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

Abstract

The application discloses two new anti-anti-thymic stromal lymphopoietin (TSLP) monoclonal antibodies, and an ELISA kit comprising the antibodies and a detection method thereof. The antibodies disclosed by the application comprise antibody heavy chain complementarity determining regions and antibody light chain complementarity determining regions, and the ELISA kit comprises two antibodies. The capture antibody, specifically, the anti-anti-thymic stromal lymphopoietin (TSLP) monoclonal antibody, is used as coating; the second antibody, specifically, the biotin-labeled anti-anti-thymic stromal lymphopoietin (TSLP) monoclonal antibody, is used as binding with the capture antibody-antibody complex. The kit can be used for successfully quantitatively detecting the anti-thymic stromal lymphopoietin (TSLP) monoclonal antibody, has high detection sensitivity and good detection specificity, and has a good development and application prospect.

Description

[0001] This case is a divisional application of the application filed on November 25, 2024, entitled "Monoclonal Antibody Against Anti-TSLP, Kit Containing the Same, and Detection Method Using the Same", with application number 202411697116.5. Technical Field

[0002] This application belongs to the field of biotechnology, specifically relating to two monoclonal antibodies against anti-thymocyte stromal lymphopoietin (TSLP) monoclonal antibodies, an ELISA kit for quantitative detection of anti-thymocyte stromal lymphopoietin (TSLP) monoclonal antibodies using these two antibodies, and a method for quantitative detection using the kit. Background Technology

[0003] Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine primarily produced by human epithelial cells. It acts on dendritic cells, T cells, B cells, and innate immune cells, playing a central role in regulating type 2 immune responses. TSLP forms a ternary complex with the thymic stromal lymphocyte receptor TSLPR and IL-7Rα chains on the surface of these cells, activating JAK1 (via IL-7Rα) and JAK2 (via TSLPR) and STATs, driving the production of downstream IL-4, IL-5, IL-9, and IL-13, thereby exerting a pro-inflammatory effect. Studies have found that the development and progression of asthma, chronic obstructive pulmonary disease (COPD), and other allergic diseases are closely related to TSLP levels. TSLP expression in the airways of asthmatic patients is higher than in healthy controls, and its level is positively correlated with the expression of Th2 cytokines and chemokines, as well as disease severity. TSLP participates in airway inflammation, airway hyperresponsiveness (AHR), and the induction of acute asthma exacerbations by inducing type 2 inflammatory responses. It can also induce AHR by directly acting on airway smooth muscle and mast cells without relying on type 2 inflammation. Therefore, inhibiting TSLP may have therapeutic effects on asthma, chronic obstructive pulmonary disease, and other allergic diseases. The anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody is a humanized IgG1κ type monoclonal antibody that specifically binds to human TSLP. Its parent antibody was obtained by screening New Zealand rabbit B cells immunized with human TSLP. The parent rabbit antibody was then humanized to obtain the anti-human TSLP monoclonal antibody.

[0004] Currently, the marketed drug targeting the same target, Tezepelumab, is a fully humanized IgG2λ monoclonal antibody. Phase II and Phase III clinical data show that Tezepelumab significantly reduces the annualized asthma exacerbation rate (AAER) regardless of baseline eosinophil levels or FeNO levels, demonstrating significant efficacy in a broad range of patients with severe uncontrolled asthma. Jiangsu Quanxin Biopharmaceutical Co., Ltd. has developed an anti-human TSLP monoclonal antibody with the trade code QX008N for drug use, which is described in Chinese patent document CN 113683694 B.

[0005] For drugs, pharmacokinetics applies the principles of kinetics and mathematical methods to quantitatively describe the dynamic changes of drugs in the body, studying the absorption, distribution, metabolism, and excretion processes of drugs entering the human body via different routes. Therefore, pharmacokinetic studies are beneficial for evaluating drug safety and efficacy. Commonly used analytical methods for biological samples include chromatography and immunoassay. Chromatography has lower sensitivity, a cumbersome experimental process, and unstable recovery rates. Immunoassay, based on specific antigen-antibody reactions, is currently the most commonly used method for detecting non-clinical and clinical biological samples. Because anti-human TSLP monoclonal antibodies are large protein molecules, they are suitable for detection using enzyme-linked immunosorbent assay (ELISA). Anti-human TSLP monoclonal antibodies bind to both a capture antibody and a biotin-labeled secondary antibody. Furthermore, horseradish peroxidase-labeled streptavidin and biotin have high specificity, significantly reducing interference from irrelevant proteins in serum, amplifying the detection signal, and improving detection sensitivity.

[0006] Sandwich ELISA involves first binding a capture antibody to an ELISA plate, then adding the target antibody which specifically binds to the capture antibody, followed by the addition of a specific secondary antibody, and finally adding an enzyme-labeled detection antibody, which then catalyzes a colorimetric reaction using substrate catalysis. In this way, the target antibody specifically binds to the capture antibody and the secondary antibody with different binding epitopes. Furthermore, the binding of horseradish peroxidase-labeled streptavidin to the biotin-labeled secondary antibody not only provides multi-stage amplification but also offers advantages such as high specificity, high sensitivity, and stability. Summary of the Invention

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

[0008] Specifically, this application relates to the following:

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

[0010] a) Antibody heavy chain complementarity-determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein:

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

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

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

[0014] b) Antibody light chain complementarity-determining regions, which include: CDR-L1, CDR-L2, and CDR-L3, wherein:

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

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

[0017] CDR-L3 has the 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 claim 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 has at least 90% identity with SEQ ID NO. 7;

[0021] LCVR has the amino acid sequence of SEQ ID NO.8: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYR ASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGY NAFGGGTEVVVK or has at least 90% identity with SEQ ID NO.8.

[0022] 3. The monoclonal antibody according to claim 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 has at least 90% identity with SEQ ID NO. 9;

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

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

[0026] a) Antibody heavy chain complementarity-determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein:

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

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

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

[0030] b) Antibody light chain complementarity-determining regions, which include: CDR-L1, CDR-L2, and CDR-L3, wherein:

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

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

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

[0034] 5. 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:

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

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

[0037] LCVR has an amino acid sequence of SEQ ID NO.18:TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENA FGGGTEVVVK or has at least 90% identity with SEQ ID NO.18.

[0038] 6. The monoclonal antibody according to claim 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 The amino acid sequence of PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK has at least 90% identity with SEQ ID NO. 19;

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

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

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

[0043] 9. As described in item 8, wherein the antibody to be tested is QX008N.

[0044] 10. The use according to item 8, wherein the capture antibody is immobilized on a solid-phase support, and the antibody to be tested binds to the capture antibody.

[0045] 11. The use according to item 10, wherein the solid-phase carrier is an enzyme-linked immunosorbent assay (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 chromogenic solution, blocking solution, sample dilution solution, 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 monoclonal antibodies against human thymic stromal lymphopoietin (TSLP), characterized in that the kit comprises a capture antibody and a biotin-labeled secondary antibody.

[0050] The capture antibody is any one of the monoclonal antibodies against anti-human thymic stromal lymphopoietin monoclonal antibody as described in items 1-3;

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

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

[0053] The second antibody is a monoclonal antibody against the anti-human thymic stromal lymphopoietin monoclonal antibody as described in any one of items 4-6;

[0054] The second antibody was conjugated with biotin protein and purified by a desalting column to obtain a biotin-labeled second antibody.

[0055] 16. The kit according to claim 15, wherein the capture antibody is immobilized on a solid-phase support, and the test antibody binds to the capture antibody.

[0056] 17. The kit according to item 16, wherein the solid-phase carrier is an enzyme-linked immunosorbent assay (ELISA) plate.

[0057] 18. The kit according to any one of claims 15 to 17, wherein the kit further comprises: horseradish peroxidase-labeled streptavidin (SA-HRP), washing buffer, substrate chromogenic solution, diluent, blocking solution, and stop solution; preferably, the washing buffer is a mixture of PBS solution and Tween 20 (V:V = 100:0.05), the substrate chromogenic solution is TMB chromogenic solution, the stop solution is a mixture of water and phosphate (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 monoclonal antibody against human thymic stromal lymphopoietin, wherein the capture antibody and biotin-labeled secondary antibody described in item 15 are used, and the detection is performed by a modified sandwich enzyme-linked immunosorbent assay (ELISA).

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

[0062] The captured antibodies were immobilized onto a solid-phase carrier and then coated and blocked.

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

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

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

[0066] The content of anti-human TSLP monoclonal antibody in the sample is calculated by substituting the OD value of the sample into the equation.

[0067] In this 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 (ELISA) method for quantitatively detecting the content of anti-human TSLP monoclonal antibodies in serum was established using rabbit anti-human TSLP monoclonal antibody as the capture antibody and secondary antibody. Second, the method established in this application has a wide effective quantitative range of 10–1000 ng / ml, with a lower limit of quantification (LOQ) of 10 ng / ml for anti-human TSLP monoclonal antibodies, and high sensitivity. Third, the method established in this application has good reproducibility, with an inter-plate repeatability coefficient of variation of 7.6%–11.8%, less than 20%. Fourth, the method established in this application has good individual selectivity, with 11 / 12 individuals meeting the acceptable range of accuracy and precision at the LQ concentration level. Fifth, this 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 assessing their safety and efficacy. Attached Figure Description

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

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

[0072] Figure 2 This is a graph showing the results of the neutralizing activity assessment 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 This is a schematic diagram of the enzyme-linked immunosorbent assay (ELISA) method of this application.

[0076] Figure 5 This is a graph of the four-parameter fitting curve for the standard curve. Detailed Implementation

[0077] The present application will now be described in further detail with reference to specific embodiments. The embodiments 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 terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0079] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and 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 group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles), which are typically present in trace amounts. Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to 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 containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies, which structurally comprise heavy and light chains, are described herein.

[0082] In this specification, CDR stands for complement determine region, also known as "hypervariant region." LCDR stands for light chain hypervariant region, and HCDR stands for heavy chain hypervariant region. Generally, both the heavy and light chains of an antibody have three CDRs, which together form the antigen-binding site of Ig. At this site, the antibody can form precise complementarity with the antigenic determinant in its spatial structure. In the following description, the specific binding of the two monoclonal antibodies claimed in this application to the anti-human TSLP monoclonal antibody in the analytical technique relies on the specific binding ability of the CDR regions defined here to the specific antigenic determinant on the anti-human TSLP monoclonal antibody.

[0083] In this specification, CVR is an abbreviation for "variable field," which is defined in contrast to "constant field." The variable fields of both heavy and light chains include the three aforementioned "complementary determination regions."

[0084] In this specification, human TSLP refers to a secretory protein derived from humans. Under stimulation by allergens, viruses, and other factors, the release of hTSLP is at the top of the inflammatory cascade, upstream of other targets in signal transduction, and plays a central role in regulating type II immune responses. hTSLP forms a ternary complex with the thymic stromal lymphocyte receptor TSLPR and the IL-7Rα chain on the cell surface, activating JAK1 (via IL-7Rα) and JAK2 (via TSLPR) and STATs, driving the production of downstream IL-4, IL-5, IL-9, and IL-13, thus exerting pro-inflammatory and immunomodulatory effects. The monoclonal antibody 6 of this application is a neutralizing antibody that specifically recognizes anti-human TSLP monoclonal antibodies, and the monoclonal antibody 11 is a non-neutralizing antibody that specifically recognizes anti-human TSLP monoclonal antibodies. Both can specifically bind to anti-human TSLP monoclonal antibodies, but their binding epitopes are different.

[0085] In this specification, "identity" refers to, in the context of amino acid sequences, the number of identical residues in two sequences when aligned for maximum correspondence. Many different algorithms known in the art exist for measuring the percentage of amino acid identity (i.e., the Basic Local Alignment Tool or...). Unless otherwise specified, use the default parameters for the specific program or algorithm.

[0086] In this specification, "anti-human TSLP monoclonal antibody" refers to a monoclonal antibody that binds to human TSLP with sufficient affinity, enabling it to be used for the identification / detection of human TSLP and as an antibody drug, for example, as a treatment for autoimmune diseases. Therefore, the sandwich ELISA method established below using the kit of this application can be used as an analytical tool for pharmacokinetic studies 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 stated, "binding affinity" as used herein refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using common methods known in the art.

[0088] In this specification, "Enzyme-Linked Immunosorbent Assay (ELISA)" refers to a detection method that utilizes the specific binding ability of antibody molecules to antigen molecules to bind free target proteins and target proteins bound to a solid-phase carrier, and then uses special labels for qualitative or quantitative analysis. The principle is as follows: antigens or antibodies can be physically adsorbed onto a solid-phase surface while maintaining their immunological activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds, while maintaining their respective immunological or enzymatic activities; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of an immune reaction can be determined by the color reaction after the addition of a substrate, and the intensity of the color reaction is directly proportional to the amount of the target antigen or antibody in the sample. Various types of detection methods can be designed depending on the substance to be detected and the available detection conditions; the sandwich method is also the most commonly used method for detecting antigens.

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

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

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

[0092] In one specific embodiment, the 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, which is an anti-human TSLP monoclonal antibody, comprises: a) an antibody heavy chain complementarity-determining region comprising: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO.11:TYYMS or has at least 90% identity with SEQ ID NO.11; CDR-H2 has the amino acid sequence of SEQ ID NO.12:YISYGGTTDYASWAKG or has at least 90% identity with SEQ ID NO.12; and CDR-H3 has the 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 complementarity-determining region comprising: CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO.14:QASQSISTKLA or has at least 90% identity with SEQ ID NO.13. NO.14 has at least 90% identity, CDR-L2 has the amino acid sequence of SEQ ID NO.15: DASDLAS or has at least 90% identity with SEQ ID NO.15, and CDR-L3 has the amino acid sequence of SEQ ID NO.16: QQGYTSYNVENA or has at least 90% identity with SEQ ID NO.16.

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

[0096] In one specific embodiment, the 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] This application relates to a kit for detecting anti-human TSLP monoclonal antibodies, wherein the kit includes a capture antibody and a biotin-labeled second antibody.

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

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

[0102] In one specific embodiment, the kit includes a capture antibody and a biotin-labeled second antibody. The capture antibody is monoclonal antibody 6, which is an anti-human TSLP monoclonal antibody. The second antibody is monoclonal antibody 11, which is an anti-human TSLP monoclonal antibody. The second antibody is conjugated with biotin protein and purified by a desalting column to obtain the biotin-labeled second antibody. The antibody to be tested is QX008N; its amino acid sequence containing the heavy chain variable region is shown in SEQ ID NO. 27; and its amino acid sequence containing 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, which is the anti-human TSLP monoclonal antibody described above. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

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

[0109] In one specific embodiment, the kit includes a capture antibody and a biotin-labeled second antibody, wherein the capture antibody is a monoclonal antibody 6 containing an anti-human TSLP monoclonal antibody; its heavy chain amino acid sequence is shown in SEQ ID NO. 9; and its light chain amino acid sequence is shown in SEQ ID NO. 10. The test antibody is QX008N, whose heavy chain amino acid sequence is shown in SEQ ID NO. 29; and its light chain amino acid sequence is 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, which is an anti-human TSLP monoclonal antibody. The second antibody is conjugated with biotin protein and purified by desalting column to obtain a biotin-labeled second antibody. Its amino acid sequence containing the heavy chain is shown in SEQ ID NO.19, and its amino acid sequence containing the light chain is shown in SEQ ID NO.20.

[0115] In one specific embodiment, the capture antibody is immobilized on a solid-phase support, and the antibody to be tested binds to the capture antibody.

[0116] In one specific embodiment, the kit further includes: horseradish peroxidase-labeled streptavidin (SA-HRP), washing buffer, substrate chromogenic solution, blocking buffer, diluent, and stop solution.

[0117] In one specific embodiment, the solid-phase carrier is an ELISA plate, preferably a 96-well ELISA plate, specifically a transparent flat-bottomed non-sterile 96-well ELISA plate from Thermo Scientific. The substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic 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 phosphate (V:V = 4:1).

[0118] In one specific implementation, the ELISA kit includes:

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

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

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

[0122] 4) Second antibody: Biotin-labeled second antibody, which is monoclonal antibody 11 against anti-human TSLP monoclonal antibody;

[0123] 5) Antibody detection: Horseradish peroxidase-labeled streptavidin (SA-HRP);

[0124] 6) Substrate: 3,3',5,5'-Tetramethylbenzidine (TMB), use after mixing equal volumes of solution A and solution B;

[0125] 7) Capture antibody coating dilution solution: phosphate buffer (1×PBS);

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

[0127] 9) Diluent / Blocking Buffer: A mixture of PBS solution, BSA powder, Tween 20 and Proclin 300 (V:W:V:V = 200:1:0.1:0.1);

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

[0129] In one 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] This application also relates to a method for quantitatively detecting the content of anti-human TSLP monoclonal antibody, wherein a capture antibody and a biotin-labeled second antibody are used, and ELISA detection is performed using a modified sandwich enzyme-linked immunosorbent assay.

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

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

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

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

[0135] (2) Blocking: Discard the coating working solution in the microplate, wash the plate 3 times with washing buffer (phosphate buffer (PBS) containing 0.05% Tween 20), and then add blocking buffer (phosphate buffer (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 hours;

[0136] (3) Sample addition: Dilute the standard and quality control samples 20 times with the minimum dilution factor (MRD) using the diluent; discard the blocking solution in the microplate, wash the plate 3 times with the washing buffer, and then add the pretreated standard and quality control samples to the microplate at a dosage of 100 μl / well, and incubate at room temperature for 2 h; the standard and quality control samples are both QX008N;

[0137] (4) Add biotin-labeled secondary antibody: Discard the standards and quality control products in the ELISA plate, wash the plate 3 times with washing buffer; dilute the biotin-labeled secondary antibody to 20 ng / ml working solution with diluent, and then add it to the ELISA plate at a dosage of 100 μl / well, and incubate at room temperature in the dark for about 1 hour.

[0138] (5) Add detection antibody: Discard the biotin-labeled secondary antibody in the ELISA plate, wash the plate 3 times with washing buffer, dilute horseradish peroxidase-labeled streptavidin (SA-HRP) with diluent to a working solution of 33.3 ng / ml, and then add it to the ELISA plate at a dosage of 100 μl / well. Incubate at room temperature in the dark for about 1 hour.

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

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

[0141] Example

[0142] Example 1: Preparation and purification of capture antibody and second antibody

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

[0144] Example 2 Biotin labeling of the second antibody

[0145] Sulfo-NHS-LC-Biotin reacts with the primary amine (-NH2) on the surface of the antibody protein to form a stable amide bond without affecting the antibody's intrinsic activity. The second antibody is mixed with Sulfo-NHS-LC-Biotin and incubated, followed by purification using a desalting column to remove unbound biotin, yielding the biotinylated second antibody. The CDR sequences of the second antibody are SEQ ID NO. 11-16, the heavy chain variable region sequence is SEQ ID NO. 17, the light chain variable region sequence 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 facilitate detection using streptavidin, exhibiting higher sensitivity and specificity.

[0146] Example 3 Performance Identification of Capture Antibody and Second Antibody

[0147] 1. Combined with activity identification

[0148] The enzyme-linked immunosorbent assay (ELISA) plates (purchased from Thermo) were coated with anti-human TSLP monoclonal antibody and human IgG as target proteins, respectively, as disclosed in CN113683694B. After blocking, serially diluted rabbit monoclonal antibody prepared in Example 1 was added, and the plates were incubated at room temperature for 2 hours. After washing, horseradish peroxidase-labeled goat anti-rabbit IgG antibody was added, and the plates were incubated at room temperature for 1 hour. Finally, after washing, the plates were developed, and the OD values ​​were read. 450 The absorbance at that point is shown in the following results. Figure 1A and Figure 1B As shown, where, Figure 1A This is a graph showing the binding results of the rabbit monoclonal antibody prepared in Example 1 with the anti-human TSLP monoclonal antibody. Figure 1B This is a graph showing the results of the binding 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 homotype IgG.

[0150] 2. Neutralization activity identification

[0151] The ELISA plate was coated with human TSLP as the antigen. After blocking, 100 ng / ml of biotin-labeled anti-human TSLP monoclonal antibody (disclosed in CN113683694B) and serially diluted rabbit monoclonal antibody prepared in Example 1 were added and incubated at room temperature for 2 hours. After washing, horseradish peroxidase-labeled streptavidin antibody was added and incubated at room temperature for 1 hour. Finally, after washing, color development was performed, and the OD was read. 450 The absorbance at that point was as follows: Figure 2 As shown, Figure 2 This is a diagram showing the results 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, which is an anti-human TSLP monoclonal antibody, blocks the binding of the anti-human TSLP monoclonal antibody to its target human TSLP and has neutralizing activity; while monoclonal antibody 11, which is an anti-human TSLP monoclonal antibody, does not block the binding of the anti-human TSLP monoclonal antibody to its target human TSLP and therefore does not have neutralizing activity.

[0153] Example 4: Identification of binding epitopes of the capture antibody and the second antibody

[0154] The rabbit monoclonal antibody prepared in Example 1 was used as a coating reagent to coat the microplates (purchased from Thermo), and the plates were washed and blocked overnight. Simultaneously, equal volumes of a fixed concentration of biotin-labeled anti-human TSLP monoclonal antibody and serially diluted rabbit monoclonal antibody prepared in Example 1 were added to the microplates, and the plates were incubated in the dark for 2 hours. After washing, horseradish peroxidase-labeled streptavidin (SA-HRP) was added, and the plates were incubated at room temperature for 1 hour. Finally, after washing, color development was performed, and the OD values ​​were read. 450 The absorbance at that point is shown in the following results. Figure 3A and Figure 3B As shown. Among them, Figure 3A This is a diagram showing the results of monoclonal antibody 6, which is an anti-human TSLP monoclonal antibody prepared in solid-phase Example 1, competing with monoclonal antibody 11, which is an anti-human TSLP monoclonal antibody, for binding to the anti-human TSLP monoclonal antibody. Figure 3B This is a diagram showing the results of monoclonal antibody 11, prepared in solid-phase Example 1, competing with monoclonal antibody 6, which is an anti-human TSLP monoclonal antibody, for binding to the anti-human TSLP monoclonal antibody.

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

[0156] Example 5 uses the antibody pairs prepared according to Example 1 to construct an ELISA kit.

[0157] The capture antibody is monoclonal antibody 6, an anti-human TSLP monoclonal antibody, immobilized on an ELISA plate; the secondary antibody is monoclonal antibody 11, an anti-human TSLP monoclonal antibody, and biotinylated; the test antibody can bind to the coated capture antibody and the subsequently added biotinylated secondary antibody, respectively; the preparation and performance determination of the capture antibody and the biotinylated secondary antibody are described in Examples 1-4; horseradish peroxidase-labeled streptavidin (SA-HRP) is used as the detection antibody. Figure 4 The method shown is used to construct an ELISA kit.

[0158] Experimental procedure:

[0159] (1) Coating: Dilute the monoclonal antibody 6 against anti-human TSLP monoclonal antibody with 1×PBS to a coating working solution of 1 μg / ml, and then add 100 μl / well to a 96-well microplate (purchased from Thermo) and coat overnight at 2-8℃.

[0160] (2) Blocking: Discard the coating working solution in the microplate, wash the plate 3 times with washing buffer (phosphate buffer (PBS) containing 0.05% Tween 20), and then add blocking solution at a rate of 200 μl / well. Block at room temperature for about 2 hours.

[0161] (3) Sample addition: The standard and quality control samples were pretreated by MRD dilution 20 times with diluent. The blocking solution in the ELISA plate was discarded. The plate was washed 3 times with washing buffer. Then, the working solution of the pretreated standard and quality control samples was added to the ELISA plate at a volume of 100 μl / well and incubated at room temperature for 2 hours. The standard and quality control samples were both the antibody to be tested, QX008N.

[0162] (4) Add secondary antibody: Discard the standard and quality control working solution in the ELISA plate, wash the plate 3 times with washing buffer, dilute the "secondary antibody" anti-human TSLP monoclonal antibody 11 prepared in Example 1 to 20 ng / ml secondary antibody working solution with diluent, and then add it to the ELISA plate at a dosage of 100 μl / well, and incubate at room temperature in the dark for about 1 hour.

[0163] (5) Add detection antibody: Discard the secondary antibody working solution in the microplate, wash the plate 3 times with washing buffer, dilute horseradish peroxidase-labeled streptavidin (SA-HRP) with diluent to 33.3 ng / ml detection antibody working solution, and then add it to the microplate at a dosage of 100 μl / well, and incubate at room temperature in the dark for about 1 hour.

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

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

[0166] The reagent information is as follows:

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

[0168] Washing solution: Add 500 μl of Tween 20 to every 1000 ml of 1×PBS;

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

[0170] TMB colorimetric solution: Take equal volumes of solution A and solution B, and mix thoroughly after they have returned to room temperature. Keep away from light and prepare fresh before use.

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

[0172] Example 6: Establishment of an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of anti-human TSLP monoclonal antibody levels in serum.

[0173] 1. Establishment of the standard curve

[0174] Anti-human TSLP monoclonal antibodies were diluted with human mixed serum (equal volumes of blank serum from 10 healthy individuals) to concentrations of 3.91, 7.81, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 ng / ml. Enzyme-linked immunosorbent assay (ELISA) was performed under the determined optimal experimental conditions according to Example 5. The detected concentration of anti-human TSLP monoclonal antibody was plotted on the x-axis, and OD... 450 Plot a four-parameter curve with the value as the ordinate, such as... Figure 5 As shown in the figure, the recovery rate was calculated, and the results are shown in Table 1.

[0175] Table 1: Recovery statistics at various concentration points after establishing the method standard curve

[0176]

[0177] The equation of the curve is: Fit:y=(0.046-3.421) / (1+(x / 208.7)^1.238)+3.421

[0178] Note: * indicates anchor points; recovery rate is not required.

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

[0180] As shown in Table 1, the standard curve concentrations for the enzyme-linked immunosorbent assay (ELISA) 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, with 3.91 ng / ml used as the anchor point to improve the fit of the standard curve (recovery rate is not required).

[0181] 2. Methodological Research

[0182] 5 2.1 Quantitative Range

[0183] Different researchers evaluated standard curves for at least three independent batches using an established enzyme-linked immunosorbent assay (ELISA) method for quantitatively detecting anti-human TSLP monoclonal antibody levels in serum on different days. The results are shown in Table 2. Table 2 shows that, except for the anchor point, at LLOQ and ULOQ concentration levels: batch-to-batch...

[0184] Accuracy (RE%) ranges from -19.4% to 5.6%, and precision (CV%) ranges from 1.0% to 21.6%. 101000 ng / ml and 10 ng / ml can be used as the upper and lower limits of quantitation for the method.

[0185] Table 2: Quantitative Range of Methods

[0186]

[0187] 2.2 Accuracy and Precision

[0188] An established enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect the content of anti-human TSLP monoclonal antibodies in serum.

[0189] The method evaluated 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 shown in Table 3, at LLOQ and ULOQ concentration levels: inter-batch accuracy (RE%) ranged from -17.3% to -3.0%, and precision (CV%) ranged from 7.6% to 11.8%. At QC concentration levels: inter-batch accuracy (RE%) ranged from -5.9% to -1.3%, and precision (CV%) ranged from 8.1% to 11.0%.

[0191] Table 3: Study on the accuracy and precision of the method

[0192]

[0193]

[0194] 2.3 Selectivity

[0195] The established enzyme-linked immunosorbent assay (ELISA) method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum was used to investigate the results by adding LLOQ concentration levels of anti-human TSLP monoclonal antibody to blank matrices from at least 10 healthy individuals. The results are shown in Table 4.

[0196] As shown in Table 4, the response values ​​of blank samples from all 12 individuals were lower than LLOQ, and the intra-batch accuracy (RE%) of LLOQ concentration level quality control samples from 11 individuals was in the range of 8.5% to 11.8%, which met the requirement of at least 8 / 10 individuals selectively passing.

[0197] Table 4: Method Selectivity Studies

[0198]

[0199] 2.4 Specificity

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

[0201] As shown in Table 5, under ULOQ concentration conditions, it can tolerate polyclonal antibodies against anti-human TSLP monoclonal antibodies at 1 μg / ml and human TSLP target antigen at 1 ng / ml; under LLOQ concentration conditions, it can tolerate polyclonal antibodies against anti-human TSLP monoclonal antibodies at 0.1 μg / ml and human TSLP target antigen at 100 pg / ml.

[0202] Table 5: Method Specificity Study

[0203]

[0204] Example 7 uses the ELISA kit constructed in Example 5 and the method established in Example 6 to determine the content of anti-human TSLP monoclonal antibody in serum.

[0205] The anti-human TSLP monoclonal antibody sample was pretreated by MRD dilution 20-fold using a diluent, and then diluted appropriately with 5% serum diluent (containing 5% human mixed serum) to fall within the quantitative range of the standard curve. The ELISA kit constructed in Example 5 and the method established in Example 6 were used to determine the content of anti-human TSLP monoclonal antibody in serum, which can accurately measure the content of anti-human TSLP monoclonal antibody in serum. In summary, the enzyme-linked immunosorbent assay (ELISA) method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum provided in this application involves forming a solid-phase carrier by coating an ELISA plate with a capture antibody, which binds to the anti-human TSLP monoclonal antibody in the biological sample to be tested, and then forming a solid-phase capture antibody-antibody-second antibody-ELISA complex through a specific second antibody and a detection antibody. Because the selected antibody pairs (capture antibody and second antibody) have high specificity, the method established in this application has high specificity, high sensitivity, and good precision and accuracy.

[0206] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

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

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. 17: The amino acid sequence of QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPGTLVTVSS; LCVR has the sequence SEQ ID NO. 18: The amino acid sequence of TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVK.

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. 19: QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPGTLVT VSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPP The amino acid sequence of ELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; The amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO. 20: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

4. A kit for detecting monoclonal antibodies against thymic stromal lymphopoietin, wherein, The kit includes a capture antibody and a biotin-labeled second antibody. The second antibody is a monoclonal antibody against the anti-thymocyte stromal lymphopoietin monoclonal antibody as described in any one of claims 1 to 3.

5. The kit according to claim 4, wherein, The amino acid sequence of the heavy chain variable region of the antibody to be tested 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.

6. The kit according to claim 4, wherein, The second antibody was conjugated with biotin protein and purified by a desalting column to obtain a biotin-labeled second antibody.

7. The kit according to claim 4, wherein, The capture antibody is immobilized on a solid-phase support, and the antibody to be tested binds to the capture antibody.

8. The kit according to claim 7, wherein, The solid support is an enzyme-labeled plate.

9. The kit according to claim 4, wherein, The kit also includes: horseradish peroxidase-labeled streptavidin, washing solution, substrate chromogenic solution, diluent, blocking solution, and stop solution.

10. The kit according to claim 4, wherein, The kit is an enzyme-linked immunosorbent assay kit.

11. The kit according to any one of claims 4 to 10, wherein, The kit is a modified sandwich enzyme-linked immunosorbent assay kit.

12. A method for quantitatively detecting the content of anti-thymic stromal lymphopoietin monoclonal antibody, wherein, ELISA detection was performed using a modified sandwich enzyme-linked immunosorbent assay (ELISA) with a capture antibody and a biotin-labeled secondary antibody. The second antibody is a monoclonal antibody against the anti-thymocyte stromal lymphopoietin monoclonal antibody as described in any one of claims 1 to 3.

13. The method according to claim 12, wherein, Includes the following steps: The captured antibody was immobilized onto a solid-phase carrier and then coated and blocked. Add the antibody to be tested, incubate to allow it to bind to the coated capture antibody, and then add the biotin-labeled second antibody for incubation. Incubate with diluted horseradish peroxidase-labeled streptavidin; Add the substrate colorimetric solution, incubate in the dark, then add the stop solution, measure the OD value, and prepare a standard curve; The content of anti-TSLP monoclonal antibody in the sample is calculated by substituting the OD value of the sample into the equation.

14. The method according to claim 12 or 13, wherein, The amino acid sequence of the heavy chain variable region of the antibody to be tested 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.

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