CN120424205A

CN120424205A - An anti-cTnI antibody and its application

Info

Publication number: CN120424205A
Application number: CN202410155057.2A
Authority: CN
Inventors: 钟冬梅; 梁碧; 唐丽娜; 孟媛
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2024-02-03
Filing date: 2024-02-03
Publication date: 2025-08-05
Also published as: WO2025162496A1

Abstract

The invention discloses an anti-cTnI antibody and application thereof, and relates to the field of antibodies. The anti-cTnI antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for the detection of cTnI, and has good affinity or activity.

Description

Anti-cTnI antibody and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-cTnI antibody and application thereof.

Background

Before the 80 s of the 20 th century, the World Health Organization (WHO) has been taking myocardial enzymatic activity as one of the diagnostic criteria for Acute Myocardial Infarction (AMI). At the end of the 80 s of the 20 th century, researchers found that troponin (troponin, tn) was more sensitive and specific than biomarkers of phosphocreatine kinase (CK), phosphocreatine kinase isozymes (CK-MB), lactate dehydrogenase and aspartate aminotransferase. Cardiac troponin (cTnI) is only present in the heart muscle and is a marker of cardiac myocytes, and its abnormal changes can affect the contractile function of the heart, and can be used for diagnosing myocardial necrosis, judging myocardial injury, etc., as one of the markers with strongest sensitivity and specificity for myocardial injury, and is a recognized main biochemical marker for rapidly diagnosing AMI and acute coronary syndrome (acute coronary syndromes, ACS) and assisting ACS risk stratification and reflecting prognosis thereof.

The cTnI content in normal human blood is generally lower than 0.3 mug/L. When the integrity of the membrane of a myocardial cell is destroyed by ischemia or hypoxia, free cTnI can rapidly penetrate the cell membrane into the blood stream. Therefore, the rapid, sensitive and accurate measurement of the cTnI and the change trend thereof in human blood at the initial stage of onset has important clinical significance for diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, monitoring of myocardial injury caused by various factors and the like. Methods for detecting cTnI levels clinically include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold, etc., and various methods have their own advantages and disadvantages, but all require antibodies against cTnI.

There is therefore a strong need in the art for anti-cTnI antibodies with good properties.

Disclosure of Invention

The application provides an anti-cTnI antibody, which provides an important raw material source for the detection of cTnI and has good activity or affinity.

In order to achieve the above object, according to one aspect of the present invention, there is provided an anti-cTnI antibody comprising three complementarity determining regions having any one of the heavy chain variable regions of amino acid sequences SEQ ID NOs 22, 23, 24, 25 and three complementarity determining regions having any one of the light chain variable regions of amino acid sequences SEQ ID NOs 30, 31, 32, 33.

In order to achieve the above object, according to a second aspect of the present invention, there is provided an anti-cTnI antibody comprising the following complementarity determining regions:

HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1;

HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;

HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No. 3;

LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4;

LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5;

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6.

In order to achieve the above object, according to a third aspect of the present invention, there is provided an anti-cTnI antibody comprising a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs 22, 23, 24, and 25 and/or a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs 30, 31, 32, and 33.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an anti-cTnI antibody comprising a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs 26, 27, 28 and 29 and/or a light chain having an amino acid sequence shown in any one of SEQ ID NOs 34, 35, 36 and 37.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a reagent or kit comprising the above antibody or the above antibody conjugate.

In order to achieve the above object, according to a seventh aspect of the present invention, there is provided use of the above antibody, antibody conjugate in the preparation of a cTnI product for detection.

In order to achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for producing the above antibody.

Detailed Description

In a first aspect, embodiments of the invention provide an anti-cTnI antibody comprising three complementarity determining regions having any one of the heavy chain variable regions of amino acid sequences SEQ ID NOs 22, 23, 24, 25 and three complementarity determining regions having any one of the light chain variable regions of amino acid sequences SEQ ID NOs 30, 31, 32, 33.

The HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody according to the first aspect, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody according to the first aspect.

For example, the HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 22, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 31.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments so long as they exhibit the desired biological activity.

Antigen binding fragments typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that the antigen binding fragments described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

Antigen binding fragments may also be obtained by recombinant genetic techniques also known to those skilled in the art or by synthesis, for example, by an automated peptide synthesizer such as that sold by Applied BioSystems and the like.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR and includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is represented by LCDR and includes LCDR1, LCDR2 and LCDR3.

CDR definition methods are well known in the art and include Kabat definition, chothia definition, IMGT definition, contact definition and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below, with slightly different definitions in the different documents. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.

TABLE 1 CDR definition ¹

CDR	Kabat	AbM2	IMGT	Chothia
					HCDR1	H31~H35³	H26~H35³	H26~H33..5⁵	H26~H32..34⁴
HCDR2	H50~H65	H50~H58	H51~H57	H52~H56
					HCDR3	H95~H102	H95~H102	H93~H102	H95~H102
LCDR1	L24~L34	L24~L34	L27~L32	L24~L34
					LCDR2	L50~L56	L50~L56	L50~L51	L50~L56
LCDR3	L89~L97	L89~L97	L89~L97	L89~L97

¹ The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of Immunological Interest" (1983).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ CDR-H1 ends at position 35 if both H35A and H35B are absent, CDR-H1 ends at position 35A if only H35A is present, and CDR-H1 ends at position 35B if both H35A and H35B are present.

⁴ CDR-H1 ends at bit 32 if both H35A and H35B are absent, at bit 33 if only H35A is present, and at bit 34 if both H35A and H35B are present.

⁵ CDR-H1 ends at position 33 if both H35A and H35B are absent, CDR-H1 ends at position 34 if only H35A is present, and CDR-H1 ends at position 35 if both H35A and H35B are present.

According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or combination of systems Kabat, chothia, IMGT, abM or contacts.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Kabat system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Chothia system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an AbM system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact system combinations.

In a second aspect, embodiments of the invention provide an anti-cTnI antibody comprising the following complementarity determining regions:

HCDR1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1.

HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 2.

HCDR3, comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 3.

LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4.

LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5.

According to an embodiment of the invention, HCDRs and LCDRs are defined by the Kabat system.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, which refers to regions of an antibody heavy chain variable region and a light chain variable region other than CDRs, wherein the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework region can be further subdivided into contiguous regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.

In an alternative embodiment, the antibody of the first or second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

In alternative embodiments, the HFR1 comprises/is as set forth in SEQ ID NO 7 or an amino acid sequence having at least 80% identity thereto;

The HFR2 comprises/has an amino acid sequence as shown in SEQ ID NO 8 or having at least 80% identity thereto;

the HFR3 comprises/has as SEQ ID NO 9 or an amino acid sequence having at least 80% identity thereto;

The HFR4 comprises/has an amino acid sequence as shown in SEQ ID NO 10 or having at least 80% identity thereto;

the LFR1 comprises/is as SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;

the LFR2 comprises/is as SEQ ID No. 12 or an amino acid sequence having at least 80% identity thereto;

The LFR3 comprises/has as SEQ ID NO 13 or an amino acid sequence having at least 80% identity thereto, and

The LFR4 comprises/is as set forth in SEQ ID No. 14 or an amino acid sequence having at least 80% identity thereto.

In other embodiments, each framework region amino acid sequence of an anti-cTnI antibody provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.

In alternative embodiments, the HFR1 comprises/is an amino acid sequence as set forth in any one of SEQ ID NOs 17, 18, 19.

In an alternative embodiment, the LFR1 comprises/is an amino acid sequence as shown in SEQ ID NO. 20.

In an alternative embodiment, the HFR2 comprises/is the amino acid sequence shown in SEQ ID NO. 21.

In an alternative embodiment, the antibody binds cTnI with an affinity of KD <4.04 x 10 ^-8 M.

In alternative embodiments, the antibody binds cTnI with an affinity of KD≤10 10 ^-8M、KD≤10^-9M、KD≤10^-10M、KD≤10^-11 M or KD≤10 10 ^-12 M.

In an alternative embodiment, the antibody binds cTnI with an affinity of KD≤4.70X10 ^-9 M.

Antibody affinity (KD) assays are widely varied and can be classified into thermodynamic, kinetic and dynamic equilibrium assays based on the principle of detection. Among them, thermodynamic detection methods are commonly known as Isothermal Titration Calorimetry (ITC), kinetic detection methods are commonly known as Surface Plasmon Resonance (SPR) and biological membrane light interferometry (BLI), and dynamic equilibrium detection methods are commonly known as enzyme-linked immunosorbent assay (ELISA).

In alternative embodiments, the KD is determined using kinetic detection methods, alternatively surface plasmon resonance, for example, by using techniques such asA biosensor system of the system.

In a third aspect, embodiments of the invention provide an anti-cTnI antibody comprising a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs 22, 23, 24 and 25, and the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs 30, 31, 32 and 33.

In an alternative embodiment, the heavy chain variable region and the light chain variable region of the first aspect or the third aspect above are selected from any combination of:

Combination of two or more kinds of materials	Heavy chain variable region	Light chain variable region
			1	SEQ ID NO:22	SEQ ID NO:30
2	SEQ ID NO:25	SEQ ID NO:30
			3	SEQ ID NO:24	SEQ ID NO:30
4	SEQ ID NO:22	SEQ ID NO:33
			5	SEQ ID NO:22	SEQ ID NO:32
6	SEQ ID NO:23	SEQ ID NO:31

。

In an alternative embodiment, the antibody of the first, second and third aspects further comprises a constant region.

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of any one of IgG, igA, igM, igE, igD or a combination of multiple constant region segments.

In alternative embodiments, the heavy chain constant region comprises CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM, and/or CH4 of IgM.

In alternative embodiments, the IgG is selected from IgG1, igG2, igG3, or IgG4.

In alternative embodiments, the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of species origin of cow, horse, cow, pig, sheep, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, nugget, or human.

In an alternative embodiment, the constant region is of ovine species origin.

In this context, the division of the variable and constant region sequences is referred to the IMGT division method, see Lefranc,the international ImMunoGeneTics database.Nucl.Acids Res.,29(1):207-209(2001).DOI:10.1093/nar/29.1.207.PMID:11125093. Martinez-Jean C.and Bosc N. OrEhrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire.the international ImMunoGenetics informationHttp:// www.imgt.org.Created:16/03/2011.Version:17/01/2020. OrEhrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire.the international ImMunoGenetics informationHttp:// www.imgt.org.Created:16/03/2011.Version:17/01/2020. The variable region divided by different methods may have a partial amino acid difference from the variable region C-terminal or constant region N-terminal divided by IMGT, and other methods known in the art may be used to divide the variable region or constant region within the scope of the present invention.

In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 15 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 16.

In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NO:15 or 16) described above.

In alternative embodiments, the antibody comprises any one of F (ab) 2, F (ab ') 2, fab', fab, fv, and scFv.

In a fourth aspect, the invention provides an anti-cTnI antibody, which comprises a heavy chain and/or a light chain, wherein the amino acid sequence of the heavy chain is shown as any one of SEQ ID NO. 26, 27, 28 and 29, and the amino acid sequence of the light chain is shown as any one of SEQ ID NO. 34, 35, 36 and 37.

In an alternative embodiment, the antibody of the first, second, third or fourth aspect above, comprises a heavy chain and a light chain in any one of the following combinations:

Combination of two or more kinds of materials	Heavy chain	Light chain
			1	SEQ ID NO:26	SEQ ID NO:34
2	SEQ ID NO:29	SEQ ID NO:34
			3	SEQ ID NO:28	SEQ ID NO:34
4	SEQ ID NO:26	SEQ ID NO:37
			5	SEQ ID NO:26	SEQ ID NO:36
6	SEQ ID NO:27	SEQ ID NO:35

In a fifth aspect, the invention provides an antibody conjugate comprising an antibody as described above.

In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

In alternative embodiments, the antibody conjugate further comprises a label or purification tag conjugated to the antibody.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18F.

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbons, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the antibody conjugate described above further comprises a solid support coupled to the antibody.

In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In a sixth aspect, the invention provides a reagent or kit comprising an antibody as described above or an antibody conjugate as described above.

As previously described, the antibodies in some embodiments or examples of the invention are capable of efficiently binding cTnI, and therefore, reagents or kits comprising the cTnI antibodies are capable of efficiently performing qualitative or quantitative detection of cTnI. The reagent or the kit provided by the invention can be used for detection of specific binding performance of cTnI and antibodies thereof, such as immunoblotting, immunoprecipitation and the like. As previously mentioned, the antibodies in some embodiments or examples of the invention have higher binding activity or affinity to cTnI, and thus the reagents or kits comprising the antibodies have higher detection sensitivity or specificity.

In a seventh aspect, the invention provides a method of detecting cTnI comprising a) contacting an antibody, antibody conjugate, reagent or kit as described above with cTnI in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immunocomplex, and b) detecting the presence of the immunocomplex, the presence of the complex being indicative of the presence of the antigen in the test sample;

In an alternative embodiment, the immune complex further comprises a second antibody, which binds to the antibody.

In an alternative embodiment, the immune complex further comprises a second antibody that binds cTnI.

In an eighth aspect, the invention provides the use of the anti-cTnI antibodies and antibody conjugates described above in the preparation of a product for detecting cTnI.

It should be noted that the products of the present invention include, but are not limited to, reagents, kits, test strips or reagent strips.

In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above antibody.

In a tenth aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In an eleventh aspect, the present invention provides a cell comprising the vector described above.

In a twelfth aspect, the invention provides a method of making an anti-cTnI antibody comprising culturing a cell as described above.

On the basis of the present invention, that the anti-cTnI antibody is prepared by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody according to any one of the above, will be readily apparent to those skilled in the art, and it is within the scope of the present invention to prepare the anti-cTnI antibody according to any one of the above techniques.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 antibody discovery of monoclonal antibodies

1. Immunization of sheep

Emulsified preparation of CTNI recombinant antigen (from fei peng organism) prepared with incomplete freund's adjuvant stimulates immune response of sheep by subcutaneous injection, and serum before and after immunization is collected on 0,14,28,42 and 69 days, respectively, and peripheral blood of sheep is extracted to prepare cell suspension.

2. Construction of phage libraries

RNA in sheep peripheral blood cells is extracted and reversely transcribed into cDNA, specifically designed sheep antibody gene amplification primers are adopted, the cDNA is used as a template to amplify VH and VL gene fragments respectively, and then the VH and VL gene fragments are sequentially inserted and connected into a phage vector V02 (constructed in the laboratory) in an enzyme digestion connection mode. Finally, electrically transforming the connected phage plasmid into TG1 competent cells, selecting monoclonal colony for PCR identification the next day, performing antibody gene sequencing, evaluating the quality of phage library, and screening phage library after passing.

3. Screening of phage libraries

Phage library TG1 bacterial liquid is inoculated into a shake flask, when the bacterial liquid is cultured to a proper bacterial liquid concentration (OD 600 is 0.8-1.0), auxiliary phage is added for infection for 1 hour, and then the culture is continued overnight. And collecting bacterial liquid in the next day, centrifuging, taking supernatant, and purifying by adopting a salting-out precipitation method to obtain the displayed phage library.

And (3) panning the phage library for 3-4 rounds by adopting a magnetic bead panning method, then selecting a monoclonal phage infected colony to express an antibody supernatant, and then screening and identifying the monoclonal phage antibody expression supernatant by adopting an ELISA screening method to obtain the anti-CTNI monoclonal phage.

4. Phage antibody gene sequencing:

And (3) carrying out antibody gene sequencing on the anti-CTNI monoclonal phage, and removing repeated and invalid sequences through sequence analysis to obtain a unique sheep monoclonal antibody sequence. And (3) carrying out eukaryotic recombinant expression verification on the obtained anti-CTNI goat monoclonal antibody sequence.

EXAMPLE 2 preparation of monoclonal antibodies

1. Construction of recombinant antibody expression plasmids

pcDNA^TM3.4Vector is constructed eukaryotic expression vector of recombinant antibody, which is modified to introduce polyclonal enzyme cutting site, 3.4A expression vector, VL and VH gene specific primers are designed separately based on the obtained variable region gene, and the two ends have restriction enzyme cutting site and protecting base, and the LIGHT CHAIN gene segment of 0.72Kb and the HEAVY CHAIN gene segment of 1.40Kb are amplified via PCR amplification process.

The HEAVY CHAIN and LIGHT CHAIN gene fragments and the 3.4A vector are respectively subjected to double enzyme digestion by adopting restriction enzymes, and HEAVY CHAIN genes and LIGHT CHAIN genes after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector to respectively obtain recombinant expression plasmids HEAVY CHAIN and LIGHT CHAIN.

2. Recombinant antibody production

Resuscitate HEK293 cells in advance, subculture to a 200ml system to enable the cell density to reach 3-5×10 ⁶ cells/ml, the cell density to reach the concentration of selected antibodies and cell viability to be more than 95%, centrifugally clean the cells, re-dissolve the cells with a culture medium, simultaneously adjust the cell density to 2.9×10 ⁶ cells/ml, re-dissolve the cells with the culture medium, and simultaneously serve as a cell dilution. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding transfection reagent diluent into plasmid DNA diluent, mixing uniformly, standing at room temperature for 15min, slowly adding the mixture into cell diluent within 1min, mixing uniformly, sampling, counting, recording and observing activity of transfected cells, culturing in a 35 ℃ constant temperature incubator at a rotating speed 120rmp and a CO2 content of 8%, and centrifuging and collecting samples after 13 days. And carrying out affinity purification on the supernatant by using a protein A affinity chromatography column to obtain the purified antibody.

The obtained antibody was designated as Anti-CTNI H5Rmb1, and the Anti-CTNI H5Rmb1 was mutated to obtain a mutated antibody, the sequences of the heavy chain (H) and the light chain (L) of which are shown in the following table:

TABLE 2 antibody sequences

Antibody name	Heavy chain	Light chain
			Anti-CTNI 8H5Rmb1	SEQ ID NO:26	SEQ ID NO:34
Anti-CTNI 8H5Rmb2	SEQ ID NO:29	SEQ ID NO:34
			Anti-CTNI 8H5Rmb3	SEQ ID NO:28	SEQ ID NO:34
Anti-CTNI 8H5Rmb4	SEQ ID NO:26	SEQ ID NO:37
			Anti-CTNI 8H5Rmb5	SEQ ID NO:26	SEQ ID NO:36
Anti-CTNI 8H5Rmb6	SEQ ID NO:27	SEQ ID NO:35

Example 2 detection of Performance of antibodies

1. Affinity analysis

And testing the binding dissociation curve of the antigen and the antibody on Biacore 8K+ equipment by utilizing a CM5 chip which is coupled with mouse anti-sheep IgG in advance, and automatically fitting by an instrument to obtain an affinity constant, a binding rate and a dissociation rate. (KD represents equilibrium dissociation constant, i.e., affinity constant; ka represents binding rate; KD represents dissociation rate)

TABLE 3 affinity data

Sample name	KD	ka	kd
				Control	4.04E-08	6.85E+03	2.77E-04
Anti-CTNI 8H5Rmb1	7.30E-10	4.15E+04	3.03E-05
				Anti-CTNI 8H5Rmb2	4.42E-10	5.23E+04	2.31E-05
Anti-CTNI 8H5Rmb3	6.04E-10	3.89E+04	2.35E-05
				Anti-CTNI 8H5Rmb4	7.47E-10	4.11E+04	3.07E-05
Anti-CTNI 8H5Rmb5	4.70E-09	1.13E+04	5.31E-05
				Anti-CTNI 8H5Rmb6	2.15E-10	1.52E+05	3.27E-05

2. Activity assay

The coating solution (main component NaHCO ₃) diluted cTnI recombinant antigen (from Phpeng organism) to 3ug/ml, 100uL per well, 4℃overnight, washing solution (main component Na ₂HPO₄ +NaCl) 2 times the next day, beating dry, adding blocking solution (20% BSA+80% PBS) 120uL per well, 37℃for 1h, beating dry, adding diluted purified and control antibodies, 100uL per well, 37℃for 30min, washing solution 5 times, beating dry, adding murine anti-goat IgG-HRP, 100uL per well, 37℃for 30min, washing solution 5 times, beating dry, adding developing solution A (50 uL per well), adding developing solution B (50 uL per well), 10min, adding stop solution, 50uL per well, reading OD value at 450nm (reference 630 nm) on a microplate reader.

Remarks are liquid A (main component citric acid+sodium acetate+acetanilide+carbamide peroxide), liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCL), and stop solution (EDTA.2Na+concentrated H ₂SO₄)

TABLE 4 Activity data

Concentration (ng/ml)	62.5	31.25	15.63	7.81	3.91	0
							Control	1.403	0.821	0.518	0.123	0.124	0.045
Anti-CTNI 8H5RMb1	1.982	1.543	0.945	0.567	0.321	0.05
							Anti-CTNI 8H5RMb2	2.001	1.449	0.865	0.545	0.29	0.051
Anti-CTNI 8H5RMb3	2.105	1.487	0.936	0.502	0.27	0.053
							Anti-CTNI 8H5RMb4	2.098	1.558	0.981	0.605	0.341	0.057
Anti-CTNI 8H5RMb5	2.013	1.553	0.842	0.554	0.263	0.052
							Anti-CTNI 8H5RMb6	2.127	1.593	0.908	0.511	0.301	0.041

3. Stability assessment

The antibodies were placed in a4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, and 7 days, 14 days and 21 days of samples were taken for status observation, and activity detection was performed on the 21 days of samples. Table 5 below shows the results of the detection of OD by the antibody Anti-CTNI H5Rmb5 on evaluation of the enzyme-free activity for 21 days.

TABLE 5 stability data

Sample concentration (ng/ml)	31.25	15.63	0
				4 ℃ 21 Day sample	1.531	0.825	0.004
-80 ℃,21 Day sample	1.527	0.843	0.035
				37 ℃ 21 Day sample	1.519	0.84	0.021

The result shows that no obvious protein state change is seen after the antibody is placed for 21 days under three examination conditions, and the activity does not decrease along with the increase of the examination temperature, thus indicating that the antibody is stable.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The partial amino acid sequences involved in the present application are shown in Table 6:

Claims

1. An anti-cTnI antibody, comprising three complementarity determining regions of a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 22, 23, 24, and 25, and three complementarity determining regions of a light chain variable region having the amino acid sequence of any one of SEQ ID NOs: 30, 31, 32, and 33.

2. The antibody according to claim 1, wherein the complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-cTnI antibody, characterized in that the antibody comprises the following complementarity determining regions:

HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5;

LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6;

Optionally, the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence at least 80% identical thereto; and

The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto;

Optionally, the antibody binds cTnI with an affinity of KD < 4.04×10 ⁻⁸ M.

4. An anti-cTnI antibody comprising a heavy chain variable region and/or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NOs: 22, 23, 24, and 25; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 30, 31, 32, and 33;

Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations:

combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:22 SEQ ID NO:30 2 SEQ ID NO:25 SEQ ID NO:30 3 SEQ ID NO:24 SEQ ID NO:30 4 SEQ ID NO:22 SEQ ID NO:33 5 SEQ ID NO:22 SEQ ID NO:32 6 SEQ ID NO:23 SEQ ID NO:31

;

Optionally, the antibody further comprises a constant region;

Optionally, the constant region includes a heavy chain constant region and/or a light chain constant region;

Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments;

Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and/or CH4 of IgM;

Optionally, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human;

Optionally, the species origin of the constant region is sheep;

Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto;

Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto;

Optionally, the antibody comprises any one of F(ab’)2, Fab’, Fab, Fv and scFv.

5. An anti-cTnI antibody comprising a heavy chain and/or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 34, 35, 36, and 37.

6. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody according to any one of claims 1 to 5;

Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody;

Optionally, the antibody conjugate further comprises a marker or purification tag coupled to the antibody;

Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels;

Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or kit, characterized in that the reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 in the preparation of a product for detecting cTnI;

Optionally, the use includes:

a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with cTnI in a sample to be tested under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

Optionally, the immune complex further comprises a second antibody, which binds to the antibody;

Optionally, the immune complex further comprises a second antibody, which binds to cTnI.

9. A nucleic acid, a vector, a cell, or a method for producing the antibody of any one of claims 1 to 5, wherein the nucleic acid encodes the antibody of any one of claims 1 to 5; the vector comprises a nucleic acid encoding the antibody of any one of claims 1 to 5; the cell comprises the nucleic acid or vector; and the method comprises the cell.