCl，pH7.0。

The PCR buffer contained 500mM KCl, 15mM MgCl₂，100mM Tris/HCl，pH9.0。

Wash buffer 1 for ELISA contained 1X PBS,0.05% tween 20.

Wash buffer 2 used for ELISA contained 1X PBS,0.1% tween 20.

Wash buffer 3 for ELISA contained water, 0.9% NaCl,0.05% tween 20.

EL-4B5 medium contained RPMI1640,10% FCS,1% glutamine/penicillin/streptomycin-mixture, 2%100mM sodium pyruvate, 1%1M HEPES buffer.

Example 2

Animal care and immunization

The experimental animals were kept according to the german animal protection law (TierSCHG) and according to the corresponding european guidelines.

Mice and hamsters from 6 to 8 weeks of age were received and immunized before 12 weeks of age. The antigen was first applied with Complete Freund's Adjuvant (CFA). Further use was made of Incomplete Freund's Adjuvant (IFA). The emulsion containing the antigen is applied subcutaneously, wherein the emulsion comprises an amount of antigen from 50 to 100 μ g depending on the weight of the subject animal.

NZW rabbits (Charles River Laboratories International, Inc.) were used for immunization. At a concentration of 1mg/ml in K₃PO₄The antigen was dissolved in buffer pH7.0 and mixed with Complete Freund's Adjuvant (CFA) (1:1) until a stable emulsion was formed. Rabbits received 2ml intradermal (i.d.) injections of the emulsion followed by a second intramuscular (i.m.) and a third subcutaneous (s.c.) injection of 1ml each at 1 week intervals. A fourth intramuscular injection of 1ml was performed 2 weeks later,followed by another 2 subcutaneous injections at 4 week intervals.

During immunization, antigen-specific assays are used to determine serum antibody titers. IC at 1:10000₅₀The blood or spleen of the immunized animal is removed. To reactivate antigen-specific B cells, 30. mu.g to 50. mu.g of antigen was applied intravenously to experimental animals 3 days before blood or spleen removal.

Example 3

Organ, blood and macrophage removal

Blood from mice and hamsters was obtained by retrobulbar venipuncture. Blood from rabbits was obtained by puncture of the auricular vein or the auricular artery (for larger volumes). Whole blood (10ml) was collected from rabbits 4-6 days after 3, 4, 5 and 6 immunizations and used for single cell sorting by FACS.

Macrophages were isolated by attachment to cell culture plastic. From mice and hamsters, approximately 3x10 can be obtained from each animal by this method⁵And macrophages are formed.

Peritoneal macrophages are isolated if a greater amount of mouse or hamster macrophages are required. For this purpose, the animals must be at least 3 months old. To remove peritoneal macrophages, the animals were sacrificed and 5ml of EL-4B5 medium at 37 ℃ was immediately injected into the peritoneal cavity. After kneading the abdomen of the animal for 5 minutes, the solution containing the cells was removed.

Example 4

Culture of EL-4B5 cells

Frozen EL-4B5 cells were flash thawed in a 37 ℃ water bath and diluted with 10ml of EL-4B5 medium. After centrifugation at 300x g for 10 minutes, the supernatant was discarded and the pellet was resuspended in culture medium. After another centrifugation step the supernatant was discarded again and the pellet resuspended in 1ml of medium.

At 3x10⁴The cell density of the cells/ml is 175cm²The flasks were inoculated with EL-4B5 cells. Cell density was measured every 2 days and adjusted to 3X10⁴Cells/ml. Cells have a doubling time of about 12 hours and must be below 5x10⁵Cells are cultured at a cell density of cells/ml, since with higher cell densities the irritation of the cells is lost.

When the total cell number is about 1.5X10⁹When the cells are cultured, the medium is removed by centrifugation. The cells were then irradiated with 50 gray (5000 rads). After determination of viable cell number by trypan blue staining, 5x10 was aliquoted⁶To 1x10⁷And frozen at-80 ℃.

For co-culture, cells were thawed and washed twice with EL-4B5 medium. To determine viable cell number, a 0.4% (w/v) trypan blue solution was used at a ratio of 1: the cell suspension was diluted 10 and 10. mu.l of the mixture was transferred to a Bowden (Neubauer) counting chamber and the number of cells was counted.

Example 5

Density gradient centrifugation

Use ofAccording to manufacturer's instructions AMammalian, cedarlane) by density gradient separation to achieve separation of Peripheral Blood Mononuclear Cells (PBMCs).

The aspirated blood was diluted 2: 1 with Phosphate Buffered Saline (PBS). The same volume of density separation medium was provided in the centrifuge tube and diluted blood was carefully added through the tube wall. The vials were centrifuged at 800Xg for 20 minutes without braking (scraping). Lymphocytes were obtained from the white intermediate layer. The removed cells were supplemented with 10ml PBS and centrifuged at 800x g for 10 min. The supernatant was discarded and the precipitate was resuspended, washed and centrifuged. Resuspend the final pellet in PBS.

Example 6

Hypotonic lysis of red blood cells

To destroy red blood cells by hypotonic lysis, ammonium chloride solution (BDLyse) was diluted 1:10 with water^TM) And added to whole blood at a ratio of 1: 16. To lyse the red blood cells, the mixture was incubated in the dark for 15 minutes. To separate cell debris from intact cells, the solution was centrifuged at 800x g for 10 minutes. The supernatant was discarded, the pellet resuspended in PBS, washed again, centrifuged and the pellet resuspended in PBS.

Example 7

Preparation of cells from internal organs of laboratory animals

To prepare spleen and thymocytes, the corresponding organs were dissected in a petri dish and cells were extracted in PBS. To remove the remaining tissue, the cell suspension was filtered through a 100 μm sieve. To obtain lymphocytes from splenocytes, density gradient centrifugation was used. No additional enrichment step is required for thymocytes.

Example 8

Removing macrophages

Macrophages and monocytes were removed by nonspecific adhesion using sterile 6-well plates (cell culture grade). The wells were coated with either KLH (keyhole limpet hemocyanin) or streptavidin and control peptide. 3ml to a maximum of 4ml of medium and a maximum of 6X10 were added to each well⁶Peripheral blood mononuclear cells from immunized rabbits were allowed to bind at 37 ℃ for 60 to 90 minutes in an incubator. The supernatant containing the lymphocytes was then transferred to a centrifuge tube and centrifuged at 800Xg for 10 minutes. Resuspend the pellet in PBS.

Using 50% of the cells in the supernatant for the panning step; the remaining 50% of cells were directly subjected to immunofluorescence staining and single cell sorting.

Example 9

Removal of KLH-specific B cells

4ml of a solution containing Keyhole Limpet Hemocyanin (KLH) at a concentration of 2. mu.g/ml were incubated overnight in the wells of the multi-well plate with the coating buffer at room temperature. Prior to the removal step, the supernatant was removed and the wells were washed 2 times with PBS. The blood cells were then adjusted to 2x10⁶Cell density of cells/ml, and 3ml per well of the multi-well plate. The multi-well plates were then incubated at 37 ℃ for 60 to 90 minutes. The supernatant was transferred to a centrifuge tube and washed 2 times with PBS and the supernatant was combined in the centrifuge tube. Cells were pelleted by centrifugation at 800Xg for 10 minutes and resuspended in PBS.

Example 10

Enrichment of antigen-specific B cells

The corresponding antigen was diluted to a final concentration of 2. mu.g/ml with coating buffer. 3ml of this solution was added to the wells of a 6-well multiwell plate and incubated overnight at room temperature. The supernatant was removed prior to use and the wells were washed 2 times with PBS. B cell solution concentration was adjusted to 2x10⁶Cells/ml and 3ml was added to each well of a 6-well multiwell plate. Plates were incubated at 37 ℃ for 60 to 90 minutes. The supernatant was removed and the wells were washed 2 to 4 times with PBS. To recover antigen-specific B cells, 1ml of trypsin/EDTA solution was added to the wells of the multi-well plate and incubated at 37 ℃ for 10 to 15 minutes. The incubation was terminated by adding medium and the supernatant was transferred to a centrifuge tube. Wash 2 wells with PBS and combine the supernatant with other supernatants. Cells were pelleted by centrifugation at 800x g for 10 minutes. Resuspend the pellet in PBS.

Example 11

Co-culture of B cells and EL-4B5 cells

a) Co-culture was performed in 96-well multi-well plates with a round bottom. Preparation of cells containing EL-4B5 (1.6X 10) in EL-4B5 Medium⁶Cells/15.2 ml) and cytokines. To each well of the multiwell plate, 200. mu.l of the base solution was added. A single B cell was added to each well by fluorescence activated cell sorting. After the addition of B cells, the plates were centrifuged at 300x g for 5 minutes. Plates were incubated at 37 ℃ for 7 days.

b) In an incubator at 37 ℃ and 5% CO₂In a 96-well plate, single sorted B cells and Pansorbin cells (1: 20000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant and Lambda-irradiated EL-4-B5 murine thymoma cells (2X 10) were cultured in a 96-well plate using 210. mu.l/well of EL-4B5 medium⁴Hole) for 7 days. B cell culture supernatants were removed for screening and cells were harvested immediately for variable region gene cloning or frozen at-80 ℃ in 100. mu.l RLT buffer (Qiagen, Hilden, Germany).

Example 12

Culture of T-cells

T cells were isolated from thymus of mice and hamsters, respectively, 3-4 weeks old, or rabbits, respectively, 4-5 weeks old. Cells were centrifuged and cultured immediately or at 3 × 10⁷Aliquots of cells were frozen. At 175cm²Minimum cell density 5x10 in culture flask⁵Cells/ml EL-4B5 medium were inoculated into thymocytes and incubated at 37 ℃ for 48 hours.

Example 13

Culture of macrophages

Macrophages were isolated from the peritoneal cavity of mice and hamsters, respectively, of at least 3 months of age. Culturing in EL-4B5In the radical of at least 1x10⁵The cell density of the cells/ml is 175cm²Peritoneal cavity macrophages from mice or hamsters, or blood mononuclear cells from rabbits were cultured in culture flasks at 37 ℃ for 1.5 hours. The medium was then removed and the unattached cells were removed from the attached macrophages by washing with warmed EL-4B5 medium followed by culture in 35ml of medium for 48 hours.

Example 14

Co-culture of T-cells and macrophages

T-cells and macrophages were cultured in separate flasks for 48 hours. T cells were centrifuged at 800x g for 10 minutes before combining the two cell populations. The supernatant was discarded and the cell pellet was resuspended in 10ml of medium. Regulation of T cells to 5x10⁵Minimum cell density of cells/ml and 10pg phorbol 12-myristate 13-acetate (PMA) and 5ng or 50ng phytohemagglutinin M (PHA-M) per ml of medium. The macrophage medium was removed and the T cell suspension was added to the macrophage containing flask. After 36 hours of co-cultivation, the medium was removed and referred to as TSN solution. To remove the remaining cells, the TSN solution was filtered through a 0.22 μm filter. The TSN solution was frozen at-80 ℃ in 4ml aliquots.

Example 15

Immunofluorescence staining

Depending on the number of cells to be stained, 100. mu.l of the medium (less than 10) were used⁶Individual cells) or 200. mu.l of medium (more than 10)⁶Individual cells) are provided. The fluorescently labeled antibody was diluted to a final volume of 100. mu.l or 200. mu.l with 5% experimental animal serum and FACS buffer, respectively. The reaction mixture was incubated at 4 ℃ in the dark for 40 min on a roller rack (roller rack). After incubation, cells were washed 2 times at 300x g for 5 min. Resuspend the pellet in 400. mu.l PBS and filter through a 70 μm sieve. The filtered solution was transferred to FACS vials,and dead cells were stained directly by the addition of propidium iodide (6.25. mu.g/ml) before the FACS experiment was started. If the labeled antibody is labeled with biotin, the antibody is detected in a second step with streptavidin-labeled Alexa Flour (R)647 (antibody 197).

Example 16

Quantification of IgG

After 7 days of co-cultivation, the co-cultured 96-well multi-well plates were centrifuged at 300x g for 5 minutes. Mu.l of the supernatant was removed and diluted 2: 1 with PBS in a second 96-well multi-well plate.

ELISA was performed as outlined in example 17.

The antibody was used at a concentration of 50 ng/ml. If the OD is 1 or more than 1 after 5 minutes incubation time, a dilution series from 0.8 to 108ng/ml IgG is detected.

Example 17

Detection of antigen-specific IgG

Antibodies produced by single deposited and co-cultured B cells or B cells obtained from immunized animals can be characterized with respect to specific antigen binding. The ELISA was performed at room temperature and the ELISA solution was incubated at 20x g on a shaker between steps. In the first step, antigens are bound to the wells of a 96-well multi-well plate. If the antigen is a protein, it has been diluted in coating buffer and applied directly to the plate. The peptide antigen is bound to biotin/streptavidin by specific binding. The wells of a multiwell plate may have been coated by the manufacturer with soluble Crotein C (CrC). If not, wells were incubated with 200. mu.l blocking buffer after antigen immobilization. After incubation with 100 μ l antigen solution/well (pre-coated multiwell plate) or 200 μ l blocking buffer, respectively, unbound antigen or blocking buffer is removed by washing with washing buffer. Diluted B cell supernatant was added at a volume of 100 μ l per well and incubated. Wells were washed after incubation. The detection antibody was then added in a volume of 100. mu.l per well. The antibody may be conjugated to horseradish peroxidase or labeled with biotin. Detection antibodies were assayed with streptavidin-horseradish peroxidase conjugate. After incubation, the well plates were washed, then 50 μ l of substrate solution containing 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) was added per well and incubated for the times as provided in table X. The enzymatic reaction was stopped by adding 50. mu.l sulfuric acid and the optical densities were measured at 450nm and 680nm using a photometer (Rainbow Thermo ELISA Reader) and Xread plus-software.

Example 18

Isolation of ribonucleic acid (RNA)

Cells from which RNA must be isolated are first pelleted by centrifugation. The cell pellet was lysed by adding 100. mu.l of RLT-buffer with 10. mu.l/ml beta-mercaptoethanol. Resuspend cells by mixing multiple times with a pipettor. The solution was transferred to wells of a multiwell plate. The plates were briefly shaken at 200x g and frozen at-20 ℃.

Use ofThe isolation of RNA was carried out with the kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Example 19

Reverse transcription polymerase chain reaction

Reverse transcription was performed in a volume of 20. mu.l. Controls with and without reverse transcriptase were performed for each reaction. Mu.l dNTPs (10 mM each), 0.4. mu.l oligo (dT) were premixed for each reaction_12-18(0.2. mu.g) and 0.6. mu.l random hexamer primer (0.03. mu.g) were then added to 8.5. mu.l of RNA in H2O. The reaction mixture was incubated at 65 ℃ for 5 minutes and then transferred directly to ice. Mu.l RT-buffer (10X), 4. mu.l MgCl2(25mM), 2. mu.l were premixed thereafterDTT (0.1M) and 1. mu.l RNAse Out^TM(40 units) and added to the ice-cold reaction mixture. After incubation at room temperature for 2 minutes, 0.5. mu.l Superscript was added^TMII reverse transcriptase (25 units). The reaction mixture was incubated at room temperature for 10 minutes.

Reverse transcription was performed at 42 ℃ for 50 min. The reverse transcriptase was inactivated by incubation at 70 ℃ for 15 minutes after reverse transcription. The cDNA was stored at-20 ℃.

Example 20

Polymerase chain reaction

The polymerase chain reaction was performed using the Taq PCR Core kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR was performed in a volume of 20. mu.l. Transfer the sample to a temperature of 95 ℃

Example 21

Sequencing

All sequences were determined by sequiserver (varstetten, germany).

Example 22

Panning antigens

a) Coating of board

Biotin/streptavidin: sterile streptavidin-coated 6-well plates (cell culture grade) were incubated with biotinylated antigen in PBS at a concentration of 0.5-2. mu.g/ml for 1 hour at room temperature. Plates were washed 3 times with sterile PBS prior to use.

Covalently bound protein: cell culture 6-well plates were coated with 2. mu.g/ml protein in carbonate buffer (0.1M sodium bicarbonate, 34mM disodium bicarbonate, pH9.55) overnight at 4 ℃. Plates were washed 3 times with sterile PBS prior to use.

b) Panning B cells on peptides

Seeding of up to 6X10 in 6-well tissue culture plates coated with the corresponding peptide⁶Cells/4 ml medium and allowed to bind for 1 hour at 37 ℃ in the incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1x PBS. The remaining adherent cells were detached by trypsin incubation at 37 ℃ for 10 minutes in an incubator and then washed 2 times in the culture medium. Cells were maintained on ice until immunofluorescent staining.

Claims (10)

1. A method of obtaining B cells, the method comprising the steps of:

a) obtaining B-cells from rabbit blood;

b) labeled IgG⁺-B-cells and/or CD138⁺-a B-cell;

c) incubating the B cells in co-culture medium at 37 ℃ for one hour before depositing the labeled B-cells as single cells;

d) co-culturing the single deposited cells with feeder cells in a co-culture medium,

e) selecting the B-cells proliferating and secreting the antibody in step d) and thereby obtaining B-cells.

2. Method according to claim 1, characterized in that it comprises a step of centrifugation of the single-cell sedimented cells before co-cultivation.

3. Method according to any one of the preceding claims, characterized in that it comprises, immediately before the marking step, the following step: ab) panning B cells with immobilized antigen.

4. Method according to claim 1 or 2, characterized in that the co-cultivation is in polystyrene multiwell plates with round-bottomed wells, said wells being at least partially coated with a non-fibrous substrate prepared from a blend of a polymeric plastic resin and amphiphilic molecules.

5. Method according to claim 1 or 2, characterized in that the B-cells are obtained from rabbit blood by density gradient centrifugation.

6. A method according to claim 1 or 2, characterized in that the feeder cells are murine EL-4B5 cells.

7. Method according to claim 1 or 2, characterized in that the co-cultivation medium comprises a feeding mixture.

8. The method according to claim 7, characterized in that the feeding mixture is thymocyte culture supernatant.

9. The method according to claim 7, characterized in that the feeding mixture comprises interleukin-1 β and tumor necrosis factor α, and at least one substance selected from the group consisting of interleukin-2, interleukin-10, staphylococcus aureus (staphylococcus aureus) Cowans strain cells, interleukin-21, BAFF, interleukin-6, and interleukin-4.

10. A method of producing an antibody, the method comprising the steps of:

a) providing a population of mature B-cells obtained from rabbit blood;

b) labeling IgG with at least one fluorescent dye⁺B cells and/or CD138⁺-a B cell;

c) incubating the B cells in a co-culture medium at 37 ℃ for one hour prior to depositing individual cells of the labeled B cell population in a separate container;

d) culturing each deposited B cell in the presence of feeder cells and a feeder mix;

e) determining the binding specificity of the antibody secreted in each B-cell culture medium,

f) determining the amino acid sequences of the variable light and heavy chain domains of the antibody specifically bound by reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining nucleic acids encoding the variable light and heavy chain domains of the monoclonal antibody,

g) introducing nucleic acids encoding the variable light and heavy chain domains of a monoclonal antibody into an expression cassette for expression of the antibody,

h) introducing a nucleic acid into a cell, introducing the nucleic acid into the cell,

i) culturing the cells and recovering the antibody from the cells or cell culture supernatant, and thereby producing the antibody.