CN114276450B

CN114276450B - Anti-human IL-17A monoclonal antibodies and uses thereof

Info

Publication number: CN114276450B
Application number: CN202011217331.2A
Authority: CN
Inventors: 殷璐; 孔永�; 李望; 梁立业; 陈卫; 陈涛
Original assignee: Jiangsu Quanxin Biomedical Co ltd
Current assignee: Jiangsu Quanxin Biomedical Co ltd
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2023-08-29
Anticipated expiration: 2040-09-27
Also published as: CN114276450A; CN112142841B; CN112142841A

Abstract

The application discloses a novel anti-human IL-17A monoclonal antibody, a human IL-17A ELISA kit containing the antibody and a detection method thereof. Specifically, the amino acid sequences of the antibodies of the application are shown in SEQ ID NO 9 and SEQ ID NO 10, and the human IL-17A ELISA kit comprises a pair of antibodies, wherein the first antibody is QX002N, which is used as a coating antibody in the modified double-antibody sandwich ELISA method, and the second antibody is the anti-human IL-17A monoclonal antibody of the application, which is used as a detection antibody in the modified double-antibody sandwich ELISA method. The kit can successfully complete the quantitative detection of the human IL-17A, has higher detection sensitivity and good detection specificity, and has good development and application prospects.

Description

Anti-human IL-17A monoclonal antibodies and uses thereof

The application relates to a novel anti-human IL-17A monoclonal antibody, a kit containing the same and a detection method thereof, which are divisional patent application of China application No. 2020110332600.

Technical Field

The application belongs to the technical field of immunoassay, and particularly relates to a novel anti-human IL-17A monoclonal antibody, a human IL-17A ELISA kit for quantitatively detecting human IL-17A by using the antibody, and a method for quantitatively detecting by using the kit.

Background

To date, six members of the Interleukin-17 (IL-17) family have been discovered: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25) and IL-17F. IL-17A is the prototype of the IL-17 family and is also the most representative member of its family. IL-17F has the highest homology (about 50%) with IL-17A, and its coding gene is located in the same segment 6p12 of the chromosome. IL-17B-E has poor homology (16% -30%) with IL-17A and is located on different chromosomes.

IL-17A is an important pro-inflammatory cytokine consisting essentially of activated memory CD4 ⁺ T lymphocytes are secreted. IL-17A, through specific binding to the receptor, plays a variety of roles in promoting the development of inflammation, immune response, hematopoiesis, and the like, and is involved in the occurrence of a variety of autoimmune diseases (such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, rheumatoid heart disease, and the like). When the body is infected or injured, the migrated lymphocytes secrete IL-17A. On the one hand, IL-17A induces the expression of inflammatory factors and chemokines, thereby recruiting more immune cells to reach the inflammatory sites to exacerbate the inflammatory response of the organism, and on the other hand, IL-17A also induces the expression of some tissue repair related factors, thereby accelerating the recovery of the organism. Although IL-17A plays a role in expanding immune defenses and protecting the body of the host during anti-infection and tissue repair, IL-17A is highly expressed in many autoimmune and oncological patients, and excessive IL-17A levels play a worsening role in the pathological progression of the disease, since it can induce the expression of many inflammatory factors. A plurality of animal experiments also prove that IL-17A deletion or antibody neutralization IL-17A can effectively inhibit the pathological degree of various autoimmune diseases.

The types of the reagent kit for measuring the IL-17A content in the current market are few, and the reagent kit is high in price. The ELISA kit can quantitatively detect the content of the IL-17A in the cell culture medium, is simple and convenient to operate, has high sensitivity and good specificity, and has important significance for researching pathogenesis of autoimmune diseases related to the IL-17A, a drug model and the like.

Disclosure of Invention

Based on the problems in the prior art, the application provides an anti-human IL-17A monoclonal antibody, an improved double-antibody sandwich ELISA kit which comprises the antibody and is developed based on an ELISA method, and the quantitative detection of human IL-17A is successfully completed by using the kit.

In particular, the application relates to the following:

1. an anti-human IL-17A monoclonal antibody, comprising:

a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

CDR-H1 has the amino acid sequence of SEQ ID NO 1:SYYYMC,

CDR-H2 has the amino acid sequence of SEQ ID NO 2: CIHTGNGYPYYANWAKG,

CDR-H3 has the amino acid sequence of SEQ ID NO 3: PVGGYDYAMDL;

b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

CDR-L1 has the amino acid sequence of SEQ ID NO 4:QASEDISQLS,

CDR-L2 has the amino acid sequence of SEQ ID NO 5: KASTLAS,

CDR-L3 has the amino acid sequence of sequence SEQ ID NO 6 QQASYFNVANT.

2. The antibody of claim 1, wherein the antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

HCVR has the sequence SEQ ID NO QSLESGGDLVQPESLALTCKADSGFSSYYYCWVRQAPGKGLEWIACIHTGYPYYANWAKFTITSTTQLTSLTVATATYFCARPVGGYDYAMDLWAGGTTLVGTLVSS amino acid sequence of (a); amino acid sequence of (a);

LCVR has the sequence SEQ ID NO Aydmtqtpassvgavagtvtincqasedistlsqqqqrpppklllliykasglasgrfrgsgtqftlttsigvatatyvqqsasyfnvantfgggtevvvk is a sequence of amino acids of (a). Is a sequence of amino acids of (a).

3. The antibody of claim 1, wherein the antibody comprises a heavy chain and a light chain, wherein:

the heavy chain amino acid sequence is selected from SEQ ID NO the method comprises the steps of 9, QSLESGGDLQPEGSLALTCKASGFQVESSQQVEQVEQVEQVEQVEQVEQVEQVEQVEQVETKQVEQVEQPQVEQTYPQVETQTYPQTYPQVEQVETKTQVETQTQTQTQTQTQTQTQTQTQTQTQTQTQVETQVETQVETQVEQVETQVEQVETQVEQVEQVEQVEQPQPQVEQPQTQTQQQTQTQQQQTQQQQTQQQTQQTQQQTQTQQTQQTQTQQQQQTQTQQQQQQQQQQTQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQTQTQTQTQTQTQTQTQQTQTQTQTQTQTQTQQTQQTQQQTtsQTtsQQQQQTtsQQQQQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVVQVQVQVVQVQVQVQVQVQVQVQVQVQVQVQQQVQQQQQQQQQQQQQV V, QV, ttsV, QV, TQV, ttstsv, tv, QV, tv, qetc., amino acid sequence of (a); amino acid sequence of (a);

the amino acid sequence of the light chain is selected from SEQ ID NO Aydmtqtpassvarangaggtvtincqasedistshqlswyqqqrpppklkluyksglastgvprfrgsgtqftlttstgvtsvquatsvqsasyfnvantfgggtevkgvkgdpvligfppamfpaadwattvtivcvankvyingdvtvtwevttqttgiensktpqnstststststslttslttsstypshkettkvttstsvvqsfnrgdc is a sequence of amino acids of (a). Is a sequence of amino acids of (a).

3-1. Use of the anti-human IL-17A monoclonal antibody of item 1 in the preparation of a kit for detecting human IL-17A.

The use according to item 3-1, wherein the kit comprises a first antibody and a second antibody, the second antibody being the anti-human IL-17A monoclonal antibody of item 1.

3-3 the use according to item 3-2, wherein the first antibody is QX002N.

The use according to item 3-2, wherein the first antibody is immobilized to a solid support and the second antibody is biotin-labeled.

The method according to item 4, wherein the solid support is an ELISA plate.

The use according to item 3-2, further comprising: peroxidase-labeled streptavidin, recombinant human IL-17A protein standard, substrate, coated antibody diluent, wash solution, blocking solution/sample diluent, and stop solution.

The use according to any one of items 3-1 to 3-6, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

The use according to any one of items 3-1 to 3-7, wherein the kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

4. A kit for detecting human IL-17A, which is characterized by comprising a first antibody and a second antibody,

the first antibody is QX002N; comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein:

CDR-H1 of the first antibody QX002N has the amino acid sequence of SEQ ID NO11:LFYMS, CDR-H2 has the amino acid sequence of SEQ ID NO 12:TIHEVASSYYASWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 13:ETYSSRYPYPNI; CDR-L1 has the amino acid sequence of SEQ ID NO 14:QASQNIGGSLA, CDR-L2 has the amino acid sequence of SEQ ID NO 15:GASSLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 16:QSYNTISTYGLA;

the second antibody is an anti-human IL-17A monoclonal antibody comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein:

CDR-H1 of the second antibody has the amino acid sequence of SEQ ID NO 1:SYYYYMC, CDR-H2 has the amino acid sequence of SEQ ID NO 2:CIHTGNGYPYYANWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 3:PVGGYYAMDL; CDR-L1 has the amino acid sequence of SEQ ID NO 4:QASEDISQLS, CDR-L2 has the amino acid sequence of SEQ ID NO 5:KASTLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 6:QQASYFNVANT.

5. The kit of claim 4, wherein the first antibody is immobilized to a solid support and the second antibody is biotin-labeled.

6. The kit of claim 5, wherein the solid support is an elisa plate.

7. The kit of item 5, further comprising: peroxidase-labeled streptavidin, recombinant human IL-17A protein standard, substrate, coated antibody diluent, wash solution, blocking solution/sample diluent, and stop solution.

8. The kit of any one of claims 4 to 7, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

9. The kit of any one of claims 4 to 8, wherein the kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

10. A method for quantitatively detecting IL-17A content, characterized in that QX002N is used as a first antibody, the antibodies described in items 1 to 3 are used as a second antibody, ELISA detection is performed by a modified double antibody sandwich method, wherein,

the QX002N comprises heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3,

and CDR-H1 has the amino acid sequence of SEQ ID NO11:LFYMS, CDR-H2 has the amino acid sequence of SEQ ID NO 12:TIHEVASSYYASWAKG, CDR-H3 has the amino acid sequence of SEQ ID NO 13:ETYSSRYPYPYNI; CDR-L1 has the amino acid sequence of SEQ ID NO 14:QASQNIGGSLA, CDR-L2 has the amino acid sequence of SEQ ID NO 15:GASSLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 16:QSYNTISTYGLA.

11. The method according to item 10, comprising the steps of:

coating a solid support with a first antibody;

adding the tested sample into the coated solid phase carrier, and incubating;

adding the second antibody marked by biotin to a solid phase carrier, and incubating;

diluting the peroxidase-labeled streptavidin, adding the diluted streptavidin into a solid phase carrier, and incubating;

adding a substrate into a solid phase carrier, incubating in a dark place, adding a stop solution, and measuring an OD value;

and fitting a standard curve by using the OD value of the recombinant human IL-17A protein standard substance, and substituting the OD value of the measured sample into an equation to calculate the content of human IL-17A in the measured sample.

The application provides a human IL-17A ELISA kit containing an anti-human IL-17A monoclonal antibody. The kit is used for measuring the IL-17A level in a sample by using an improved double-antibody sandwich ELISA kit, and carrying out Biotin labeling on an anti-human IL-17A monoclonal antibody, so that a Biotin-Avidin System (BAS) can be formed by the kit and an enzyme-labeled secondary antibody, and the high-affinity firm combination of the kit can play a role in multilevel amplification of biological reaction, so that the kit has high detection sensitivity, good detection specificity and good development and application prospects.

Drawings

FIG. 1 shows the binding of QX002N (primary antibody) and an anti-human IL-17A monoclonal antibody (secondary antibody) to human IL-17A.

FIG. 2 shows the competitive inhibition of QX002N (primary antibody) and anti-human IL-17A monoclonal antibody (secondary antibody) with the antigen human IL-17A binding site.

FIG. 3 shows that human IL-17A is at OD ₄₅₀ Is a standard graph of absorbance of (a).

FIG. 4 is a human IL-17A ELISA kit specificity study.

Detailed Description

The present application is described in further detail below in conjunction with the detailed description of the application, examples are given to provide a better understanding of the present application and to fully convey the scope of the application to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description proceeds with reference to the general principles of the description. The scope of the application is defined by the appended claims.

Technical and scientific terms used in the present specification have the same meaning as commonly understood by one of ordinary skill in the art, and if so conflict, the present specification will control.

Generally, terms used in the present specification have the following meanings.

In the present specification, "monoclonal antibody" means an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of monoclonal antibody preparations), which are typically present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the application can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies are described herein.

In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K _D ) And (3) representing. Affinity can be measured by common methods known in the art.

In the present specification, human interleukin 17A (Human Interleukin-17A, hIL-17A) represents a protein derived from human. IL-17A is a pro-inflammatory cytokine produced by Th17 cells, which, by binding to its receptor, stimulates the cell to produce cytokines, and is involved in and contributes to a variety of diseases mediated by aberrant immune responses. The monoclonal antibodies of the application are neutralizing antibodies that specifically recognize IL-17A, which are capable of specifically binding to IL-17A.

In the present specification, "anti-human IL-17A monoclonal antibody" means such a monoclonal antibody: which is capable of binding human interleukin 17A with sufficient affinity such that the monoclonal antibody can be used to identify/detect human interleukin 17A.

In the present specification, "enzyme-linked immunosorbent assay (EILSA)" refers to a detection method in which a free hetero protein is bound to a target protein bound to a solid carrier by utilizing the characteristic that an antibody molecule can specifically bind to an antigen molecule, and qualitative or quantitative analysis is performed using a specific label. The principle is as follows: the antigen or antibody can be physically adsorbed on the solid surface and maintain the immunological activity thereof; the antigen or antibody is capable of forming an enzyme conjugate with the enzyme via a covalent bond while maintaining the respective immunological or enzymatic activity; after binding the enzyme conjugate to the corresponding antigen or antibody, the occurrence of an immune response can be determined by a color reaction of the added substrate, the color reaction being in direct proportion to the amount of the corresponding antigen or antibody in the sample. According to the substance to be detected and the conditions of detection, various detection methods of different types can be designed, and the double antibody sandwich method is the most common method for detecting antigens. The antiserum containing known antibody is adsorbed in a small hole on a micro-titer plate and washed once; adding an antigen to be detected, if the antigen and the antigen are specific, combining, and then washing out redundant antibodies; adding an enzyme-linked antibody which specifically reacts with an antigen to be detected to form a sandwich; the addition of the substrate for the enzyme indicates the presence of the corresponding antigen if colored enzymatic products are observed.

In the present specification, "avidin" is a glycoprotein consisting of 4 subunits per molecule, and can be closely bound to 4 biotin molecules. More streptavidin (streptavidin) extracted from Streptomyces is used.

In the present specification, "biotin" is also called vitamin H. Chemically prepared derivatives, biotin-hydroxysuccinimide (BNCHS), can form biotinylated products with various types of large and small molecules such as proteins, carbohydrates and enzymes. The binding of avidin and biotin is not immune, but has strong specificity and high affinity, and once the avidin and biotin are combined, the binding is extremely stable. Since 1 avidin molecule has 4 binding sites for biotin molecules, more biotinylated molecules can be attached to form a lattice-like complex. Thus, combining avidin and biotin with ELISA can greatly increase the sensitivity of ELISA.

The biotin-avidin system is used in ELISA in various forms, and can be used for indirect coating and final reaction amplification. The enzyme-labeled antibody in conventional ELISA can also be replaced with biotinylated antibody, followed by ligation of avidin-enzyme conjugates to amplify the reaction signal.

The application relates to an anti-human IL-17A monoclonal antibody, which is characterized in that the antibody comprises: a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO 1:SYYYMC, CDR-H2 has the amino acid sequence of SEQ ID NO 2:CIHTGNGYPYYANWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 3:PVGGYYAMDL; b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 4:QASEDISQLS, CDR-L2 has the amino acid sequence of SEQ ID NO 5:KASTLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 6:QQASYFNVANT.

In a specific embodiment, the antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: HCVR has the sequence SEQ ID NO QSLESGGDLVQPESLALTCKADSGFSSYYYCWVRQAPGKGLEWIACIHTGYPYYANWAKFTITSTTQLTSLTVATATYFCARPVGGYDYAMDLWAGGTTLVGTLVSS amino acid sequence of (a); amino acid sequence of (a); LCVR has the sequence SEQ ID NO Aydmtqtpassvgavagtvtincqasedistlsqqqqrpppklllliykasglasgrfrgsgtqftlttsigvatatyvqqsasyfnvantfgggtevvvk is a sequence of amino acids of (a). Is a sequence of amino acids of (a).

In a specific embodiment, the antibody comprises a heavy chain and a light chain, wherein:

the heavy chain amino acid sequence is selected from SEQ ID NO the method comprises the steps of 9, QSLESGGDLQPEGSLALTCKASGFQVESSQQVEQVEQVEQVEQVEQVEQVEQVEQVEQVETKQVEQVEQPQVEQTYPQVETQTYPQTYPQVEQVETKTQVETQTQTQTQTQTQTQTQTQTQTQTQTQTQVETQVETQVETQVEQVETQVEQVETQVEQVEQVEQVEQPQPQVEQPQTQTQQQTQTQQQQTQQQQTQQQTQQTQQQTQTQQTQQTQTQQQQQTQTQQQQQQQQQQTQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQTQTQTQTQTQTQTQTQQTQTQTQTQTQTQTQQTQQTQQQTtsQTtsQQQQQTtsQQQQQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVVQVQVQVVQVQVQVQVQVQVQVQVQVQVQVQQQVQQQQQQQQQQQQQV V, QV, ttsV, QV, TQV, ttstsv, tv, QV, tv, qetc., amino acid sequence of (a); amino acid sequence of (a); the amino acid sequence of the light chain is selected from SEQ ID NO Aydmtqtpassvarangaggtvtincqasedistshqlswyqqqrpppklkluyksglastgvprfrgsgtqftlttstgvtsvquatsvqsasyfnvantfgggtevkgvkgdpvligfppamfpaadwattvtivcvankvyingdvtvtwevttqttgiensktpqnstststststslttslttsstypshkettkvttstsvvqsfnrgdc is a sequence of amino acids of (a). Is a sequence of amino acids of (a).

The application relates to a kit for detecting human IL-17A, which is characterized by comprising a first antibody and a second antibody, wherein the first antibody is QX002N; comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: CDR-H1 of the first antibody QX002N has the amino acid sequence of SEQ ID NO11:LFYMS, CDR-H2 has the amino acid sequence of SEQ ID NO 12:TIHEVASSYYASWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 13:ETYSSRYPYPNI; CDR-L1 has the amino acid sequence of SEQ ID NO 14:QASQNIGGSLA, CDR-L2 has the amino acid sequence of SEQ ID NO 15:GASSLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 16:QSYNTISTYGLA; the second antibody is an anti-human IL-17A monoclonal antibody comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: CDR-H1 of the second antibody has the amino acid sequence of SEQ ID NO 1:SYYYYMC, CDR-H2 has the amino acid sequence of SEQ ID NO 2:CIHTGNGYPYYANWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 3:PVGGYYAMDL; CDR-L1 has the amino acid sequence of SEQ ID NO 4:QASEDISQLS, CDR-L2 has the amino acid sequence of SEQ ID NO 5:KASTLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 6:QQASYFNVANT.

In a specific embodiment, the kit comprises a first antibody and a second antibody, the first antibody being QX002N; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO 17; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO 18.

SEQ ID NO 17:

EVQLQESGPGLVKPSETLSLTCTVSGIDLSLFYMSWIRQPPGKGLEWIGTIHEVASSYYASWAKGRVTISKDTSKNQFSLKLSSVTAADTAVYYCARETYSSRYPYPNIWGQGTLVTVSS

SEQ ID NO 18:

DIQMTQSPSSVSASVGDRVTITCQASQNIGGSLAWYQQKPGKAPKLLIYGASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYNTISTYGLAFGGGTKVEIK

The second antibody is an anti-human IL-17A monoclonal antibody, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO 8.

SEQ ID NO 7:

QSLEESGGDLVQPEGSLALTCKASGFDLSSYYYMCWVRQAPGKGLEWIACIHTGNGYPYYANWAKGRFTISKTSSTTVTLQLTSLTVADTATYFCARPVGGYDYAMDLWGPGTLVTVSS

SEQ ID NO 8:

AYDMTQTPASVSAAVGGTVTINCQASEDISSQLSWYQQRPGQPPKLLIYKASTLASGVPSRFRGSGSGTQFTLTISGVQCADAATYYCQQSASYFNVANTFGGGTEVVVK

In a specific embodiment, the kit comprises a first antibody and a second antibody, the first antibody being QX002N; the amino acid sequence of the heavy chain is shown as SEQ ID NO 19; the amino acid sequence of the light chain is shown as SEQ ID NO 20.

SEQ ID NO 19:

EVQLQESGPGLVKPSETLSLTCTVSGIDLSLFYMSWIRQPPGKGLEWIGTIHEVASSYYASWAKGRVTISKDTSKNQFSLKLSSVTAADTAVYYCARETYSSRYPYPNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO 20:

DIQMTQSPSSVSASVGDRVTITCQASQNIGGSLAWYQQKPGKAPKLLIYGASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYNTISTYGLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The second antibody is an anti-human IL-17A monoclonal antibody, and the amino acid sequence of the heavy chain of the second antibody is shown as SEQ ID NO 9; the amino acid sequence of the light chain is shown in SEQ ID NO 10.

SEQ ID NO 9:

QSLEESGGDLVQPEGSLALTCKASGFDLSSYYYMCWVRQAPGKGLEWIACIHTGNGYPYYANWAKGRFTISKTSSTTVTLQLTSLTVADTATYFCARPVGGYDYAMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

SEQ ID NO 10:

AYDMTQTPASVSAAVGGTVTINCQASEDISSQLSWYQQRPGQPPKLLIYKASTLASGVPSRFRGSGSGTQFTLTISGVQCADAATYYCQQSASYFNVANTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

In a specific embodiment, the first antibody is immobilized to a solid support and the second antibody is biotin-labeled.

In a specific embodiment, the kit further comprises: peroxidase-labeled streptavidin, recombinant human IL-17A protein standard, enzyme-labeled secondary antibody, substrate, coated antibody diluent, washing liquid, blocking liquid/sample diluent and stop solution.

In a specific embodiment, the solid support is an elisa plate. Preferably 96-well ELISA plates, in particular polystyrene plastic plates of Corning Coster, U.S.A. The biotin is as follows: biotin. The peroxidase is horseradish peroxidase (HRP), and the labeled streptavidin is streptavidin-horseradish peroxidase conjugate (SA-HRP). The substrate is 3,3', 5' -tetramethyl benzidine (TMB); the coated antibody diluent is Phosphate Buffer (PBS) pH7.4; the wash solution was Phosphate Buffered Saline (PBS) containing 0.05% Tween20; blocking/sample dilutions were Phosphate Buffered Saline (PBS) containing 0.5% bsa, 0.05% tween20 and 0.05% proclin300; the stop solution is phosphoric acid.

In a specific embodiment, the ELISA kit comprises:

1) Solid phase carrier: the ELISA plate is 1 block;

2) Coating an antibody: primary antibody QX002N,10 μg/tube, 1 tube;

3) Detection of antibodies: the secondary antibody anti-human IL-17A monoclonal antibody (labeled with biotin), 10 μg/tube, 1 tube;

4) Standard substance: recombinant human IL-17A protein, 10 ng/tube, 1 tube;

5) Peroxidase-labeled streptavidin: streptavidin-horseradish peroxidase conjugate (strepavidin-HRP, SA-HRP for short), 500 ng/tube, 1 tube;

6) A substrate: 3,3', 5' -tetramethyl benzidine (TMB), 5 ml/tube, 1 tube each for liquid A and liquid B;

7) Coating antibody diluent: phosphate Buffered Saline (PBS), pH7.4, 50 ml/bottle, 1 bottle;

8) Washing liquid: phosphate Buffered Saline (PBS) containing 0.05% Tween20, 200 ml/bottle, 1 bottle;

9) Blocking/sample dilution: phosphate Buffered Saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300, 200 ml/bottle, 1 bottle;

10 Termination liquid: 1M phosphoric acid, 10 ml/tube, 1 tube.

In a specific embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit. Preferably, the kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

The application also relates to a method for quantitatively detecting the content of IL-17A, which is characterized in that QX002N is used as a first antibody, an anti-human IL-17A monoclonal antibody is used as a second antibody, and an improved double antibody sandwich method is used for ELISA detection, and the method comprises the following steps: coating a solid support with a first antibody; adding the tested sample into the coated solid phase carrier, and incubating; adding the second antibody marked by biotin to a solid phase carrier, and incubating; diluting the peroxidase-labeled streptavidin, adding the diluted streptavidin into a solid phase carrier, and incubating; adding a substrate into an ELISA plate, incubating in a dark place, adding a stop solution, and measuring an OD value; and fitting a standard curve by using the OD value of the recombinant human IL-17A protein standard substance, and substituting the OD value of the measured sample into an equation to calculate the content of human IL-17A in the measured sample.

In one embodiment, the ELISA kit of the application can be used for quantitatively detecting the content of human IL-17A in a sample, and comprises the following steps:

1) Coating: preparing a coated antibody working solution with the concentration of 1 mug/ml by adopting a coated antibody diluent, namely a coated antibody QX002N (primary antibody), adding the working solution into an ELISA plate (96-hole Coster ELISA plate) according to the dosage of 50 mug/hole, and standing overnight at the temperature of 2-8 ℃;

2) Closing: the coated antibody working solution in the ELISA plate is discarded, the plate is washed 3 times by washing solution (phosphate buffer solution (PBS) containing 0.05% Tween 20), then a blocking solution (phosphate buffer solution (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin 300) is added according to the dosage of 100 mu l/hole, and the plate is blocked for 2 hours at room temperature in a shaking table (120 rpm);

3) Protein standard preparation: 8 EP tubes were taken and numbered sequentially, 100 μl of sample diluent was added per tube from tube 2 and placed on the EP tube rack; preparing recombinant human IL-17A protein standard into liquid with the concentration of 10ng/ml by using a sample diluent, sucking 200 mu l of the liquid into a 1 st EP pipe, sucking 100 mu l of the liquid from the 1 st EP pipe, adding the liquid into a 2 nd EP pipe for double dilution, and the like until reaching a 7 th EP pipe (the concentration of the last pipe is 0);

(4) Preparing a quality control sample: taking 5 EP tubes, sampling from 25ng/ml respectively, and preparing quality control samples of 12.5ng/ml, 10ng/ml, 5ng/ml, 0.2ng/ml and 0.098 ng/ml;

(5) Sample adding: removing the sealing liquid in the ELISA plate, washing the plate 3 times by using a washing liquid, adding recombinant human IL-17A protein standard liquid and a quality control sample according to the dosage of 50 μl/hole, and placing the plates in a shaking table (120 rpm) for incubation for 2 hours at room temperature;

(6) Adding a detection antibody: discarding a protein standard solution and a sample to be detected in the ELISA plate, washing the plate for 3 times by using a washing liquid, preparing a detection antibody anti-human IL-17A monoclonal antibody (biotin labeling) into a detection antibody working solution with the concentration of 0.2 mug/ml by using a sample diluent, adding the detection antibody working solution into the ELISA plate according to the dosage of 50 mug/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation reaction for 2 hours;

(7) Adding enzyme-labeled secondary antibodies: discarding the detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing liquid, preparing an ELISA secondary antibody (streptavidin-horseradish peroxidase conjugate (SA-HRP)) into an ELISA secondary antibody working solution with the concentration of 100ng/ml by using a sample diluent, adding the ELISA plate according to the dosage of 50 μl/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation for 1 hour at room temperature;

(8) Color development: mixing substrate solution A and solution B (3, 3', 5' -tetramethyl benzidine (TMB), 5 ml/tube, 1 tube each of solution A and solution B) in equal volume; discarding the enzyme-labeled secondary antibody working solution in the enzyme-labeled plate, washing the plate for 3 times by using washing liquid, adding a substrate according to the dosage of 50 mu l/hole, and carrying out light-shielding reaction for 5-10 minutes at room temperature;

(9) Terminating the color development: and adding a termination solution into the ELISA plate according to the dosage of 50 mu l/hole to terminate the reaction, measuring the OD value of each hole in the ELISA plate by using an ELISA reader at the wavelength of 450nm, fitting a standard curve according to the OD value of a standard substance, substituting the OD value of a quality control sample into an equation, and calculating the concentration of the quality control sample.

Examples

EXAMPLE 1 preparation of QX002N (primary antibody) and anti-human IL-17A monoclonal antibody (secondary antibody)

The primary antibody is QX002N, and the preparation method is as follows: CN 1086409918.

The second antibody is an anti-human IL-17A monoclonal antibody. The preparation method comprises the following steps: the correct sequence secondary antibody heavy chain expression plasmid and light chain expression plasmid were co-transfected into the ExpiCHO-S cells. The day before the transfection,dilution of ExpiCHO-S cells to 3X 10 ⁶ Each cell/ml was passaged before transfection. On the day of transfection, cell densities were diluted to 6X 10 ⁶ 25ml of cell suspension were shake-bottled at each cell/ml, 125ml, and waiting for transfection. Mu.l of OptiPRO SFM (available from Gibco) was added to wells 1 and 2, respectively, followed by 12.5. Mu.g of heavy and light chain plasmids in well 1 and 80. Mu. l ExpiFectamine CHO reagent (available from Gibco) in well 2. Transfer well 2 to well 1, mix well, stand for 1 minute and add to the cells. On the first day after transfection, 150. Mu.l of enhancement (from Gibco) and 6ml of Feed broth (from Gibco) were added. The sixth day after transfection, the culture supernatant was harvested and treated with Protein A @A, purchased from Merck) was purified in one step, and the obtained secondary antibody was obtained. The second antibody obtained by the application is a rabbit monoclonal antibody, and is not subjected to humanized transformation.

The complete sequence of the heavy and light chain of the secondary antibody was obtained by sequencing (entrusting the organism) and then the CDR sequence was obtained according to the Kabat rule (ref: kabat E.A., wu T., bilofsky H.Attempts to locate residues in complementarity-determining regions of antibody combining sites that make contact with anti.Proc.Natl. Acad. Sci.USA.1976; 73:617-619).

EXAMPLE 2 characterization of primary and secondary antibodies

1. Binding experiments of primary and secondary antibodies to human IL-17A:

the elisa plate (from Costar) was coated with primary and secondary antibodies, respectively, and after blocking, a gradient of human IL-17A (His-tag, from Novoprotein) was added and incubated for 2 hours at room temperature. After washing the plate, adding detection antibody HRP-labeled Anti-his antibody (purchased from Genscript) and finally washing the plate, developing and reading OD ₄₅₀ Absorbance at. As a result, as shown in FIG. 1, both the primary antibody and the secondary antibody were able to specifically bind to human IL-17A.

2. Competitive inhibition experiments of antibody recognition antigen sites:

coating the ELISA plate with the first antibody, closing, and adding the same bodyThe primary and secondary antibodies, as well as a concentration of human IL-17A, were incubated for 2 hours at room temperature. After washing the plate, the Anti-his antibody labeled with the detection antibody HRP was added and incubated at room temperature for 1 hour. Finally, the plate is washed and developed, and OD is read ₄₅₀ Absorbance at. As a result, as shown in FIG. 2, the primary antibody and the secondary antibody do not compete with the binding site of the human IL-17A antigen, and the secondary antibody can be used as a detection antibody of the human IL-17A ELISA kit.

3. Detection antibody (secondary antibody) biotin label:

use of EZ-for detection of antibodies for biotin labellingSulfo-NHS-LC-Biotin kit (available from Thermo), the specific steps were performed strictly according to the kit instructions. After the labeling was completed, ztba was used ^TM The spin desalting column (Spin Desalting Colum) was filtered to remove free biotin, and the resulting solution was a biotin-labeled detection antibody (secondary antibody).

Example 3 quantitative detection kit for human IL-17A ELISA

The composition, specification, source and storage conditions of the human IL-17A ELISA quantitative determination kit are shown in Table 1 below.

TABLE 1

EXAMPLE 4 quantitative detection of IL-17A in cell culture Using the human IL-17A ELISA kit of example 3

1. Sample treatment:

the recombinant human IL-17A protein standard was diluted with a sample diluent and a cell culture solution (90% RPMI 1640 (available from Hyclone) +10% FBS (available from Gibco)), respectively, and a standard curve was fitted according to the absorbance of the recombinant human IL-17A protein standard diluted with the sample diluent using the recombinant human IL-17A protein standard diluted with the cell culture solution as a sample to be examined. Substituting the OD value of the sample to be detected into a standard curve to obtain a theoretical concentration value of human IL-17A in the sample to be detected, and then calculating the recovery rate of the sample to be detected (recovery rate=theoretical concentration value/actual concentration value: 100), thereby analyzing the feasibility of the ELISA kit in example 3 for quantitative detection of human IL-17A in the cell culture fluid.

2. Quantitative detection operation steps:

(1) Coating: preparing coated antibody into coated antibody working solution with concentration of 1 mu g/ml by adopting coated antibody diluent, adding the coated antibody working solution into an ELISA plate according to the dosage of 50 mu l/hole, and standing at 4 ℃ overnight;

(2) Closing: discarding the coated antibody working solution in the ELISA plate, washing the plate with washing solution for 3 times, adding a sealing solution according to the dosage of 100 μl/hole, and sealing for 2 hours at room temperature in a shaking table (120 rpm);

(3) Recombinant human IL-17A protein standard preparation: 8 EP tubes were taken and numbered sequentially, 100 μl of sample diluent was added per tube from tube 2 and placed on the EP tube rack; preparing standard protein into a liquid with the concentration of 10ng/ml by using a sample diluent, sucking 200 mu l of the liquid into a 1 st EP pipe, sucking 100 mu l of the liquid from the 1 st EP pipe, adding the liquid into a 2 nd EP pipe for double dilution, and the like to a 7 th EP pipe (the concentration of the last pipe is 0);

(4) Adding a sample to be tested and a standard substance: removing the sealing liquid in the ELISA plate, washing the plate with washing liquid for 3 times, adding protein standard liquid and a sample to be tested according to the dosage of 50 mu l/hole, and placing the mixture in a shaking table (120 rpm) for incubation for 2 hours at room temperature;

(5) Adding a detection antibody: removing protein standard solution and sample to be detected in the ELISA plate, washing the plate for 3 times by using washing liquid, preparing detection antibody (biotin labeling) into detection antibody working solution with the concentration of 1 mu g/ml by using sample diluent, adding the detection antibody working solution into the ELISA plate according to the dosage of 50 mu l/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation reaction for 2 hours;

(6) Adding enzyme-labeled secondary antibodies: discarding the detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing liquid, preparing the ELISA secondary antibody into an ELISA secondary antibody working solution with the concentration of 50ng/ml by using a sample diluent, adding the ELISA secondary antibody working solution into the ELISA plate according to the dosage of 50 μl/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation for 1 hour at room temperature;

(7) Color development: mixing the substrate A and B in equal volume; discarding the enzyme-labeled secondary antibody working solution in the enzyme-labeled plate, washing the plate for 3 times by using washing liquid, adding a substrate according to the dosage of 50 mu l/hole, and carrying out light-shielding reaction for 10-15 minutes at room temperature;

(8) Terminating the color development: and adding a termination solution into the ELISA plate according to the dosage of 50 mu l/hole to terminate the reaction, measuring the OD value of each hole in the ELISA plate by using an ELISA apparatus at the wavelength of 450nm, fitting a standard curve according to the OD value of a standard substance, substituting the OD value of a sample to be detected into an equation, and calculating the concentration of human IL-17A in the sample to be detected, thereby finishing quantitative detection.

3. Detection result:

(1) The detection data for recombinant human IL-17A standard are shown in Table 2 below:

TABLE 2

(2) Curve equation: 4-P Fit: y= (0.105-2.3)/(1+ (x/2.28)/(1.31+2.3), R ² =0.998 (see fig. 3).

(3) The test data of the samples to be tested diluted with the cell culture fluid are shown in the following table 3:

TABLE 3 Table 3

The recovery rate of each concentration point is between 95% and 105%, which shows that the cell culture solution has no influence on the experimental result, and the ELISA method can be suitable for the content measurement of human IL-17A in a cell culture solution system.

4. Accuracy investigation:

(1) In3 different experiments, three human IL-17A (8, 2, 0.5 ng/ml) with known concentration diluted by cell culture solution is taken as a sample to be tested, 2 compound holes are respectively arranged, quantitative detection of the human IL-17A is carried out, and the accuracy of the kit is analyzed. Accuracy is expressed as coefficient of variation CV%, equal to SD/average x 100.

(2) Results and discussion table 4 below:

TABLE 4 Table 4

The coefficient of variation CV is less than 10%, which shows that the detection method has good accuracy.

5. Specificity investigation:

different concentration gradients of human IL-17A, human IL-6 and human IL-23 proteins (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 ng/ml) were added to the detection wells of the primary antibody pre-coated ELISA kit, respectively, and absorbance of each protein at different concentrations was determined using the ELISA assay established in example 4. The results are shown in FIG. 4, OD of human IL-17A proteome ₄₅₀ Exhibit significant dose-dependent effects, OD, between different concentrations ₄₅₀ The absorbance increased with increasing protein concentration, whereas the absorbance of both human IL-6 and human IL-23 protein groups was close to 0, with no dose-dependent effects. The experimental results show that the detection method using the human IL-17A ELISA kit of example 3 has good specificity.

The above description is only of the preferred embodiments of the present application, and is not intended to limit the present application in any way. Any person skilled in the art may make variations or modifications to the equivalent embodiments using the teachings disclosed above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Sequence listing

<110> Jiangsu Xin biological medicine Co., ltd

<120> anti-human IL-17A monoclonal antibodies and uses thereof

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Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

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Arg Pro Val Gly Gly Tyr Asp Tyr Ala Met Asp Leu Trp Gly Pro Gly

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Thr Leu Val Thr Val Ser Ser

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Leu Ser Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Lys Leu Leu Ile

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Gly Ala Ser Ser Leu Ala Ser

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Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Thr Tyr Ser Ser Arg Tyr Pro Tyr Pro Asn Ile Trp Gly Gln

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115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

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Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

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Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

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Gly Lys

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Gly Gly Ser

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Gly Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Asn Thr Ile Ser Thr

85 90 95

Tyr Gly Leu Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

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Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. An anti-human IL-17A monoclonal antibody, comprising:

CDR-H1 is the amino acid sequence of sequence SEQ ID NO1 SYYYMC,

CDR-H2 is the amino acid sequence of SEQ ID NO 2: CIHTGNGYPYYANWAKG,

CDR-H3 is the amino acid sequence of SEQ ID NO 3: PVGGYDYAMDL;

CDR-L1 is the amino acid sequence of SEQ ID NO 4: QASEDISSQLS,

CDR-L2 is the amino acid sequence of the sequence SEQ ID NO 5 KASTLAS,

CDR-L3 is the amino acid sequence of SEQ ID NO 6: QQSASYFNVANT,

wherein the antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

HCVR has the amino acid sequence of SEQ ID NO 7: QSLEESGGDLVQPEGSLALTCKASGFDLSSYYYMCWVRQAPGKGLEWIACIHTGNGYPYYANWAKGRFTISKTSSTTVTLQLTSLTVADTATYFCARPVGGYDYAMDLWGPGTLVTVSS;

LCVR has the amino acid sequence of SEQ ID NO 8: AYDMTQTPASVSAAVGGTVTINCQASEDISSQLSWYQQRPGQPPKLLIYKASTLASGVPSRFRGSGSGTQFTLTISGVQCADAATYYCQQSASYFNVANTFGGGTEVVVK.

2. Use of an anti-human IL-17A monoclonal antibody according to claim 1 for the preparation of a kit for detecting human IL-17A.

3. The use of claim 2, wherein the kit comprises a first antibody and a second antibody, the second antibody being an anti-human IL-17A monoclonal antibody of claim 1.

4. The use of claim 3, wherein the first antibody is QX002N.

5. The use according to claim 3, wherein the first antibody is immobilized to a solid support and the second antibody is biotin-labeled.

6. The use according to claim 5, wherein the solid support is an elisa plate.

7. Use according to claim 3, further comprising: peroxidase-labeled streptavidin, recombinant human IL-17A protein standard, substrate, coated antibody diluent, wash solution, blocking solution/sample diluent, and stop solution.

8. The use according to any one of claims 2 to 7, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

9. The use according to claim 8, wherein the kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.