CN114773466A

CN114773466A - Anti-human interleukin 23, kit comprising same and detection method thereof

Info

Publication number: CN114773466A
Application number: CN202210299843.0A
Authority: CN
Inventors: 王波; 殷璐; 徐蕾; 孔永�; 陈涛; 陈卫
Original assignee: Jiangsu Quanxin Biomedical Co ltd
Current assignee: Jiangsu Quanxin Biomedical Co ltd
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2022-07-22
Anticipated expiration: 2040-11-26
Also published as: CN113156134B; CN112625129A; CN113156134A; CN114773466B; CN112625129B

Abstract

The application provides an anti-human interleukin 23 monoclonal antibody, an ELISA kit containing the antibody and a detection method of the ELISA kit. Specifically, the ELISA kit described herein comprises a pair of such antibodies, each of which is used as a coating antibody and a detection antibody in a modified double antibody sandwich ELISA method. The kit provided by the application can complete the quantitative detection of human interleukin 23, has higher detection sensitivity, and has good development and application prospects.

Description

Anti-human interleukin 23, kit comprising same and detection method thereof

The application is a divisional application of an invention patent application with the application date of 26/11/2020 and the application number of 202011347406.9, namely 'anti-human interleukin 23, a kit containing the same and a detection method thereof'.

Technical Field

The application belongs to the technical field of immunoassay, and relates to an ELISA kit for combining an antibody of human interleukin 23 and quantitatively detecting the human interleukin 23, and a method for quantitatively detecting by using the kit.

Background

Interleukins are a group of cytokines with important functions, are produced by immune cells or non-immune cells, and with the rapid development of molecular biology and cell biology technologies, at least 38 interleukins are found at present, are respectively named as IL-1-IL-38, have complex functions, form networks and are overlapped in a complex way; plays an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and also participates in various physiological and pathological reactions of the body. Interleukin 23(IL-23), an interleukin discovered in 2000, has now been found to have a variety of biological functions and to play a role in a variety of diseases.

IL-23 is mainly produced by activated dendritic cells, macrophages, monocytes and the like, is a new member of the IL-12 heterodimer cytokine family, and mainly consists of two subunits of IL-23p19 and IL-12/IL-23p40, wherein IL-12/IL-23p40 is the subunit which is commonly contained with IL-12. IL-23p19 and IL-12/IL-23p40 subunit alone, without biological function, only two connected to form a homodimer, can play a biological function.

IL-23 activates downstream signaling pathways to perform biological functions, primarily through interaction with its receptors. IL-23 acts mainly on Th17 cells, plays an important role in the proliferation and stabilization of Th17 cells, and can promote Th17 cells to produce cytokines such as IL-23, IL-17F and IL-22, and these inflammatory factors act on keratinocytes, resulting in the activation and excessive proliferation of keratinocytes. The activated keratinocytes can recruit and activate immune cells such as T cells by producing a large amount of cytokines, chemokines, antimicrobial peptides and the like, and form a cascade effect of immune response, so that the psoriasis lesions are generated. IL-23 is also a promoting factor in joint autoimmune inflammation, such as in rheumatoid arthritis, IL-23 promotes IL-17 secretion, resulting in joint destruction. Therefore, in the clinical treatment process, the detection of the IL-23 level and the change thereof in the serum of a patient has very important significance.

At present, the kit for measuring the content of the interleukin 23 in the market has few types and high price. The ELISA kit can quantitatively detect the content of the interleukin 23 in the cell culture medium, has simple and convenient operation, high sensitivity and good specificity, and has important significance for researching pathogenesis of autoimmune diseases related to the interleukin 23, medicine models and the like.

Disclosure of Invention

Based on the problems in the prior art, the invention provides a human interleukin 23ELISA kit which is developed based on an improved double-antibody sandwich ELISA method and aims at the high affinity of human interleukin 23, and the kit comprises an anti-human interleukin 23 monoclonal antibody. The kit can realize the quantitative detection of the human interleukin 23.

In particular, the invention relates to the following:

1. an isolated monoclonal antibody against human interleukin 23, comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

a) the amino acid sequence of CDR-H1 (CDR-H1 denotes heavy chain CDR1 in the present specification) is shown as SEQ ID No. 1(SYWMN) or SEQ ID No. 2 (SYGIS);

b) the amino acid sequence of CDR-H2 (CDR-H2 in this specification means heavy chain CDR2) is shown as SEQ ID No. 3(YIIPITGYTEYNQKFKD) or SEQ ID No. 4 (KISPRSVNAYYNEKFKG);

c) the amino acid sequence of CDR-H3 (CDR-H3 in this specification represents the heavy chain CDR3) is shown as SEQ ID No. 5(GGGNLPY) or SEQ ID No. 6 (DYSNLIFDY);

d) the amino acid sequence of CDR-L1 (CDR-L1 in this specification means light chain CDR1) is shown as SEQ ID No. 7(SASSSVSFTYLY) or SEQ ID No. 8 (SVSSSISSSNLH);

e) the amino acid sequence of CDR-L2 (CDR-L2 denotes the light chain CDR2 in the present specification) is shown as SEQ ID No. 9(SISNLAS) or SEQ ID No. 10 (GTSSLAS);

f) the amino acid sequence of CDR-L3 (CDR-L3 in this specification refers to light chain CDR3) is shown as SEQ ID No. 11(QQWSSNPPIT) or SEQ ID No. 12 (QQWSSYPLT).

2. The monoclonal antibody of claim 1, wherein the CDR-H1 has an amino acid sequence as set forth in SEQ ID No. 1(SYWMN), the CDR-H2 has an amino acid sequence as set forth in SEQ ID No. 3(YIIPITGYTEYNQKFKD), the CDR-H3 has an amino acid sequence as set forth in SEQ ID No. 5(GGGNLPY), the CDR-L1 has an amino acid sequence as set forth in SEQ ID No. 7(SASSSVSFTYLY), the CDR-L2 has an amino acid sequence as set forth in SEQ ID No. 9(SISNLAS), and the CDR-L3 has an amino acid sequence as set forth in SEQ ID No. 11 (QQWSSNPPIT).

3. The monoclonal antibody of claim 1, wherein the CDR-H1 has an amino acid sequence as shown in SEQ ID No. 2(SYGIS), the CDR-H2 has an amino acid sequence as shown in SEQ ID No. 4(KISPRSVNAYYNEKFKG), the CDR-H3 has an amino acid sequence as shown in SEQ ID No. 6(DYSNLIFDY), the CDR-L1 has an amino acid sequence as shown in SEQ ID No. 8(SVSSSISSSNLH), the CDR-L2 has an amino acid sequence as shown in SEQ ID No. 10(GTSSLAS), and the CDR-L3 has an amino acid sequence as shown in SEQ ID No. 12 (QQWSSYPLT).

4. The monoclonal antibody of any one of claims 1-3, wherein the monoclonal antibody comprises an antibody Heavy Chain Variable Region (HCVR) and an antibody Light Chain Variable Region (LCVR), wherein:

the amino acid sequence of HCVR is shown in SEQ ID No. 13

(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEWIGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCARGGGNLPYWGQGTLVTVSA) or SEQ ID No:14

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSS);

LCVR has an amino acid sequence as set forth in SEQ ID No. 15

(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWIYSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITFGAGTKLELK) or SEQ ID No:16

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELK).

5. The monoclonal antibody of claim 4, wherein the antibody heavy chain variable region HCVR has the sequence of SEQ ID No 13

(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEWIGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCARGGGNLPYWGQGTLVTVSA), wherein the antibody light chain variable region LCVR has the amino acid sequence as shown in SEQ ID No. 15

(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWIYSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITFGAGTKLELK) in the presence of a protease.

6. The monoclonal antibody of claim 4, wherein the antibody heavy chain variable region HCRV has the sequence as shown in SEQ ID No 14

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSS), wherein the antibody light chain variable region LCVR has the amino acid sequence as shown in SEQ ID No:16

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELK) in the sequence listing.

7. The monoclonal antibody of any one of claims 1-6, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

the amino acid sequence of the heavy chain is shown as SEQ ID No. 17

(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEWIGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCARGGGNLPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK) or SEQ ID No:18

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK);

the amino acid sequence of the light chain is shown as SEQ ID No. 19

(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWIYSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC) or SEQ ID No:20

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC);

8. the monoclonal antibody of claim 7, wherein the monoclonal antibody is anti-human interleukin 23 monoclonal antibody # 1, and the heavy chain thereof has the amino acid sequence shown in SEQ ID No. 17

(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEWIGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCARGGGNLPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK) and the light chain has the amino acid sequence shown as SEQ ID No. 19

(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWIYSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC) in the presence of a protease.

9. The monoclonal antibody of claim 7, wherein the monoclonal antibody is anti-human interleukin 23 monoclonal antibody # 2, and the heavy chain thereof has the amino acid sequence shown in SEQ ID No. 18

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK) and the light chain has the amino acid sequence shown as SEQ ID No. 20

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC) in the sequence listing.

10. An ELISA kit for detecting human interleukin 23, characterized in that the ELISA kit comprises:

a first antibody (coating antibody) that has been immobilized or is to be immobilized to a solid phase carrier; and

a biotin-labeled secondary antibody (detection antibody);

wherein the first antibody and the second antibody are monoclonal antibodies according to any one of claims 1 to 9.

11. The ELISA kit of claim 10 wherein the first antibody is anti-human interleukin 23 monoclonal antibody # 1 and the second antibody is anti-human interleukin 23 monoclonal antibody # 2.

12. The ELISA kit according to claim 10 or 11, further comprising peroxidase-labeled streptavidin, interleukin 23 standard, substrate, coated antibody diluent, wash solution, blocking/sample diluent, and stop buffer.

13. The ELISA kit of any one of claims 10 to 12 wherein the kit is a modified double antibody sandwich ELISA kit.

14. Use of the ELISA kit of any of claims 10-13 to detect interleukin 23 in cell culture fluid, human serum.

15. A method for detecting the content of interleukin 23 in a sample, which is characterized in that the antibody of any one of claims 1 to 9 is used for performing ELISA detection by using a modified double-antibody sandwich method.

16. The method of claim 15, comprising the steps of,

coating an enzyme label plate with an anti-human interleukin 23 monoclonal antibody 1 #;

adding the sample to be detected into the coated ELISA plate, and incubating;

adding the monoclonal antibody 2# of the anti-human interleukin 23 marked by the biotin into an enzyme label plate, and incubating;

diluting peroxidase-labeled streptavidin, adding the diluted streptavidin to an ELISA plate, and incubating;

adding a substrate into an enzyme label plate, incubating in a dark place, adding a stop solution, and measuring an OD value;

fitting a standard curve by using the OD value of the interleukin 23 standard substance, and substituting the OD value of the detected sample into an equation to calculate the content of the interleukin 23 in the detected sample.

17. The method of claim 15 or 16, wherein said interleukin 23 is one of human interleukin 23, human murine chimeric interleukin 23, or human rabbit chimeric interleukin 23.

Effects of the invention

The application provides a novel anti-human interleukin 23 monoclonal antibody and a human interleukin 23ELISA kit containing the same. The kit applies an improved double-antibody sandwich enzyme-labeled immunoassay method to determine the level of interleukin 23 in a sample, and carries out Biotin labeling on an anti-human interleukin 23 antibody, so that a Biotin-Avidin System (Biotin-Avidin-System, BAS) can be formed with an enzyme-labeled secondary antibody, and the firm combination of high affinity of the Biotin-Avidin-System and BAS can play a role in multistage amplification of biochemical reaction, so that the kit has higher detection sensitivity. Meanwhile, the kit can also detect human-mouse chimeric interleukin 23 and human-rabbit chimeric interleukin 23, and has good development and application prospects.

Drawings

FIG. 1 is a schematic diagram showing the competitive effect of anti-human interleukin monoclonal antibodies 1# and 2# on the binding site of antigen human IL-23

FIG. 2 is a graph showing the standard curve of the detection of human IL-23 concentration

FIG. 3 shows the results of detection of chimeric human-mouse IL-23

FIG. 4 shows the results of detection of chimeric human rabbit IL-23

Detailed Description

The following detailed description of the present application is provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

Technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art, and in case of conflict, the definitions set forth herein will control.

In general, the terms used in the present specification have the following meanings.

In the present specification, an "isolated" antibody is an antibody that has been separated from components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). Methods for assessing antibody purity are well known in the art and can be found, for example, in: flatman et al, j.chromatogr.b848: 79-87(2007).

In the present specification, "monoclonal antibody" means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants generally being present in minor amounts, except possibly for variant antibodies (e.g., containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation). 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 the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained 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 in accordance with the invention 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 in this specification refers to an intrinsic 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 (4) showing. Affinity can be measured by common methods known in the art.

In the present specification, "Human interleukin 23 (hIL-23) means a protein derived from Human, and is a heterodimer composed of two subunits, p19 and p 40. The amino acid sequence of p19 is shown in SEQ ID NO. 21, the amino acid sequence of p40 is shown in SEQ ID NO. 22, wherein the underlined part indicates the signal peptide.

SEQ ID No:21：

MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP

SEQ ID No:22:

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

In the present specification, the "anti-human interleukin 23 monoclonal antibody" means a monoclonal antibody: which is capable of binding human interleukin 23 with sufficient affinity such that the monoclonal antibody is useful as a diagnostic and/or therapeutic agent targeting human interleukin 23.

The anti-human interleukin 23(IL-23) monoclonal antibody of the present application does not bind to a target-independent protein. Here, "irrelevant protein" means a protein other than human interleukin 23 as a target; here, "not to bind" means: the anti-human interleukin 23(IL-23) monoclonal antibody of the present application has a binding ability to the unrelated protein of less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0, where the binding ability of the anti-human interleukin 23(IL-23) monoclonal antibody of the present invention to human interleukin 23 as its target is taken as 100%.

The anti-human interleukin 23(IL-23) monoclonal antibody of the present application does not bind to interleukin 23 of other animal species. Here, "other animal species" means other animal species than humans, such as rhesus monkey, cynomolgus monkey, rat, mouse, and the like; here, "not to bind" means: the binding ability of the anti-human interleukin 23(IL-23) monoclonal antibody of the present invention to interleukin 23 of another animal species is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0, assuming that the binding ability of the anti-human interleukin 23(IL-23) monoclonal antibody of the present invention to human interleukin 23 as its target is 100%.

The monoclonal antibody against human interleukin 23(IL-23) of the present application has an equilibrium dissociation constant (K) of not more than 1 μ M, not more than 100nM, not more than 50nM, not more than 40nM_D)。

In the present specification, ELISA (enzyme linked immunosorbent assay, abbreviated as ELISA), refers to a detection method in which free hetero protein is bound to a target protein bound to a solid phase carrier by using the characteristic that an antibody molecule can specifically bind to an antigen molecule, and the target protein is qualitatively or quantitatively analyzed by using a specific marker. The principle is that antigen or antibody can be physically adsorbed on the solid phase surface and keep the immunological activity; the antigen or antibody is capable of forming an enzyme conjugate with the enzyme via a covalent bond while retaining the respective immunological or enzymatic activity; the enzyme conjugate, when bound to the corresponding antigen or antibody, can determine the occurrence of an immune reaction by a color reaction of the added substrate, the color reaction being proportional to the amount of the corresponding antigen or antibody in the sample. Various different types of detection methods can be designed according to the substance to be detected and the detection conditions, and the double antibody sandwich method is the most common method for detecting the antigen. The antiserum containing known antibodies is adsorbed in small holes on a microtiter plate and washed once; adding antigen to be detected, if the antigen and the antigen are specific, combining, and then washing off redundant antibody; adding enzyme-linked antibody which reacts specifically with antigen to be detected to form a sandwich; addition of the substrate for the enzyme, if a colored cleavage product is observed, indicates the presence of the corresponding antigen.

In the present specification, "avidin" is a glycoprotein, each molecule consisting of 4 subunits, which can be tightly bound to 4 biotin molecules. More used is streptavidin (streptavidin) extracted from streptomyces.

In this specification, biotin is a small molecule growth factor, also known as coenzyme R or vitamin H, that is widely distributed in animals and plants. Avidin is a basic glycoprotein extracted from ovalbumin and has a very high affinity for biotin. The avidin molecule combined with enzyme reacts with the biotin molecule combined with specific antibody, which not only plays a role of multi-stage amplification, but also develops color due to the catalytic action of enzyme when meeting corresponding substrate, thereby achieving the function of measuring the amount of the combined antibody. Therefore, the combination of avidin and biotin with ELISA can greatly improve the sensitivity of ELISA. The biotin-avidin system has many applications in ELISA, both for indirect coating and for final reaction amplification. The enzyme-labeled antibody in a conventional ELISA may also be replaced with a biotinylated antibody, which is then conjugated with an avidin-enzyme conjugate to amplify the reaction signal.

Specifically, the present application relates to an isolated monoclonal antibody against human interleukin 23 comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of CDR-H1 is shown in SEQ ID No. 1(SYWMN) or SEQ ID No. 2 (SYGIS); the amino acid sequence of CDR-H2 is shown as SEQ ID No. 3(YIIPITGYTEYNQKFKD) or SEQ ID No. 4 (KISPRSVNAYYNEKFKG); the amino acid sequence of CDR-H3 is shown in SEQ ID No. 5(GGGNLPY) or SEQ ID No. 6 (DYSNLIFDY); the amino acid sequence of CDR-L1 is shown in SEQ ID No. 7(SASSSVSFTYLY) or SEQ ID No. 8 (SVSSSISSSNLH); the amino acid sequence of CDR-L2 is shown in SEQ ID No. 9(SISNLAS) or SEQ ID No. 10 (GTSSLAS); the amino acid sequence of CDR-L3 is shown in SEQ ID No. 11(QQWSSNPPIT) or SEQ ID No. 12 (QQWSSYPLT).

In a specific embodiment, the antibody provided herein has CDR-H1 having the amino acid sequence shown as SEQ ID No. 1(SYWMN), CDR-H2 having the amino acid sequence shown as SEQ ID No. 3(YIIPITGYTEYNQKFKD), CDR-H3 having the amino acid sequence shown as SEQ ID No. 5(GGGNLPY), CDR-L1 having the amino acid sequence shown as SEQ ID No. 7(SASSSVSFTYLY), CDR-L2 having the amino acid sequence shown as SEQ ID No. 9(SISNLAS), CDR-L3 having the amino acid sequence shown as SEQ ID No. 11 (QQWSSNPPIT).

In a specific embodiment, the monoclonal antibody provided herein has CDR-H1 having the amino acid sequence shown as SEQ ID No. 2(SYGIS), CDR-H2 having the amino acid sequence shown as SEQ ID No. 4(KISPRSVNAYYNEKFKG), CDR-H3 having the amino acid sequence shown as SEQ ID No. 6(DYSNLIFDY), CDR-L1 having the amino acid sequence shown as SEQ ID No. 8(SVSSSISSSNLH), CDR-L2 having the amino acid sequence shown as SEQ ID No. 10(GTSSLAS), and CDR-L3 having the amino acid sequence shown as SEQ ID No. 12 (QQWSSYPLT).

In a specific embodiment, the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: the amino acid sequence of HCVR is shown as SEQ ID No. 13

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSS); the amino acid sequence of LCVR is shown in SEQ ID No. 15

In a specific embodiment, the HCVR of the monoclonal antibody provided herein has the sequence of SEQ ID No. 13

(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEWIGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCARGGGNLPYWGQGTLVTVSA) wherein the LCVR has the amino acid sequence as set forth in SEQ ID No. 15

(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWIYSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITFGAGTKLELK) in the sequence listing.

In a specific embodiment, the HCRV of a monoclonal antibody provided herein has the sequence as SEQ ID No:14

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSS) and the LCVR has the amino acid sequence as shown in SEQ ID No. 16

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELK) in the presence of a protease.

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

the amino acid sequence of the heavy chain is shown as SEQ ID No. 17

(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEWIGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK); the amino acid sequence of the light chain is shown as SEQ ID No. 19

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC).

In a specific embodiment, the anti-human interleukin 23(IL-23) monoclonal antibody of the present application is anti-human interleukin 23 monoclonal antibody # 1, and its heavy chain has the amino acid sequence shown in SEQ ID No:17

In a specific embodiment, the anti-human interleukin 23(IL-23) monoclonal antibody of the present application is anti-human interleukin 23 monoclonal antibody # 2, and its heavy chain has the amino acid sequence shown in SEQ ID No:18

(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIFGTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC) in the presence of a protease.

The experimental results show that the monoclonal antibody against human interleukin 23(IL-23) can specifically bind to the p19 subunit of human interleukin 23 (IL-23).

The monoclonal antibody for resisting human interleukin 23(IL-23) is equivalent to or superior to the similar monoclonal antibody products on the market in various aspects of biological activity. The biological activity can inhibit the activity of STAT3 phosphorylation in cells induced by IL-23, the activity of IL-17A release of mouse spleen cells induced by IL23 and the activity of IFN-gamma release of human NK cells induced by IL-23.

Furthermore, the monoclonal antibodies against human interleukin 23 in the present application are all murine monoclonal antibodies.

The application also relates to an ELISA kit for detecting human interleukin 23, which is characterized in that the ELISA kit comprises: a first antibody (coating antibody) which has been immobilized to a solid phase carrier or is to be immobilized to the solid phase carrier; biotin-labeled secondary antibody (detection antibody).

In a specific embodiment, the anti-human interleukin monoclonal antibody in the ELISA kit described herein comprises a first antibody and a second antibody, wherein the first antibody is a coating antibody which is fixed to or is to be fixed on a solid-phase carrier; the second antibody is a detection antibody, which is labeled with biotin. Preferably, the solid phase carrier is an enzyme label plate. More preferably, the enzyme-labeled plate is a 96-well plate, specifically, polystyrene plastic plate of Corning Coster, USA.

Specifically, the first antibody and the second antibody are anti-human interleukin 23 monoclonal antibodies provided by the application.

The kit provided by the application is a double-antibody sandwich determination kit, namely, a double-antibody sandwich method is utilized to detect interleukin 23 substances in cells and/or serum in a sample. Specifically, a known amount of a first antibody, i.e., a coating antibody, is bound to the surface of a solid support. The test sample is then applied to the surface such that any interleukin-23 substance present therein in the cells and/or serum is captured by the immobilized first antibody. Unbound material is preferably removed by one or more washing steps. A second antibody, the detection antibody, labelled with biotin is then added and allowed to bind to interleukin 23 species in any cells and/or serum captured by the first antibody, allowing each unit of interleukin 23 to bind both antibodies simultaneously to form a "sandwich". The amount of bound secondary antibody is then determined in a direct or indirect detection method. In particular, the label or enzyme may be linked to the second antibody directly or indirectly via a linkage such as a biotin-streptavidin or biotin-avidin linkage.

In a specific embodiment, the first antibody in the ELISA kit is anti-human interleukin 23 monoclonal antibody # 1, the heavy chain has the amino acid sequence shown as SEQ ID No. 17, and the light chain has the amino acid sequence shown as SEQ ID No. 19; the second antibody is anti-human interleukin 23 monoclonal antibody 2#, the heavy chain has the amino acid sequence shown in SEQ ID No. 18, and the light chain has the amino acid sequence shown in SEQ ID No. 20.

Further, the ELISA kit also comprises a device or a reagent which is necessary for detecting interleukin 23.

Specifically, the ELISA kit also comprises peroxidase-labeled streptavidin, an interleukin 23 standard substance, a substrate, a coated antibody diluent, a washing solution, a confining solution/sample diluent and a stop solution.

In a specific embodiment, the peroxidase is preferably horseradish peroxidase (HRP), and the labeled streptavidin is a streptavidin-horseradish peroxidase conjugate (SA-HRP).

In a specific embodiment, the interleukin 23 standard in the kit is human IL-23 protein; the substrate is 3,3 ', 5, 5' -tetramethyl benzidine (TMB); the coating antibody diluent is Phosphate Buffered Saline (PBS) pH7.4; the washing solution is Phosphate Buffered Saline (PBS) containing 0.05 percent of Tween 20; blocking solution/sample diluent Phosphate Buffered Saline (PBS) containing 0.5% BSA, 0.05% Tween20, and 0.05% Proclin 300; the stop solution is phosphoric acid.

Preferably, the ELISA kit comprises the following components:

1) solid phase carrier: an ELISA plate 1;

2) coating antibody: anti-human interleukin-23 monoclonal antibody # 1 (i.e., primary antibody), 10 μ g/tube, 1 tube;

3) and (3) detecting an antibody: anti-human interleukin-23 monoclonal antibody # 2 (i.e., secondary antibody) (labeled with biotin), 10 μ g/tube, 1 tube;

4) and (3) standard substance: human IL-23 protein, 10 ng/tube, 1 tube;

5) streptavidin-horseradish peroxidase conjugate (Streptavidin-HRP, SA-HRP for short), 500 ng/tube, 1 tube;

6) substrate: 3,3 ', 5, 5' -Tetramethylbenzidine (TMB), 5 ml/tube, 1 tube for each of solution A and solution B;

7) coating antibody dilution: phosphate Buffered Saline (PBS), pH7.4, 50 ml/vial, 1 vial;

8) washing solution: phosphate Buffered Saline (PBS), containing 0.05% Tween20, 200 ml/vial, 1 vial;

9) blocking solution/sample diluent: phosphate Buffered Saline (PBS) containing 0.5% BSA, 0.05% Tween20, and 0.05% Proclin300, 200 ml/vial, 1 vial;

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

The application also relates to a method for detecting the content of interleukin 23 in a sample, which is characterized in that the anti-human interleukin 23 monoclonal antibody provided by the application is used for ELISA detection by using an improved double-antibody sandwich method.

Specifically, the method comprises the following steps,

coating an enzyme label plate by using an anti-human interleukin 23 monoclonal antibody 1# (namely a first antibody);

adding the sample to be detected into the coated enzyme label plate, and incubating;

adding the biotin-labeled anti-human interleukin 23 monoclonal antibody 2# (namely a second antibody) into an enzyme label plate, and incubating;

diluting peroxidase-labeled streptavidin, adding the diluted streptavidin to an enzyme label plate, and incubating;

In a specific embodiment, the ELISA kit described herein can be used to quantitatively detect the amount of human interleukin 23 in a sample, comprising the following steps:

(1) coating: preparing a coated antibody working solution with the concentration of 1 mu g/ml by using a coated antibody diluent (phosphate buffer solution (PBS), pH 7.4) and using a coated antibody anti-human interleukin 23 monoclonal antibody 1# (namely a first antibody), adding the coated antibody working solution into an enzyme label plate (96-hole Coster plate) according to the dosage of 50 mu l/hole, and standing overnight at the temperature of 2-8 ℃;

(2) and (3) sealing: discarding the coated antibody working solution in the ELISA plate, washing the plate 3 times with a washing solution (phosphate buffered saline (PBS) containing 0.05% Tween20), adding a blocking solution (phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300) according to the dosage of 100 μ l/well, and placing the plate in a shaker (120rpm) for blocking at room temperature for 2 hours;

(3) preparing a protein standard product: taking 8 EP tubes and numbering in sequence, adding 200 mul of sample diluent into each tube from the 2 nd tube and placing the sample diluent on an EP tube frame; preparing a standard protein (human IL-23 protein) into a liquid with a concentration of 25ng/ml by using a sample diluent, sucking 400 mu l of the liquid, adding the liquid into a 1 st EP tube, sucking 200 mu l of the liquid from the 1 st EP tube, adding the liquid into a 2 nd EP tube, performing double dilution, and so on until reaching a 10 th EP tube, wherein a point of 25ng/ml and 0.049ng/ml is used as an anchor point;

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

(5) sample adding: discarding the confining liquid in the ELISA plate, washing the plate with a washing solution for 3 times, adding a protein standard solution and a quality control sample according to the dosage of 50 mul/hole, and incubating for 2 hours at room temperature in a shaking table (120 rpm);

(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 solution, preparing a detection antibody (anti-human interleukin 23 monoclonal antibody 2# (namely a second antibody) by using a sample diluent into a detection antibody working solution with the concentration of 0.2 mu g/ml, 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 (120rpm) for incubation reaction for 2 hours;

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

(8) color development: mixing the solution A and the solution B (3,3 ', 5, 5' -Tetramethylbenzidine (TMB), 5 ml/tube, 1 tube for each solution A and B) of the substrate in equal volume; discarding the enzyme-labeled secondary antibody working solution in the ELISA plate, washing the plate for 3 times by using washing liquor, then adding a substrate according to the dosage of 50 mu l/hole, and reacting for 5-10 minutes at room temperature in a dark place;

(9) and (3) stopping color development: adding the stop solution (1M phosphoric acid) into the ELISA plate according to the dosage of 50 mu l/hole to stop reaction, then measuring the OD value of each hole in the ELISA plate under the wavelength of 450nm by using an ELISA reader, fitting a standard curve according to the OD value of the standard substance, substituting the OD value of the quality control sample into an equation, and calculating the concentration of the quality control sample.

The application also relates to application of the anti-human interleukin 23 monoclonal antibody, an ELISA kit containing the anti-human interleukin 23 monoclonal antibody and the method for detecting the interleukin 23 content in a sample in detecting interleukin 23 in cell culture fluid and human serum.

Compared with the prior art, the human IL-23ELISA kit has higher detection sensitivity, can detect human-mouse chimeric IL-23 and human-rabbit chimeric IL-23, and has good development and application prospects.

Examples

The present disclosure will be described below in conjunction with specific embodiments, but the scope of the present disclosure is not limited thereto.

The reagents and apparatus used in the following embodiments are all conventional in the art and are commercially available, unless otherwise specified. The methods used are all conventional experimental methods, and the person skilled in the art can, without any doubt, carry out the protocols and obtain the corresponding results according to the contents of the examples.

Example 1Anti-human interleukinScreening and preparation of monoclonal antibody of element 23

Preparing human-mouse chimeric interleukin 23(IL-23), using it to immunize Balb/c mouse, using hybridoma technique to make cell fusion, screening out monoclonal cell strain, and further making extensive culture to obtain monoclonal antibody. First, cell supernatants were examined by Binding ELISA, clones Binding to IL-23 were selected, and then examined by Blocking ELISA and the cell method, and clones having IL-23 inhibitory activity were selected, and the first antibody and the second antibody of the present invention were obtained by screening. The first antibody and the second antibody obtained by the invention are mouse monoclonal antibodies and are not subjected to humanization modification. The above immunization and screening procedures are delegated to intelligent chemistry.

The complete sequences of the heavy and light chains of the first and second antibodies were obtained by sequencing, and then the CDR sequences were obtained according to the Kabat rules (reference: Kabat E.A., Wu T.T., Bilofsky H.Attempts to locate reactions in complementary-determining regions of antibody combining sites that was used in the USA 1976; 73: 617. 619.).

Example 2Characterization of anti-human interleukin-23 monoclonal antibody

1. Competition experiments for antibody recognition of antigenic sites:

the ELISA plates were coated with primary and secondary antibodies, respectively, overnight at 2-8 ℃ and blocked for 2h at room temperature the next day. Gradient diluted Bio IL-23 was incubated with equal volumes of primary antibody and secondary antibody of 10. mu.g/ml, 1. mu.g/ml, 0. mu.g/ml for 1.5 hours at room temperature. After washing, the incubated antibody and Bio IL-23 complex was added and incubated for 2h at room temperature. After washing the plate, diluted enzyme-labeled secondary antibody was added and incubated at room temperature for 1 hour. Finally, the plate was washed, developed and OD read₄₅₀Absorbance of (d) in (d). As shown in FIG. 1, the first antibody and the second antibody did not compete with the binding site of human IL-23 antigen, and in the experiment in which the first antibody was coated on the microplate, the addition of the second antibody did not affect the binding between the first antibody and Bio IL-23, and in the same experiment in which the second antibody was coated on the microplate, the addition of the first antibody did not affect the binding between the second antibody and Bio IL-23. The second antibody can be used as a detection antibody of a human IL-23 kitA body.

2. Second antibody biotin labeling:

biotin labelling of the second antibody (detection antibody)

The Sulfo-NHS-LC-Biotin kit comprises the following specific steps strictly according to the kit instructions. After labeling is complete, use ZTba^TMAnd filtering by using Spin Deasling columns to remove free biotin, wherein the obtained solution is the biotin-labeled secondary antibody.

Example 3Composition of human IL-23ELISA quantitative detection kit

The first antibody was used as a coating antibody, and the second antibody (labeled with biotin) was used as a detection antibody to prepare an ELISA kit, and the information of the reagent composition, specification, source, storage conditions, and the like in this kit is shown in table 1.

TABLE 1 ELISA kit Components

Example 4Quantitative detection of human IL-23 by human IL-23ELISA kit

The human IL-23 in the quality control samples was quantitatively detected by using the human IL-23ELISA kit in example 3.

1. Sample treatment:

diluting the human IL-23 protein with a sample diluent to prepare a protein standard, and diluting the human IL-23 protein with a cell culture solution to prepare a quality control sample as a sample to be detected. And fitting a standard curve according to the light absorption value of the protein standard. Substituting the OD value of the sample to be tested into the standard curve to obtain the theoretical concentration of human IL-23 in the sample to be tested, and then calculating the recovery rate (theoretical concentration/actual concentration) of the sample to be tested (100), thereby analyzing the feasibility of the ELISA kit in example 3 for quantitative detection of human IL-23 in the cell culture fluid.

2. Quantitative detection operation steps:

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

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

(3) preparing a protein standard product: taking 8 EP tubes and numbering in sequence, adding 200 mul of sample diluent into each tube from the 2 nd tube and placing the sample diluent on an EP tube frame; preparing a standard protein into liquid with the concentration of 25ng/ml by using the sample diluent, sucking 400 mu l of the liquid, adding the liquid into the 1 st EP tube, sucking 200 mu l of the liquid from the 1 st EP tube, adding the liquid into the 2 nd EP tube for double dilution, and so on until reaching the 10 th EP tube, wherein the point of 25ng/ml and 0.049ng/ml is used as an anchoring point;

(5) sample adding: discarding the confining liquid in the ELISA plate, washing the plate with a washing solution for 3 times, adding a protein standard solution and a quality control sample according to the dosage of 50 mu l/hole, and incubating at room temperature for 2 hours in a shaking table (120 rpm);

(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 solution, preparing a detection antibody (biotin label) into a detection antibody working solution with the concentration of 0.2 mu g/ml by using a 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 (120rpm) for incubation reaction for 2 hours;

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

(8) color development: mixing the solution A and the solution B of the substrate in equal volume; discarding the enzyme-labeled secondary antibody working solution in the ELISA plate, washing the plate for 3 times by using washing liquor, then adding a substrate according to the dosage of 50 mu l/hole, and carrying out a light-shielding reaction at room temperature for 5-10 minutes;

(9) and (3) stopping color development: adding the stop solution into the ELISA plate according to the dosage of 50 mu l/hole to stop reaction, then measuring the OD value of each hole in the ELISA plate under the wavelength of 450nm by using an ELISA reader, fitting a standard curve according to the OD value of the standard substance, substituting the OD value of the quality control sample into an equation, and calculating the concentration of the quality control sample.

3. And (3) detection results:

(1) the detection data of the human IL-23 standard are shown in Table 2:

TABLE 2 detection data for human IL-23 standard

The detection curve of human IL-23 was recovered at a concentration range of 0.049-25 ng/ml.

(2) The curve equation: 4-P Fit: y ═ 2.36 (0.0603)/(1 + (x/4.22) ^1.1+2.36, R²1 (see fig. 2).

(3) The detection data of the samples to be detected are shown in table 3:

TABLE 3 data of the samples to be tested

4. And (3) examining accuracy and precision:

(1) in 2 different experiments, each assay lot contained 2 sets of samples to be tested, each set containing 5 concentrations (LLOQ 0.098, LQC 0.2, MQC 5, HQC 10 and ULOQ 12.5 ng/mL). The precision of the method is expressed by coefficient of variation CV%, where CV% is SD/average × 100. Accuracy is expressed as the relative error RE%, (average observed concentration-nominal concentration)/nominal concentration × 100. The detection accuracy of the kit is analyzed through the parameters. The analysis results are shown in Table 4.

TABLE 4 quantitative determination accuracy and precision test data for human IL-23

(2) Results and discussion:

the results in Table 4 show that the 5 concentration levels of the intra-batch coefficient of variation CV% ≦ 13.238%. The relative error RE% within batches of ULOQ, HQC, MQC, and LQC ranges from-4.949% to 15.000%. The relative error RE% within the batch of LLOQ ranges from 0% to 2.551%. The results show that the human IL-23 quantitatively detecting the sample by using the human IL-23ELISA kit provided by the application can meet the requirement that both the intra-batch variation coefficient and the relative error are less than or equal to 20% (LLOQ and ULOQ are less than or equal to 25%), and the detection method provided by the application has good accuracy.

Example 5Detection of human-mouse chimeric IL-23 and human-rabbit chimeric IL-23 by ELISA kit

1. Description of the samples:

in the experiment, the human-mouse chimeric IL-23 is formed by chimeric human p19 subunit and mouse p40 subunit, the human-rabbit chimeric IL-23 is formed by chimeric human p19 subunit and rabbit p40 subunit (two chimeric proteins are from Jiangsu Quanxin biomedicine, Inc.), the diluted solution is used for diluting to 5 mu g/ml to be used as the first hole concentration, 1/5 is used for carrying out gradient dilution on 11 holes, and the last hole is zero for standby; the commercially available kit is the Human IL-23DuoSet ELISA kit (Cat: DY1290, Lot: P148670) from R & D. Specific information is shown in table 5:

TABLE 5 Human IL-23DuoSet ELISA kit composition

Name(s)	Specification of	Working concentration	Lot#
				Capture	360μg	6.00μg/ml	JM1417051
Detection	24.0μg	50.0ng/ml	CCEM0417051
				Standard of reference	520ng	125-8000pg/ml	1346567
Streptavidin-HRP	NA	Diluting by 40 times	P141286

2. A detection operation step:

(1) coating: preparing a coated antibody working solution with the concentration of 1 mu g/ml by adopting a coated antibody diluent, taking a capture antibody in an R & D kit, diluting to 6 mu g/ml, adding the capture antibody into an ELISA plate according to the dosage of 50 mu l/hole, and standing overnight at 4 ℃;

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

(4) sample adding: discarding the confining liquid in the ELISA plate, washing the plate with a washing solution for 3 times, adding diluted human-mouse chimeric IL-23 and human-rabbit chimeric IL-23 according to the dosage of 50 mul/hole, and incubating at room temperature for 2 hours in a shaking table (120 rpm);

(6) adding a detection antibody: discarding a protein standard solution and a sample to be detected in an enzyme label plate, washing the plate for 3 times by using a washing solution, preparing a Detection antibody (biotin label) into a Detection antibody working solution with the concentration of 0.2 mu g/ml by using a sample diluent, and diluting the Detection antibody working solution to 50ng/ml in an R & D kit; then adding the mixture into an enzyme label plate according to the dosage of 50 mul/hole, and placing the enzyme label plate in a shaking table (120rpm) for incubation reaction for 2 hours;

(7) adding an enzyme-labeled secondary antibody: discarding a detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing solution, preparing an ELISA secondary antibody into an ELISA secondary antibody working solution with the concentration of 100ng/ml by using a sample diluent, taking SA-HRP in an R & D kit, diluting by 40 times to the working concentration, adding the SA-HRP into the ELISA plate according to the dosage of 50 mu l/hole, and incubating for 1 hour at room temperature in a shaking table (120 rpm);

(9) and (3) stopping color development: adding the stop solution into the ELISA plate according to the dosage of 50 mul/hole to stop reaction, then measuring the OD value of each hole in the ELISA plate under the wavelength of 450nm by using an ELISA reader, and fitting a curve according to the OD value of the standard substance.

The detection results are shown in fig. 3 and 4.

3. And (3) detection results:

as shown in the detection results of FIG. 3, the method of the present application can detect the chimeric IL-23 of human and mouse, and the R & D kit fails to detect the chimeric IL-23 of human and mouse; as shown in the detection result of FIG. 3, the method of the present application can detect the chimeric IL-23 of the human rabbit, and the detection sensitivity is much higher than that of the R & D kit.

The human-mouse chimeric IL-23 is human IL-23p 19-mouse IL-23p40, the human-rabbit chimeric IL-23 is human IL-23p 19-rabbit IL-23p40, and the first antibody and the second antibody in the method can both specifically recognize human IL-23p19, so the method can be used for detecting the human-mouse chimeric IL-23 and the human-rabbit chimeric IL-23.

The above description is only for the preferred embodiment of the present application and should not be taken as limiting the present application in any way. Any person skilled in the art may apply the above disclosed technical content to change or modify equivalent embodiments with equivalent variations. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Sequence listing

<110> Jiangsu Quanxin biomedical products, Ltd

<120> human interleukin 23, kit comprising same and detection method thereof

<130> TPD01056D2

<160> 20

<170> PatentIn version 3.5

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Gly Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Ser Pro Arg Ser Val Asn Ala Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Tyr Ser Asn Leu Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 15

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 15

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

1 5 10 15

Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Phe Thr

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Thr Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro

85 90 95

Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 16

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser

20 25 30

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

35 40 45

Ile Phe Gly Thr Ser Ser Leu Ala Ser Gly Val Pro Val Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro

85 90 95

Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 17

<211> 439

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 17

Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp

20 25 30

Met Asn Trp Ile Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

35 40 45

Tyr Ile Ile Pro Ile Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Lys

50 55 60

Asp Met Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met

65 70 75 80

Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Gly Asn Leu Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro

115 120 125

Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val

130 135 140

Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser

145 150 155 160

Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu

165 170 175

Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Pro Arg Pro Ser

180 185 190

Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val

195 200 205

Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys

210 215 220

Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val

245 250 255

Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp

260 265 270

Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe

275 280 285

Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys

325 330 335

Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys

340 345 350

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

355 360 365

Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys

370 375 380

Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser

385 390 395 400

Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr

405 410 415

Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser

420 425 430

Leu Ser His Ser Pro Gly Lys

435

<210> 18

<211> 442

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 18

Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Met Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Ser Pro Arg Ser Val Asn Ala Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Tyr Ser Asn Leu Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Pro

180 185 190

Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

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

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 19

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 19

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

1 5 10 15

Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Phe Thr

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Thr Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro

85 90 95

Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp

100 105 110

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

115 120 125

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

130 135 140

Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly

145 150 155 160

Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn

180 185 190

Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val

195 200 205

Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 20

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser

20 25 30

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

35 40 45

Ile Phe Gly Thr Ser Ser Leu Ala Ser Gly Val Pro Val Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro

85 90 95

Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala

100 105 110

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

115 120 125

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

130 135 140

Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val

145 150 155 160

Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met

165 170 175

Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser

180 185 190

Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys

195 200 205

Ser Phe Asn Arg Asn Glu Cys

210 215

Claims

a) the amino acid sequence of CDR-H1 is shown in SEQ ID No. 2;

b) the amino acid sequence of CDR-H2 is shown in SEQ ID No. 4;

c) the amino acid sequence of CDR-H3 is shown in SEQ ID No. 6;

d) the amino acid sequence of CDR-L1 is shown in SEQ ID No. 8;

e) the amino acid sequence of CDR-L2 is shown in SEQ ID No. 10;

f) the amino acid sequence of CDR-L3 is shown in SEQ ID No. 12.

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

the amino acid sequence of HCVR is shown in SEQ ID No. 14;

the amino acid sequence of LCVR is shown in SEQ ID No. 16.

3. The monoclonal antibody of any one of claims 1 or 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

the amino acid sequence of the heavy chain is shown as SEQ ID No. 18;

the amino acid sequence of the light chain is shown as SEQ ID No. 20.