Monoclonal antibody against anti-TSLP monoclonal antibody, kit comprising same, and detection method using same

Abstract

The invention discloses two novel monoclonal antibodies for resisting a thymic stromal lymphopoietin (TSLP) monoclonal antibody, an ELISA (enzyme-linked immuno sorbent assay) kit containing the antibodies and a detection method of the ELISA kit. The antibody comprises an antibody heavy chain complementarity determining region and an antibody light chain complementarity determining region, and the ELISA kit comprises two antibodies. Wherein the capture antibody is specifically a monoclonal antibody of a monoclonal antibody against thymic stromal lymphopoietin (TSLP), and is used for coating; and the second antibody is specifically a biotin-labeled monoclonal antibody against a thymic stromal lymphopoietin (TSLP) monoclonal antibody, and is used for being combined with a capture antibody-antibody compound to be detected. The kit can be used for successfully and quantitatively detecting the anti-thymic stromal lymphopoietin (TSLP) monoclonal antibody, has higher detection sensitivity and good detection specificity, and has good development and application prospects.

Description

[0001] This case is a divisional application of a case with an application date of November 25, 2024, an invention name of monoclonal antibodies, kits containing the same, and detection methods using the same, and an application number of 202411697116.5. Technical Field

[0002] The present application belongs to the field of biotechnology, and specifically, relates to two monoclonal antibodies against anti-thymic stromal lymphopoietin (TSLP) monoclonal antibodies, an ELISA kit for quantitatively detecting anti-thymic stromal lymphopoietin (TSLP) monoclonal antibodies using the two antibodies, and a method for quantitative detection using the kit. Background Art

[0003] Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine that is mainly produced by human epithelial cells. It can act on dendritic cells, T cells, B cells, and innate immune cells, and plays a central role in regulating type 2 immune responses. TSLP forms a ternary complex with the thymic stromal lymphocyte receptor TSLPR and IL-7Rα chain on the surface of the above cells, activates JAK1 (through IL-7Rα) and JAK2 (through TSLPR) and STATs, and drives the production of downstream IL-4, IL-5, IL-9, and IL-13, thereby exerting a pro-inflammatory effect. Studies have found that the occurrence and development of asthma, chronic obstructive pulmonary disease, and other allergic diseases are closely related to TSLP levels. The expression of TSLP in the airways of asthmatic patients is higher than that in healthy controls, and its level is positively correlated with the expression of Th2 cytokines and chemokines and the severity of the disease. TSLP participates in airway inflammation, airway hyperresponsiveness (AHR) and acute asthma attacks by inducing type 2 inflammatory response. It can also directly act on airway smooth muscle and mast cells to induce AHR independently of type 2 inflammation. Therefore, inhibition of TSLP may have therapeutic effects on asthma, chronic obstructive pulmonary disease and other allergic diseases. Anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody is an IgG1κ type humanized monoclonal antibody that specifically binds to human TSLP. Its parent antibody is obtained by screening New Zealand rabbit B cells immunized with human TSLP. The parent rabbit antibody is humanized to obtain anti-human TSLP monoclonal antibody.

[0004] Currently, the marketed drug with the same target, Tezepelumab, is a fully humanized IgG2λ monoclonal antibody. Phase II and Phase III clinical data showed that Tezepelumab significantly reduced the annualized asthma exacerbation rate (AAER) without being affected by the patient's baseline eosinophil level and FeNO level, and showed significant efficacy in a wide range of patients with severe uncontrolled asthma. Jiangsu Quanxin Biopharmaceutical Co., Ltd. has developed an anti-human TSLP monoclonal antibody with the trade name QX008N for use as a drug, which is described in the Chinese patent document CN 113683694 B.

[0005] For drugs, pharmacokinetics is the application of the principles of kinetics and mathematical processing methods to quantitatively describe the laws of dynamic changes of drugs in the body and study the absorption, distribution, metabolism and excretion of drugs that enter the human body through different pathways. Therefore, the study of pharmacokinetics will be conducive to the evaluation of drug safety and effectiveness. Commonly used analytical methods for biological samples include chromatography and immunoassay. Chromatography has low sensitivity, cumbersome experimental process, and unstable recovery rate; immunoassay is an analytical method based on specific antigen-antibody reaction, which is currently the most commonly used method for non-clinical and clinical biological sample detection. Since anti-human TSLP monoclonal antibodies belong to protein macromolecules, they are suitable for determination using enzyme-linked immunosorbent assay, in which anti-human TSLP monoclonal antibodies are respectively combined with capture antibodies and biotin-labeled secondary antibodies, and horseradish peroxidase-labeled streptavidin and biotin have high specificity, which greatly reduces the interference of irrelevant proteins in serum, can amplify the detection signal, and improve the detection sensitivity.

[0006] Sandwich ELISA means that the capture antibody is first bound to the ELISA plate, then the antibody to be detected is added to specifically bind to the capture antibody, then the specific second antibody is added, and finally the enzyme-labeled detection antibody is added and the substrate is used to catalyze the color reaction. In this way, the antibody to be detected specifically binds to the capture antibody and the second antibody with different binding epitopes, and the horseradish peroxidase-labeled streptavidin binds to the biotin-labeled second antibody, which not only plays a multi-stage amplification role, but also has the advantages of high specificity, high sensitivity, and stability. Summary of the invention

[0007] Based on the needs existing in the above-mentioned existing research process, the present application provides two monoclonal antibodies against human TSLP monoclonal antibodies, an improved sandwich ELISA kit based on an ELISA platform containing the above-mentioned antibodies, and the quantitative detection of anti-human TSLP monoclonal antibodies was successfully completed using the kit.

[0008] Specifically, this application involves the following:

[0009] 1. A monoclonal antibody against human thymic stromal lymphopoietin monoclonal antibody, wherein the monoclonal antibody comprises:

[0010] a) an antibody heavy chain complementarity determining region, comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

[0011] CDR-H1 has an amino acid sequence of SEQ ID NO.1: STYWIC or has at least 90% identity with SEQ ID NO.1,

[0012] CDR-H2 has an amino acid sequence of SEQ ID NO.2: CIYAGFTGNTYYASWVNG or has at least 90% identity with SEQ ID NO.2,

[0013] CDR-H3 has an amino acid sequence of SEQ ID NO.3: NAHDDSGEYIPHNF or has at least 90% identity with SEQ ID NO.3;

[0014] b) an antibody light chain complementary determining region, comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

[0015] CDR-L1 has an amino acid sequence of SEQ ID NO.4: QASEDIYSNLA or has at least 90% identity with SEQ ID NO.4,

[0016] CDR-L2 has an amino acid sequence of SEQ ID NO.5: RASTLAS or has at least 90% identity with SEQ ID NO.5,

[0017] CDR-L3 has an amino acid sequence of SEQ ID NO.6: LGVASYISVDGYNA or has at least 90% identity with SEQ ID NO.6.

[0018] 2. The monoclonal antibody according to item 1, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0019] HCVR has the sequence SEQ ID NO.7:

[0020] The amino acid sequence of QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSS or has at least 90% identity with SEQ ID NO.7;

[0021] The LCVR has an amino acid sequence of SEQ ID NO. 8: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVK or has at least 90% identity to SEQ ID NO. 8.

[0022] 3. The monoclonal antibody according to item 1 or 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0023] The heavy chain amino acid sequence is SEQ ID NO.9:QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGE YIPHNFWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTC The amino acid sequence of SKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or having at least 90% identity with SEQ ID NO.9;

[0024] The amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO.10: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO.10.

[0025] 4. A monoclonal antibody against human thymic stromal lymphopoietin monoclonal antibody, wherein the monoclonal antibody comprises:

[0026] a) an antibody heavy chain complementarity determining region, comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

[0027] CDR-H1 has an amino acid sequence of SEQ ID NO.11:TYYMS or has at least 90% identity with SEQ ID NO.11,

[0028] CDR-H2 has an amino acid sequence of SEQ ID NO.12: YISYGGTTDYASWAKG or has at least 90% identity with SEQ ID NO.12,

[0029] CDR-H3 has an amino acid sequence of SEQ ID NO.13: GYGDGFNP or has at least 90% identity with SEQ ID NO.13;

[0030] b) an antibody light chain complementary determining region, comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

[0031] CDR-L1 has an amino acid sequence of SEQ ID NO.14: QASQSISTKLA or has at least 90% identity with SEQ ID NO.14,

[0032] CDR-L2 has an amino acid sequence of SEQ ID NO.15: DASDLAS or has at least 90% identity with SEQ ID NO.15,

[0033] CDR-L3 has an amino acid sequence of SEQ ID NO.16:QQGYTSYNVENA or has at least 90% identity to SEQ ID NO.16.

[0034] 5. The monoclonal antibody according to item 1, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0035] HCVR has the sequence SEQ ID NO.17:

[0036] The amino acid sequence of QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARG YGDGFNPWGPGTLVTVSS or has at least 90% identity with SEQ ID NO. 17;

[0037] The LCVR has an amino acid sequence of SEQ ID NO. 18:TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVK or has at least 90% identity to SEQ ID NO. 18.

[0038] 6. The monoclonal antibody according to item 4 or 5, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0039] The heavy chain amino acid sequence is SEQ ID NO.19:QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPG TLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK, or an amino acid sequence that is at least 90% identical to SEQ ID NO. 19;

[0040] The amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO.20:TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO.20.

[0041] 7. The monoclonal antibodies against anti-human thymic stromal lymphopoietin monoclonal antibodies described in Items 1 and 4 are used to prepare a kit for detecting anti-human thymic stromal lymphopoietin monoclonal antibodies.

[0042] 8. The use according to item 7, wherein the kit comprises a capture antibody and a biotin-labeled second antibody, wherein the capture antibody is a monoclonal antibody of the anti-human thymic stromal lymphopoietin monoclonal antibody described in item 1; and the second antibody is a monoclonal antibody of the anti-human thymic stromal lymphopoietin monoclonal antibody described in item 4.

[0043] 9. The use according to item 8, wherein the antibody to be tested is QX008N.

[0044] 10. The use according to item 8, wherein the capture antibody is fixed to a solid carrier, and the antibody to be detected is bound to the capture antibody.

[0045] 11. The use according to item 10, wherein the solid phase carrier is an ELISA plate.

[0046] 12. The use according to item 8, characterized in that the kit further comprises: horseradish peroxidase-labeled streptavidin (SA-HRP), washing solution, substrate color development solution, blocking solution, sample diluent and stop solution.

[0047] 13. The use according to any one of items 7 to 12, characterized in that the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0048] 14. The use according to any one of items 7 to 13, characterized in that the kit is a modified sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0049] 15. A kit for detecting anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody, characterized in that the kit comprises a capture antibody and a biotin-labeled secondary antibody,

[0050] The capture antibody is a monoclonal antibody against human thymic stromal lymphopoietin monoclonal antibody described in any one of items 1 to 3;

[0051] The antibody to be tested is QX008N; it comprises heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein:

[0052] The CDR-H1 of the antibody to be tested QX008N has an amino acid sequence of SEQ ID NO.21: SYYMS, CDR-H2 has an amino acid sequence of SEQ ID NO.22: FISYGGSAYHATWAQG, and CDR-H3 has an amino acid sequence of SEQ ID NO.23: EFRSMTYGAEWGI; CDR-L1 has an amino acid sequence of SEQ ID NO.24: QASESIYDTLA, CDR-L2 has an amino acid sequence of SEQ ID NO.25: SASSLAS, and CDR-L3 has an amino acid sequence of SEQ ID NO.26: QQGYTMPDVDKNP;

[0053] The second antibody is a monoclonal antibody against human thymic stromal lymphopoietin monoclonal antibody according to any one of items 4 to 6;

[0054] The second antibody is coupled to the Biotin protein and purified through a desalting column to obtain a biotin-labeled second antibody.

[0055] 16. The kit according to item 15, wherein the capture antibody is fixed to a solid support, and the antibody to be detected is bound to the capture antibody.

[0056] 17. The kit according to item 16, wherein the solid phase carrier is an ELISA plate.

[0057] 18. A kit according to any one of items 15 to 17, wherein the kit further comprises: horseradish peroxidase-labeled streptavidin (SA-HRP), a washing solution, a substrate colorimetric solution, a diluent, a blocking solution and a stop solution, preferably, the washing solution is a mixture of PBS solution and Tween 20 (V:V=100:0.05), the substrate colorimetric solution is TMB colorimetric solution, the stop solution is a mixture of water and phosphoric acid (V:V=4:1), and the diluent / blocking solution is a mixture of PBS solution, BSA powder, Tween 20 and Proclin 300 (V:W:V:V=200:1:0.1:0.1).

[0058] 19. The kit according to any one of items 15 to 18, characterized in that the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0059] 20. The kit according to any one of items 15 to 19, wherein the kit is a modified sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0060] 21. A method for quantitatively detecting the content of anti-human thymic stromal lymphopoietin monoclonal antibody, wherein the capture antibody involved in item 15 and the biotin-labeled second antibody are used to perform ELISA detection using a modified sandwich enzyme-linked immunosorbent assay.

[0061] 22. The method according to claim 21, characterized in that it comprises the following steps:

[0062] Fixing the capture antibody to the solid phase carrier for coating and blocking;

[0063] Add the antibody to be tested and incubate it to react with the coated capture antibody, then add the biotin-labeled secondary antibody and incubate;

[0064] Add diluted horseradish peroxidase-labeled streptavidin (SA-HRP) and incubate;

[0065] Add substrate colorimetric solution, incubate in dark, add stop solution, measure OD value, and prepare standard curve;

[0066] Substitute the OD value of the sample to be tested into the equation to calculate the content of anti-human TSLP monoclonal antibody in the sample to be tested.

[0067] In the present application, the OD value is the OD value at 450 nm.

[0068] This application has the following beneficial effects:

[0069] First, an enzyme-linked immunosorbent assay method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum was established using rabbit anti-anti-human TSLP monoclonal antibodies as capture antibodies and secondary antibodies; second, the method established in the present application has a wide effective quantitative range of 10 to 1000 ng / ml, and the lower limit of quantification of anti-human TSLP monoclonal antibodies is 10 ng / ml, with high sensitivity; third, the method established in the present application has good repeatability, and the coefficient of variation of repeated experiments between plates is 7.6% to 11.8%, which is less than 20%; fourth, the method established in the present application has good individual selectivity, and 11 / 12 individuals meet the accuracy and precision within the acceptable range at the concentration level of the lower limit of quantification; fifth, the present application can be used to detect the content of anti-human TSLP monoclonal antibodies in serum, and can be used for clinical evaluation of the pharmacokinetic characteristics of anti-human TSLP monoclonal antibodies, thereby evaluating their safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1A This is a graph showing the binding results of the monoclonal antibody prepared in Example 1 with the anti-human TSLP monoclonal antibody.

[0071] Figure 1B This is a graph showing the binding results of the monoclonal antibody prepared in Example 1 to human IgG.

[0072] Figure 2 This is a diagram showing the results of identifying the neutralizing activity of the monoclonal antibody prepared in Example 1.

[0073] Figure 3A This is a diagram showing the epitope identification results of the monoclonal antibody prepared in Example 1.

[0074] Figure 3B This is a diagram showing the epitope identification results of the monoclonal antibody prepared in Example 1.

[0075] Figure 4 Schematic diagram of the enzyme-linked immunosorbent assay method of the present application.

[0076] Figure 5 This is the four-parameter fitting curve of the standard curve. DETAILED DESCRIPTION

[0077] The present application is further described in detail below in conjunction with specific implementation methods. The examples given are intended to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0078] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0079] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0080] Generally speaking, the terms used in this specification have the following meanings.

[0081] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous antibody population, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody population, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present 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 all or part of transgenic animals comprising human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein, and monoclonal antibodies structurally include heavy chains and light chains.

[0082] In this specification, CDR is the abbreviation of complement determine region, also known as "hypervariable region". LCDR is the abbreviation of light chain hypervariable region, and HCDR is the abbreviation of heavy chain hypervariable region. Generally, the heavy chain and light chain of an antibody have three CDRs, which together constitute the antigen-binding site of Ig. In this site, the antibody can form a precise complement with the antigenic determinant in terms of spatial structure. In the following, the two monoclonal antibodies claimed in this application are used in the analytical technology as "capture antibodies" and "anti-antibodies" to specifically bind to the anti-human TSLP monoclonal antibody, that is, they rely on the specific binding ability of the CDR region defined herein to specific antigenic determinants on the anti-human TSLP monoclonal antibody.

[0083] In this specification, CVR is the abbreviation of "variable domain", which is defined relative to "constant domain". The variable domains of heavy and light chains both include the three "complementarity determining regions" mentioned above.

[0084] In this specification, human TSLP refers to a secretory protein derived from humans. Under the stimulation of allergens, viruses and other factors, the release of hTSLP is located at the top of the inflammatory cascade reaction, upstream of other targets in signal transduction, and plays a core role in regulating type II immune response. hTSLP forms a ternary complex with the thymic stromal lymphocyte receptor TSLPR and IL-7Rα chain on the cell surface, activates JAK1 (through IL-7Rα) and JAK2 (through TSLPR) and STATs, drives the production of downstream IL-4, IL-5, IL-9 and IL-13, and exerts pro-inflammatory and immunomodulatory effects. Monoclonal antibody 6 of the anti-TSLP monoclonal antibody of the present application is a neutralizing active antibody that specifically recognizes anti-human TSLP monoclonal antibodies, and monoclonal antibody 11 of the anti-TSLP monoclonal antibody is a non-neutralizing antibody that specifically recognizes anti-human TSLP monoclonal antibodies. Both can specifically bind to anti-human TSLP monoclonal antibodies and have different binding epitopes.

[0085] In this specification, "identity" refers to the number of identical residues in two sequences when aligned for maximum correspondence in the context of amino acid sequences. There are many different algorithms known in the art that can be used to measure the percentage of amino acid identity (i.e., the Basic Local Alignment Tool or ). Unless otherwise indicated, default parameters for specific programs or algorithms were used.

[0086] In this specification, "anti-human TSLP monoclonal antibody" means a monoclonal antibody that can bind to human TSLP with sufficient affinity, so that the monoclonal antibody can be used to identify / detect human TSLP, and can also be used as an antibody drug, for example, as a drug for treating autoimmune diseases. Therefore, the sandwich ELISA method established below using the kit of the present application can be used as an analytical means for the pharmacokinetic study of such antibody drugs.

[0087] In this specification, "affinity" refers to the strength of the non-covalent interaction 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 the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0088] In this specification, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules, combines free target proteins with target proteins bound to solid phase carriers, and uses special markers to conduct qualitative or quantitative analysis. The principle is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of immune reactions can be determined by the color reaction after adding the substrate, and the depth of the color reaction is proportional to the amount of target antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The sandwich method is also the most commonly used method for detecting antigens.

[0089] In this specification, "standard curve" refers to the linear relationship between the content of the sample to be tested and the measurable value (such as absorbance value) of the analytical method, so as to achieve quantitative analysis of the sample to be tested.

[0090] The present application relates to monoclonal antibody 6 against anti-human TSLP monoclonal antibody and monoclonal antibody 11 against anti-human TSLP monoclonal antibody. Wherein, the monoclonal antibody 6 of the anti-human TSLP monoclonal antibody comprises: a) an antibody heavy chain complementary determining region, which comprises: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has an amino acid sequence of SEQ ID NO.1: STYWIC or has at least 90% identity with SEQ ID NO.1, CDR-H2 has an amino acid sequence of SEQ ID NO.2: CIYAGFTGNTYYASWVNG or has at least 90% identity with SEQ ID NO.2, CDR-H3 has an amino acid sequence of SEQ ID NO.3: NAHDDSGEYIPHNF or has at least 90% identity with SEQ ID NO.3; b) an antibody light chain complementary determining region, which comprises: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has an amino acid sequence of SEQ ID NO.4: QASEDIYSNLA or has at least 90% identity with SEQ ID NO.4 has at least 90% identity, CDR-L2 has an amino acid sequence of SEQ ID NO.5: RASTLAS or has at least 90% identity with SEQ ID NO.5, and CDR-L3 has an amino acid sequence of SEQ ID NO.6: LGVASYISVDGYNA or has at least 90% identity with SEQ ID NO.6.

[0091] In a specific embodiment, the above-mentioned antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: HCVR has an amino acid sequence of SEQ ID NO.7: QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSS or has at least 90% identity with SEQ ID NO.7; LCVR has an amino acid sequence of SEQ IDNO.8: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYR ASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGY NAFGGGTEVVVK or has at least 90% identity with SEQ ID NO.8.

[0092] In a specific embodiment, the above-mentioned antibody comprises a heavy chain and a light chain, wherein:

[0093] The heavy chain amino acid sequence is SEQ ID NO.9:QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGE YIPHNFWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTC SKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG The amino acid sequence of QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or with SEQ ID NO.9 has at least 90% identity; the amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO.10: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO.10.

[0094] The monoclonal antibody 11 of the anti-human TSLP monoclonal antibody comprises: a) an antibody heavy chain complementary determining region, which comprises: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has an amino acid sequence of SEQ ID NO.11: TYYMS or has at least 90% identity with SEQ ID NO.11, CDR-H2 has an amino acid sequence of SEQ ID NO.12: YISYGGTTDYASWAKG or has at least 90% identity with SEQ ID NO.12, and CDR-H3 has an amino acid sequence of SEQ ID NO.13: GYGDGFNP or has at least 90% identity with SEQ ID NO.13; b) an antibody light chain complementary determining region, which comprises: CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 has an amino acid sequence of SEQ ID NO.14: QASQSISTKLA or has at least 90% identity with SEQ ID NO.14 has at least 90% identity, CDR-L2 has an amino acid sequence of SEQ ID NO.15: DASDLAS or has at least 90% identity with SEQ ID NO.15, and CDR-L3 has an amino acid sequence of SEQ ID NO.16: QQGYTSYNVENA or has at least 90% identity with SEQ ID NO.16.

[0095] In a specific embodiment, the above-mentioned antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: HCVR has an amino acid sequence of SEQ ID NO.17: QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYI SYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGD GFNPWGPGTLVTVSS or has at least 90% identity with SEQ ID NO.17; LCVR has an amino acid sequence of SEQ ID NO.18: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYD ASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVK or has at least 90% identity with SEQ ID NO.18.

[0096] In a specific embodiment, the above-mentioned antibody comprises a heavy chain and a light chain, wherein:

[0097] The heavy chain amino acid sequence is SEQ ID NO.19:QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPG TLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPL The amino acid sequence of EPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or with SEQ ID NO.19 has at least 90% identity; the amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO.20:TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO.20.

[0098] In this specification, having at least 90% identity means having at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity.

[0099] The present application relates to a kit for detecting anti-human TSLP monoclonal antibodies, wherein the kit comprises a capture antibody and a biotin-labeled secondary antibody.

[0100] In a specific embodiment, the capture antibody is the monoclonal antibody 6 of the anti-human TSLP monoclonal antibody described above. In a specific embodiment, the second antibody is the monoclonal antibody 11 of the anti-human TSLP monoclonal antibody described above. In a specific embodiment, the antibody to be tested is QX008N, which comprises heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: CDR-H1 of the antibody to be tested QX008N has an amino acid sequence of SEQ ID NO.21: SYYMS, CDR-H2 has an amino acid sequence of SEQ ID NO.22: FISYGGSAYHATWAQG, and CDR-H3 has an amino acid sequence of SEQ ID NO.23: EFRSMTYGAEWGI; CDR-L1 has an amino acid sequence of SEQ ID NO.24: QASESIYDTLA, CDR-L2 has an amino acid sequence of SEQ ID NO.25: SASSLAS, and CDR-L3 has an amino acid sequence of SEQ ID NO.26: QQGYTMPDVDKNP.

[0101] In a specific embodiment, the capture antibody is monoclonal antibody 6 of the anti-human TSLP monoclonal antibody described above; the second antibody is monoclonal antibody 11 of the anti-human TSLP monoclonal antibody described above, and the second antibody is coupled to the Biotin protein and purified by a desalting column to obtain a biotin-labeled second antibody. The antibody to be tested is QX008N, which comprises heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: CDR-H1 of the antibody to be tested QX008N has an amino acid sequence of SEQ ID NO.21: SYYMS, CDR-H2 has an amino acid sequence of SEQ ID NO.22: FISYGGSAYHATWAQG, and CDR-H3 has an amino acid sequence of SEQ ID NO.23: EFRSMTYGAEWGI; CDR-L1 has an amino acid sequence of SEQ ID NO.24: QASESIYDTLA, CDR-L2 has an amino acid sequence of SEQ ID NO.25: SASSLAS, and CDR-L3 has an amino acid sequence of SEQ ID NO.26: QQGYTMPDVDKNP.

[0102] In a specific embodiment, the kit includes a capture antibody and a biotin-labeled second antibody, wherein the capture antibody is monoclonal antibody 6 of anti-human TSLP monoclonal antibody; the second antibody is monoclonal antibody 11 of anti-human TSLP monoclonal antibody, the second antibody is coupled to Biotin protein and purified by a desalting column to obtain a biotin-labeled second antibody. The antibody to be tested is QX008N; the amino acid sequence of the heavy chain variable region thereof is shown in SEQ ID NO.27; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.28.

[0103] SEQ ID NO.27:

[0104] EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWGQGTLVTVSS

[0105] SEQ ID NO.28:

[0106] AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVEIK

[0107] The capture antibody is monoclonal antibody 6 of the anti-human TSLP monoclonal antibody described above, which comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO.7; and the amino acid sequence of the light chain variable region as shown in SEQ ID NO.8.

[0108] The second antibody is the monoclonal antibody 11 of the anti-human TSLP monoclonal antibody described above, which comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO.17; and the amino acid sequence of the light chain variable region as shown in SEQ ID NO.18.

[0109] In a specific embodiment, the kit includes a capture antibody and a biotin-labeled secondary antibody, wherein the capture antibody is monoclonal antibody 6 of anti-human TSLP monoclonal antibody, which comprises a heavy chain amino acid sequence as shown in SEQ ID NO.9; and a light chain amino acid sequence as shown in SEQ ID NO.10. The antibody to be tested is QX008N, which comprises a heavy chain amino acid sequence as shown in SEQ ID NO.29; and a light chain amino acid sequence as shown in SEQ ID NO.30.

[0110] SEQ ID NO.29:

[0111] EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0112] SEQ ID NO.30:

[0113] AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0114] The second antibody is monoclonal antibody 11 of anti-human TSLP monoclonal antibody. The second antibody is coupled with Biotin protein and purified through a desalting column to obtain a biotin-labeled second antibody; the amino acid sequence of the heavy chain is shown in SEQ ID NO.19; and the amino acid sequence of the light chain is shown in SEQ ID NO.20.

[0115] In a specific embodiment, the capture antibody is fixed to a solid support, and the antibody to be detected binds to the capture antibody.

[0116] In a specific embodiment, the kit further comprises: streptavidin labeled with horseradish peroxidase (SA-HRP), washing solution, substrate color developing solution, blocking solution, diluent and stop solution.

[0117] In a specific embodiment, the solid phase carrier is an ELISA plate, preferably a 96-well ELISA plate, specifically a transparent flat-bottom non-sterile 96-well ELISA plate from Thermo. The substrate colorimetric solution is a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution; the washing solution is a mixture of PBS solution and Tween 20 (V:V=100:0.05); the diluent / blocking solution is a mixture of PBS solution, BSA powder, Tween 20 and Proclin 300 (V:W:V:V=200:1:0.1:0.1); the stop solution is a mixture of water and phosphoric acid (V:V=4:1).

[0118] In a specific embodiment, the ELISA kit comprises:

[0119] 1) Solid phase carrier: ELISA plate, 1 piece;

[0120] 2) Capture antibody: monoclonal antibody 6 against human TSLP monoclonal antibody;

[0121] 3) Standard / quality control products of the antibody to be tested, QX008N;

[0122] 4) Second antibody: a biotin-labeled second antibody, wherein the second antibody is monoclonal antibody 11 against anti-human TSLP monoclonal antibody;

[0123] 5) Detection antibody: horseradish peroxidase-labeled streptavidin (SA-HRP);

[0124] 6) Substrate: 3,3',5,5'-tetramethylbenzidine (TMB), mix equal volumes of solution A and solution B before use;

[0125] 7) Capture antibody coating diluent: phosphate buffered saline (1×PBS);

[0126] 8) Washing solution: a mixture of PBS solution and Tween 20 (V:V = 100:0.05);

[0127] 9) Dilution / blocking solution: a mixed solution of PBS solution, BSA powder, Tween 20 and Proclin 300 (V:W:V:V=200:1:0.1:0.1);

[0128] 10) Stop solution: a mixture of water and phosphoric acid (V:V = 4:1).

[0129] In a specific embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit. Preferably, the kit is a modified sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0130] The present application also relates to a method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies, wherein a capture antibody and a biotin-labeled second antibody are used to perform ELISA detection using a modified sandwich enzyme-linked immunosorbent assay.

[0131] In a specific embodiment, the capture antibody is the monoclonal antibody 6 of the anti-human TSLP monoclonal antibody described above. In a specific embodiment, the second antibody is the monoclonal antibody 11 of the anti-human TSLP monoclonal antibody described above. In a specific embodiment, the antibody to be tested is QX008N, which comprises heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: CDR-H1 of the antibody to be tested QX008N has an amino acid sequence of SEQ ID NO.21: SYYMS, CDR-H2 has an amino acid sequence of SEQ ID NO.22: FISYGGSAYHATWAQG, and CDR-H3 has an amino acid sequence of SEQ ID NO.23: EFRSMTYGAEWGI; CDR-L1 has an amino acid sequence of SEQ ID NO.24: QASESIYDTLA, CDR-L2 has an amino acid sequence of SEQ ID NO.25: SASSLAS, and CDR-L3 has an amino acid sequence of SEQ ID NO.26: QQGYTMPDVDKNP.

[0132] In a specific embodiment, monoclonal antibody 6 of anti-human TSLP monoclonal antibody is used as capture antibody to bind to the antibody to be tested (such as QX008N), and a biotin-labeled second antibody is used as an anti-antibody to bind to the antibody to be tested, and the second antibody is a monoclonal antibody 11 of anti-human TSLP monoclonal antibody. ELISA detection is performed using a modified sandwich enzyme-linked immunosorbent assay, and the method includes the following steps: fixing the capture antibody to a solid phase carrier and coating and blocking it; adding the antibody to be tested for incubation; then adding the biotin-labeled second antibody for incubation; adding diluted horseradish peroxidase-labeled streptavidin (SA-HRP) for incubation; adding a substrate colorimetric solution, incubating in the dark, then adding a stop solution, measuring the OD value, and preparing a standard curve; substituting the OD value of the sample to be tested into the equation to calculate the content of the anti-human TSLP monoclonal antibody in the sample to be tested.

[0133] In one embodiment, the ELISA kit described in the present application can be used to quantitatively detect the content of anti-human TSLP monoclonal antibodies in a sample, comprising the following steps:

[0134] (1) Coating: Dilute the capture antibody with 1× PBS to a coating working solution of 1 μg / ml, then add 100 μl / well to the ELISA plate (96-well Thermo ELISA plate) and coat overnight at 2-8°C;

[0135] (2) Blocking: Discard the coating working solution in the ELISA plate, wash the plate three times with washing solution (phosphate buffered saline (PBS) containing 0.05% Tween 20), then add blocking solution (phosphate buffered saline (PBS) containing 0.5% BSA powder, 0.05% Tween 20 and 0.05% Proclin 300) at a volume of 200 μl / well, and block at room temperature for about 2 h;

[0136] (3) Sample addition: The standard and quality control samples were uniformly diluted 20 times with diluent at the minimum dilution multiple (MRD) for pretreatment; the blocking solution in the ELISA plate was discarded, the plate was washed 3 times with washing solution, and then the pretreated standard and quality control samples were added to the ELISA plate at a volume of 100 μl / well and incubated at room temperature for 2 h; the standard and quality control samples were both QX008N;

[0137] (4) Add biotin-labeled secondary antibody: discard the standard and quality control samples in the ELISA plate, wash the plate three times with washing solution; dilute the biotin-labeled secondary antibody to a working solution of 20 ng / ml with diluent, then add 100 μl / well to the ELISA plate and incubate at room temperature in the dark for about 1 h;

[0138] (5) Adding detection antibody: discard the biotin-labeled secondary antibody in the ELISA plate, wash the plate three times with washing solution, dilute the horseradish peroxidase-labeled streptavidin (SA-HRP) with diluent to 33.3 ng / ml of detection antibody working solution, and then add 100 μl / well of the ELISA plate and incubate at room temperature in the dark for about 1 h;

[0139] (6) Color development: Mix the A solution and B solution of the substrate (3,3',5,5'-tetramethylbenzidine (TMB)) in equal volumes as required; discard the detection antibody working solution in the ELISA plate, wash the plate three times with washing solution, and then add the substrate at a volume of 100 μl / well, and color develop at room temperature in the dark for 10-15 min;

[0140] (7) Termination and reading: Add 100 μl / well of the stop solution to the ELISA plate to terminate the reaction. Then use a microplate reader to measure the OD value of each well in the ELISA plate at a wavelength of 450 nm. Fit the standard curve based on the OD value of the standard. Substitute the OD value of the quality control sample into the equation to calculate the back-calculated concentration and recovery rate of the quality control sample.

[0141] Example

[0142] Example 1 Preparation and purification of capture antibody and secondary antibody

[0143] Rabbits were immunized using anti-human TSLP monoclonal antibody (F(ab)2) as an antigen, and neutralizing and / or non-neutralizing rabbit monoclonal antibodies that could bind to the anti-human TSLP monoclonal antibody and did not cross-react with other isotype human IgG were screened out using B cell cloning technology (i.e., capture antibodies and secondary antibodies were obtained). Sequencing was performed using conventional methods in the art, and the CDR sequences of the capture antibodies were SEQ ID NOs.1-6, the sequence of the heavy chain variable region was SEQ ID NO.7, the sequence of the light chain variable region was SEQ ID NO.8, the heavy chain sequence was SEQ ID NO.9, and the light chain sequence was SEQ ID NO.10; the CDR sequences of the second antibody were SEQ ID NOs.11-16, the sequence of the heavy chain variable region was SEQ ID NO.17, the sequence of the light chain variable region was SEQ ID NO.18, the heavy chain sequence was SEQ ID NO.19, and the light chain sequence was SEQ ID NO.20.

[0144] Example 2 Biotin Labeling of Secondary Antibody

[0145] Sulfo-NHS-LC-biotin can react with the primary amine (-NH2) on the surface of the antibody protein to form a stable amide bond without affecting the activity of the antibody itself. Sulfo-NHS-LC-biotin is mixed and incubated with the second antibody, and then purified by a desalting column to remove unbound biotin to obtain a biotinylated second antibody. The CDR sequences of the second antibody are SEQ ID NO.11-16, the sequence of the heavy chain variable region is SEQ ID NO.17, the sequence of the light chain variable region is SEQ ID NO.18, the heavy chain sequence is SEQ ID NO.19, and the light chain sequence is SEQ ID NO.20. Biotin-labeled antibodies are convenient for detection using streptavidin and have higher sensitivity and specificity.

[0146] Example 3 Performance evaluation of capture antibody and secondary antibody

[0147] 1. Binding activity identification

[0148] The anti-human TSLP monoclonal antibody and human IgG disclosed in CN113683694B were used as target proteins to coat the ELISA plate (purchased from Thermo), and after blocking, the rabbit monoclonal antibody prepared in Example 1 was added in gradient dilutions and incubated at room temperature for 2 hours. After washing the plate, goat anti-rabbit IgG antibody labeled with horseradish peroxidase was added and incubated at room temperature for 1 hour. Finally, the plate was washed and color was developed, and the OD was read. 450 The absorbance at Figure 1A and Figure 1B As shown, Figure 1A 1 is a graph showing the binding result of the rabbit monoclonal antibody prepared in Example 1 and the anti-human TSLP monoclonal antibody. Figure 1B This is a graph showing the binding results of the rabbit monoclonal antibody prepared in Example 1 to human IgG.

[0149] from Figure 1A and Figure 1B It can be seen that the rabbit monoclonal antibody has good binding activity with the anti-human TSLP monoclonal antibody and does not cross-react with the same type of IgG.

[0150] 2. Neutralization activity identification

[0151] Human TSLP was used as an antigen to coat the ELISA plate. After blocking, 100 ng / ml biotin-labeled anti-human TSLP monoclonal antibody disclosed in CN113683694B and a gradient-diluted mixture of rabbit monoclonal antibodies prepared in Example 1 were added and incubated at room temperature for 2 hours. After washing the plate, horseradish peroxidase-labeled streptavidin antibody was added and incubated at room temperature for 1 hour. Finally, the plate was washed and color was developed, and the OD was read. 450 The absorbance at Figure 2 As shown, Figure 2 This is a graph showing the result of the rabbit monoclonal antibody prepared in Example 1 blocking the binding of the anti-human TSLP monoclonal antibody to human TSLP.

[0152] from Figure 2 It can be seen that monoclonal antibody 6 of anti-anti-human TSLP monoclonal antibody blocks the binding between anti-human TSLP monoclonal antibody and its target human TSLP, and has neutralizing activity; monoclonal antibody 11 of anti-anti-human TSLP monoclonal antibody does not block the binding between anti-human TSLP monoclonal antibody and its target human TSLP, and therefore does not have neutralizing activity.

[0153] Example 4 Identification of Binding Epitopes of Capture Antibody and Secondary Antibody

[0154] The rabbit monoclonal antibody prepared in Example 1 was used as a coating reagent to coat the ELISA plate (purchased from Thermo), and the plate was washed and blocked overnight. At the same time, an equal volume of a fixed concentration of biotin-labeled anti-human TSLP monoclonal antibody and a gradient dilution of the rabbit monoclonal antibody prepared in Example 1 were added to the ELISA plate and incubated for 2 hours in the dark. After washing the plate, horseradish peroxidase-labeled streptavidin (SA-HRP) was added and incubated at room temperature for 1 hour. Finally, the plate was washed for color development and the OD was read. 450 The absorbance at Figure 3A and Figure 3B As shown. Among them, Figure 3A This is a diagram showing the result of monoclonal antibody 6 against anti-human TSLP monoclonal antibody prepared in solid phase Example 1 competing with monoclonal antibody 11 against anti-human TSLP monoclonal antibody for binding to anti-human TSLP monoclonal antibody; Figure 3B This is a diagram showing the result of monoclonal antibody 11 against anti-human TSLP monoclonal antibody prepared in solid phase Example 1 competing with monoclonal antibody 6 against anti-anti-human TSLP monoclonal antibody for binding to anti-human TSLP monoclonal antibody.

[0155] from Figure 3A and Figure 3B It can be seen that the monoclonal antibody 6 of the anti-anti-human TSLP monoclonal antibody and the monoclonal antibody 11 of the anti-anti-human TSLP monoclonal antibody prepared in Example 1 do not overlap in binding epitopes with the anti-human TSLP monoclonal antibody.

[0156] Example 5: Constructing an ELISA kit using the "antibody pair" prepared in Example 1

[0157] The capture antibody is monoclonal antibody 6 of anti-human TSLP monoclonal antibody and is fixed on the ELISA plate; the second antibody is monoclonal antibody 11 of anti-human TSLP monoclonal antibody and is biotinylated; the antibody to be tested can bind to the coated capture antibody and the biotinylated second antibody added later respectively; the preparation and performance determination of the capture antibody and the biotinylated second antibody are shown in Examples 1-4; horseradish peroxidase-labeled streptavidin (SA-HRP) is used as the detection antibody, and Figure 4 The ELISA kit was constructed according to the method shown.

[0158] Experimental process:

[0159] (1) Coating: Anti-human TSLP monoclonal antibody monoclonal antibody 6 was diluted with 1× PBS to a coating working solution of 1 μg / ml, and then added to a 96-well ELISA plate (purchased from Thermo) at a dosage of 100 μl / well and coated overnight at 2-8°C;

[0160] (2) Blocking: Discard the coating working solution in the ELISA plate, wash the plate three times with washing solution (phosphate buffered saline (PBS) containing 0.05% Tween 20), then add blocking solution at a volume of 200 μl / well and block at room temperature for about 2 h;

[0161] (3) Sample loading: The standard and quality control samples were uniformly pre-treated with MRD dilution 20 times with diluent, the blocking solution in the ELISA plate was discarded, the plate was washed 3 times with washing solution, and then the pre-treated standard and quality control working solutions were added to the ELISA plate at a volume of 100 μl / well and incubated at room temperature for 2 h; the standard and quality control samples were both the test antibody QX008N;

[0162] (4) Adding secondary antibody: discard the standard and quality control working solutions in the ELISA plate, wash the plate three times with washing solution, dilute the "secondary antibody" anti-human TSLP monoclonal antibody monoclonal antibody 11 prepared in Example 1 with diluent to a secondary antibody working solution of 20 ng / ml, and then add 100 μl / well to the ELISA plate, and incubate at room temperature in the dark for about 1 hour;

[0163] (5) Adding detection antibody: discard the secondary antibody working solution in the ELISA plate, wash the plate three times with washing solution, dilute the horseradish peroxidase-labeled streptavidin (SA-HRP) with diluent to 33.3 ng / ml detection antibody working solution, then add 100 μl / well to the ELISA plate and incubate at room temperature in the dark for about 1 h;

[0164] (6) Color development: Mix the A solution and B solution of the substrate (3,3',5,5'-tetramethylbenzidine (TMB)) in equal volumes as needed; discard the detection antibody working solution in the ELISA plate, wash the plate three times with washing solution, and then add the substrate at a volume of 100 μl / well, and color develop at room temperature in the dark for 10-15 min;

[0165] (7) Termination and reading: Add 100 μl / well of the stop solution to the ELISA plate to terminate the reaction. Then use a microplate reader to measure the OD value of each well in the ELISA plate at a wavelength of 450 nm. Fit the standard curve based on the OD value of the standard. Substitute the OD value of the quality control sample into the equation to calculate the back-calculated concentration and recovery rate of the quality control sample.

[0166] Among them, the reagent information is:

[0167] Phosphate buffer (1×PBS): 23.48 g PBS powder was dissolved in 2000 ml ultrapure water;

[0168] Washing solution: add 500 μl Tween 20 per 1000 ml 1× PBS;

[0169] Diluent / blocking solution: Weigh 2.5 g BSA powder, dissolve it in 500 ml 1×PBS, add 250 μl Tween20 and 250 μl Proclin 300, and mix thoroughly;

[0170] TMB colorimetric solution: Take equal volumes of solution A and solution B, mix thoroughly after returning to room temperature, and use immediately after preparation; protect from light;

[0171] Stop solution: 100 ml H3PO4, added to 400 ml ultrapure water;

[0172] Example 6 Establishment of an ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum

[0173] 1. Establishment of standard curve

[0174] The anti-human TSLP monoclonal antibody was diluted with mixed human serum (10 healthy individuals blank serum equal volume mixed) to 3.91, 7.81, 15.625, 31.25, 62.5, 125, 250, 500, 1000 ng / ml, and the ELISA experiment was performed according to Example 5 under the determined optimal experimental conditions. The concentration of the detected anti-human TSLP monoclonal antibody was taken as the horizontal axis, and the OD 450 Draw a four-parameter curve with the value as the ordinate, such as Figure 5 The recovery rate was calculated and the results are shown in Table 1.

[0175] Table 1: Recovery statistics at each concentration point established by the method standard curve

[0176]

[0177] Curve equation: Fit:y=(0.046-3.421) / (1+(x / 208.7)^1.238)+3.421

[0178] Note: * marks the anchor point, and the recovery rate is not required

[0179] from Figure 5 It can be seen that the anti-human TSLP monoclonal antibody concentration is in the range of 7.81 to 1000 ng / ml, and the curve has a good recovery rate.

[0180] As can be seen from Table 1, the concentrations of the standard curve of the enzyme-linked immunosorbent assay method established in this application are 3.91 (lower anchor point), 7.81, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 ng / ml. The standard curve range is 7.81-1000 ng / ml, and 3.91 ng / ml is used as an anchor point to improve the fitting of the standard curve (recovery rate is not required).

[0181] 2. Methodological research

[0182] 5 2.1 Quantitative range

[0183] Different experimenters evaluated at least three independent batches of standard curves on different days using the established ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum. The results are shown in Table 2. It can be seen from Table 2 that, except for the anchor point, at the LLOQ and ULOQ concentration levels:

[0184] The accuracy RE% is in the range of -19.4% to 5.6%, and the precision CV% is in the range of 1.0% to 21.6%. 101000ng / ml and 10ng / ml can be used as the upper and lower limits of quantitation of the method.

[0185] Table 2: Method quantitative range study

[0186]

[0187] 2.2 Accuracy and precision

[0188] ELISA for quantitative detection of anti-human TSLP monoclonal antibody in serum

[0189] The method was evaluated for at least three independent batches of quality control samples (including LLOQ, LQC, MQC, 15HQC, and ULOQ), and the results are shown in Table 3.

[0190] As can be seen from Table 3, at the LLOQ and ULOQ concentration levels: the inter-batch accuracy RE% is in the range of -17.3% to -3.0%, and the precision CV% is in the range of 7.6% to 11.8%. At the QC concentration level: the inter-batch accuracy RE% is in the range of -5.9% to -1.3%, and the precision CV% is in the range of 8.1% to 11.0%.

[0191] 20Table 3: Method accuracy and precision study

[0192]

[0193]

[0194] 2.3 Selectivity

[0195] The established ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum was used to investigate the addition of anti-human TSLP monoclonal antibodies at LLOQ concentration levels to blank matrices from at least 10 healthy individuals. The results are shown in Table 4.

[0196] As can be seen from Table 4, the blank sample response values ​​of the 12 individual sources were all lower than the LLOQ, and the intra-batch accuracy RE% of the LLOQ concentration level quality control samples of the 11 individual sources was in the range of 8.5% to 11.8%, meeting the requirement of at least 8 / 10 individual selectivity passing.

[0197] Table 4: Method selectivity studies

[0198]

[0199] 2.4 Specificity

[0200] The established ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum was used to investigate by adding gradient concentration levels of interfering substances (including human TSLP and polyclonal antibodies against anti-human TSLP monoclonal antibodies) at blank samples, LLOQ and ULOQ concentration levels. The results are shown in Table 5.

[0201] As can be seen from Table 5, under the ULOQ concentration condition, 1 μg / ml of polyclonal antibody against anti-human TSLP monoclonal antibody and 1 ng / ml of human TSLP target antigen can be tolerated; under the LLOQ concentration condition, 0.1 μg / ml of polyclonal antibody against anti-human TSLP monoclonal antibody and 100 pg / ml of human TSLP target antigen can be tolerated.

[0202] Table 5: Method specificity studies

[0203]

[0204] Example 7: Determination of anti-human TSLP monoclonal antibody content in serum using the ELISA kit constructed in Example 5 and the method established in Example 6

[0205] Use diluent to pre-treat the anti-human TSLP monoclonal antibody sample to be tested by MRD dilution 20 times, and then use 5% serum diluent (the diluent contains 5% human mixed serum) to dilute it by an appropriate multiple to fall within the quantitative range of the standard curve; use the ELISA kit constructed in Example 5 and the method established in Example 6 to determine the content of anti-human TSLP monoclonal antibodies in serum, which can accurately measure the content of anti-human TSLP monoclonal antibodies in serum. In summary, the enzyme-linked immunosorbent assay method provided in the present application for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum is a method of forming a solid phase carrier by coating an ELISA plate with a capture antibody, combining with the anti-human TSLP monoclonal antibody in the biological sample to be tested, and then forming a solid phase capture antibody-antibody-second antibody-enzyme-labeled antibody complex through a specific second antibody and a detection antibody. Since the antibody pair (capture antibody and second antibody) obtained by screening has a strong specificity, the method established in the present application has a strong specificity, high sensitivity, and good precision and accuracy.

[0206] The above is only a preferred embodiment of the present application and does not limit the present application in any form. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the content of the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A monoclonal antibody against anti-thymic stromal lymphopoietin monoclonal antibody, wherein: The monoclonal antibody comprises: a) an antibody heavy chain complementarity determining region, comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has an amino acid sequence of SEQ ID NO.1: STYWIC, CDR-H2 has an amino acid sequence of SEQ ID NO.2:CIYAGFTGNTYYASWVNG, CDR-H3 has an amino acid sequence of SEQ ID NO.3: NAHDDSGEYIPHNF; b) an antibody light chain complementary determining region, comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has an amino acid sequence of SEQ ID NO.4: QASEDIYSNLA, CDR-L2 has an amino acid sequence of SEQ ID NO.5: RASTLAS, CDR-L3 has an amino acid sequence of SEQ ID NO.6: LGVASYISVDGYNA.

2. The monoclonal antibody according to claim 1, wherein The monoclonal 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.7: The amino acid sequence of QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAP GKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAA DTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSS; LCVR has the sequence SEQ ID NO.8: The amino acid sequence of ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISV DGYNAFGGGTEVVVK.

3. The monoclonal antibody according to claim 1 or 2, wherein The monoclonal antibody comprises a heavy chain and a light chain, wherein: The heavy chain amino acid sequence is SEQ ID NO.9: QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIP HNFWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCS The amino acid sequence of KPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; The amino acid sequence of the light chain is SEQ ID NO.10: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVV Amino acid sequence of KGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

4. A monoclonal antibody against anti-thymic stromal lymphopoietin monoclonal antibody, wherein: The monoclonal antibody comprises: a) an antibody heavy chain complementarity determining region, comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has an amino acid sequence of SEQ ID NO.11: TYYMS, CDR-H2 has an amino acid sequence of SEQ ID NO.12:YISYGGTTDYASWAKG or SEQ ID NO., CDR-H3 has an amino acid sequence of SEQ ID NO.13: GYGDGFNP; b) an antibody light chain complementary determining region, comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has an amino acid sequence of SEQ ID NO.14: QASQSISTKLA, CDR-L2 has an amino acid sequence of SEQ ID NO.15: DASDLAS, CDR-L3 has the amino acid sequence of SEQ ID NO.16:QQGYTSYNVENA.

5. The monoclonal antibody according to claim 4, wherein The monoclonal 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.17: The amino acid sequence of QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARG YGDGFNPWGPGTLVTVSS; LCVR has the sequence SEQ ID NO.18: The amino acid sequence of TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVK.

6. The monoclonal antibody according to claim 4 or 5, wherein The monoclonal antibody comprises a heavy chain and a light chain, wherein: The heavy chain amino acid sequence is SEQ ID NO.19: QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPGTLVT VSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPP The amino acid sequence of ELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; The amino acid sequence of the light chain is SEQ ID NO.20: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVK Amino acid sequence of GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

7. A kit for detecting anti-thymic stromal lymphopoietin monoclonal antibody, wherein: The kit comprises a capture antibody and a biotin-labeled secondary antibody, wherein the capture antibody is a monoclonal antibody of the anti-thymic stromal lymphopoietin monoclonal antibody according to any one of claims 1 to 3.

8. The kit according to claim 7, wherein The second antibody is the monoclonal antibody against anti-thymic stromal lymphopoietin monoclonal antibody according to any one of claims 4 to 6.

9. The kit according to claim 7 or 8, wherein The antibody to be tested comprises heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: The CDR-H1 of the antibody to be tested has an amino acid sequence of SEQ ID NO.21: SYYMS, CDR-H2 has an amino acid sequence of SEQ ID NO.22: FISYGGSAYHATWAQG, and CDR-H3 has an amino acid sequence of SEQ ID NO.23: EFRSMTYGAEWGI; CDR-L1 has an amino acid sequence of SEQ ID NO.24: QASESIYDTLA, CDR-L2 has an amino acid sequence of SEQ ID NO.25: SASSLAS, and CDR-L3 has an amino acid sequence of SEQ ID NO.26: QQGYTMPDVDKNP.

10. The kit according to any one of claims 7 to 9, wherein The second antibody is coupled to the Biotin protein and purified through a desalting column to obtain a biotin-labeled second antibody.

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