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

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to two kinds of 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 quantitatively detecting by using the kit. BACKGROUND

[0002] Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine mainly produced by human epithelial cells, which can act on dendritic cells, T cells, B cells and innate immune cells, and plays a key role in regulating type 2 immune responses. TSLP forms a ternary complex with TSLPR and IL-7Rα chain on the surface of the above-mentioned 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 playing a pro-inflammatory role. Studies have found that the occurrence and development of asthma, chronic obstructive pulmonary disease and other allergic diseases are closely related to the level of TSLP. The expression of TSLP in the airway of asthma patients is higher than that in the healthy control group, 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 induction of acute asthma attack by inducing type 2 inflammatory response, and can also directly act on airway smooth muscle and mast cells to induce AHR without relying on type 2 inflammation. Therefore, inhibition of TSLP may have a therapeutic effect 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. The parental antibody is obtained by screening New Zealand rabbit B cells immunized with human TSLP, and the anti-human TSLP monoclonal antibody is obtained by humanization of the parental rabbit antibody.

[0003] At present, the marketed drug with the same target Tezepelumab is a fully humanized IgG2λ monoclonal antibody. Phase II and III clinical data show that Tezepelumab significantly reduces asthma annual exacerbation rate (AAER) without being affected by the baseline level of eosinophils and FeNO level, and shows significant efficacy in a wide range of severe uncontrolled asthma patients. Jiangsu Quanxin Biomedicine Co., Ltd. has developed an anti-human TSLP monoclonal antibody with a trade code of QX008N for use as a drug. The anti-human TSLP monoclonal antibody is described in Chinese patent document CN 113683694 B.

[0004] For drugs, pharmacokinetics is the application of the principles of kinetics and mathematical processing methods to quantitatively describe the dynamic changes of drugs in the body, and to study the absorption, distribution, metabolism and excretion of drugs entering the human body by different routes. Therefore, the study of pharmacokinetics will be beneficial to the evaluation of drug safety and effectiveness. Common analysis methods for biological samples include chromatography and immunization. Chromatography has low sensitivity, a cumbersome test process, and unstable recovery rate; immunization is an analysis method based on specific antigen-antibody reaction, and is the most commonly used method in current non-clinical and clinical biological sample detection methods. Since the anti-human TSLP monoclonal antibody is a protein macromolecule, it is suitable for determination by enzyme-linked immunosorbent assay, in which the anti-human TSLP monoclonal antibody is combined with the capture antibody and the biotin-labeled secondary antibody, and the horseradish peroxidase-labeled streptavidin has high specificity with biotin, greatly reducing the interference of irrelevant proteins in the serum, amplifying the detection signal, and improving the detection sensitivity.

[0005] Sandwich ELISA refers to first binding the capture antibody to the ELISA plate, then adding the antibody to be detected to specifically bind with the capture antibody, then adding a specific secondary antibody, and finally adding an enzyme-labeled detection antibody and utilizing the substrate to catalyze the color development reaction. In this way, the antibody to be detected is specifically combined with the capture antibody and the secondary antibody of different binding epitopes, and the horseradish peroxidase-labeled streptavidin is combined with the biotin-labeled secondary antibody, not only playing a multi-level amplification role, but also having high specificity, high sensitivity, stability and other advantages. SUMMARY

[0006] Based on the above-mentioned needs existing in the prior research process, the present application provides two kinds of anti-anti-human TSLP monoclonal antibody monoclonal antibodies, an improved sandwich ELISA kit based on the ELISA platform comprising the above-mentioned antibodies, and the quantitative detection of anti-human TSLP monoclonal antibody is successfully completed by using the kit.

[0007] Specifically, the present application relates to the following:

[0008] 1. An anti-anti-human thymic stromal lymphopoietin monoclonal antibody monoclonal antibody, wherein the monoclonal antibody comprises:

[0009] a) an antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

[0010] CDR-H1 has an amino acid sequence of SEQ ID NO. 1: STYWIC or at least 90% identity with SEQ ID NO. 1,

[0011] CDR-H2 has the amino acid sequence of SEQ ID NO. 2: CIYAGFTGNTYYASWVNG or at least 90% identity to SEQ ID NO. 2,

[0012] CDR-H3 has the amino acid sequence of SEQ ID NO. 3: NAHDDSGEYIPHNF or at least 90% identity to SEQ ID NO. 3;

[0013] b) an antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

[0014] CDR-L1 has the amino acid sequence of SEQ ID NO. 4: QASEDIYSNLA or at least 90% identity to SEQ ID NO. 4,

[0015] CDR-L2 has the amino acid sequence of SEQ ID NO. 5: RASTLAS or at least 90% identity to SEQ ID NO. 5,

[0016] CDR-L3 has the amino acid sequence of SEQ ID NO. 6: LGVASYISVDGYNA or at least 90% identity to SEQ ID NO. 6.

[0017] 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:

[0018] HCVR has the amino acid sequence of SEQ ID NO. 7:

[0019] QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSS or at least 90% identity to SEQ ID NO. 7;

[0020] LCVR has the amino acid sequence of SEQ ID NO. 8: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYR ASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGY NAFGGGTEVVVK or at least 90% identity to SEQ ID NO. 8.

[0021] 3. The monoclonal antibody of item 1 or 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0022] the heavy chain amino acid sequence is the amino acid sequence of SEQ ID NO. 9: QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or at least 90% identity to SEQ ID NO. 9;

[0023] the light chain amino acid sequence is the amino acid sequence of SEQ ID NO. 10: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or at least 90% identity to SEQ ID NO. 10.

[0024] 4. A monoclonal antibody against a monoclonal antibody against human thymic stromal lymphopoietin, wherein the monoclonal antibody comprises:

[0025] a) an antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

[0026] CDR-H1 has an amino acid sequence of SEQ ID NO. 11: TYYMS or at least 90% identity to SEQ ID NO. 11,

[0027] CDR-H2 has an amino acid sequence of SEQ ID NO. 12: YISYGGTTDYASWAKG or at least 90% identity to SEQ ID NO. 12,

[0028] CDR-H3 has an amino acid sequence of SEQ ID NO. 13: GYGDGFNP or at least 90% identity to SEQ ID NO. 13;

[0029] b) an antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

[0030] CDR-L1 has an amino acid sequence of SEQ ID NO. 14: QASQSISTKLA or at least 90% identity to SEQ ID NO. 14,

[0031] CDR-L2 has an amino acid sequence of SEQ ID NO. 15: DASDLAS or at least 90% identity to SEQ ID NO. 15,

[0032] CDR-L3 has an amino acid sequence of SEQ ID NO. 16: QQGYTSYNVENA or at least 90% identity to SEQ ID NO. 16.

[0033] 5. The monoclonal antibody of item 1, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0034] HCVR has an amino acid sequence of SEQ ID NO. 17:

[0035] QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWI GYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCAR GYGDGFNPWGPGTLVTVSS or at least 90% identity to SEQ ID NO. 17;

[0036] The LCVR has an amino acid sequence of SEQ ID NO. 18: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYD ASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENA FGGGTEVVVK or at least 90% identity to SEQ ID NO. 18.

[0037] 6. The monoclonal antibody according to item 4 or 5, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0038] the heavy chain amino acid sequence is SEQ ID NO. 19: QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or at least 90% identity to SEQ ID NO. 19;

[0039] The amino acid sequence of the light chain is SEQ ID NO. 20: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or an amino acid sequence having at least 90% identity with SEQ ID NO. 20.

[0040] 7. The monoclonal antibody of the anti-anti-human thymic stromal lymphopoietin monoclonal antibody of item 1 and item 4 for preparing a kit for detecting the anti-anti-human thymic stromal lymphopoietin monoclonal antibody.

[0041] 8. The use of item 7, wherein the kit comprises a capture antibody and a biotin-labeled secondary antibody, the capture antibody being the monoclonal antibody of the anti-anti-human thymic stromal lymphopoietin monoclonal antibody of item 1; and the secondary antibody being the monoclonal antibody of the anti-anti-human thymic stromal lymphopoietin monoclonal antibody of item 4.

[0042] 9. The use of item 8, wherein the antibody to be detected is QX008N.

[0043] 10. The use of item 8, wherein the capture antibody is immobilized on a solid support, and the antibody to be detected is combined with the capture antibody.

[0044] 11. The use of item 10, wherein the solid support is an enzyme-labeled plate.

[0045] 12. The use of item 8, further comprising horseradish peroxidase-labeled streptavidin (SA-HRP), a washing solution, a substrate developing solution, a blocking solution, a sample diluent, and a termination solution.

[0046] 13. The use of any one of items 7-12, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0047] 14. The use of any one of items 7-13, wherein the kit is a modified sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0048] 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,

[0049] the capture antibody is a monoclonal antibody against the anti-human thymic stromal lymphopoietin monoclonal antibody according to any one of items 1-3;

[0050] the antibody to be tested is QX008N; and comprises heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein:

[0051] the CDR-H1 of the antibody to be tested QX008N has an amino acid sequence of SEQ ID NO. 21: SYYMS, the CDR-H2 has an amino acid sequence of SEQ ID NO. 22: FISYGGSAYHATWAQG, the CDR-H3 has an amino acid sequence of SEQ ID NO. 23: EFRSMTYGAEWGI; the CDR-L1 has an amino acid sequence of SEQ ID NO. 24: QASESIYDTLA, the CDR-L2 has an amino acid sequence of SEQ ID NO. 25: SASSLAS, and the CDR-L3 has an amino acid sequence of SEQ ID NO. 26: QQGYTMPDVDKNP;

[0052] the secondary antibody is a monoclonal antibody against the anti-human thymic stromal lymphopoietin monoclonal antibody according to any one of items 4-6;

[0053] the secondary antibody is coupled with Biotin protein and purified by a desalting column to obtain a biotin-labeled secondary antibody.

[0054] 16. The kit according to item 15, wherein the capture antibody is immobilized on a solid support, and the antibody to be tested binds to the capture antibody.

[0055] 17. The kit according to item 16, wherein the solid support is an enzyme-labeled plate.

[0056] 18. The kit according to any one of claims 15-17, wherein the kit further comprises horseradish peroxidase-labeled streptavidin (SA-HRP), a washing solution, a substrate developing solution, a dilution solution, a blocking solution and a termination solution, preferably the washing solution is a mixture of PBS solution and Tween 20 (V:V=100:0.05), the substrate developing solution is TMB developing solution, the termination solution is a mixture of water and phosphoric acid (V:V=4:1), and the dilution / blocking solution is a mixed solution of PBS solution, BSA powder, Tween 20 and Proclin 300 (V:W:V:V=200:1:0.1:0.1).

[0057] 19. The kit according to any one of claims 15-18, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0058] 20. The kit according to any one of claims 15-19, wherein the kit is a modified sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0059] 21. A method for quantitatively detecting the content of anti-human thymic stromal lymphopoietin monoclonal antibody, wherein the modified sandwich enzyme-linked immunosorbent assay is used to perform ELISA detection by using the capture antibody and the biotin-labeled secondary antibody according to claim 15.

[0060] 22. The method according to claim 21, comprising the following steps:

[0061] fixing the capture antibody to a solid-phase carrier for coating and blocking;

[0062] adding the antibody to be detected, incubating to react with the coated capture antibody, and then adding the biotin-labeled secondary antibody for incubation;

[0063] adding diluted horseradish peroxidase-labeled streptavidin (SA-HRP) for incubation;

[0064] adding the substrate developing solution, incubating in the dark, then adding the termination solution, measuring the OD value, and preparing a standard curve;

[0065] substituting the OD value of the sample to be detected into the equation to calculate the content of anti-human TSLP monoclonal antibody in the sample to be detected.

[0066] In the present application, the OD value is the OD value at 450 nm.

[0067] The present application has the following beneficial effects:

[0068] 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

[0069] 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.

[0070] Figure 1B This is a graph showing the results of the monoclonal antibody prepared in Example 1 binding with human IgG.

[0071] Figure 2 This is a graph showing the results of the neutralizing activity assessment of the monoclonal antibody prepared in Example 1.

[0072] Figure 3A This is a diagram showing the epitope identification results of the monoclonal antibody prepared in Example 1.

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

[0074] Figure 4 This is a schematic diagram of the enzyme-linked immunosorbent assay (ELISA) method of this application.

[0075] Figure 5 This is a graph of the four-parameter fitting curve for the standard curve. Detailed Implementation

[0076] 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.

[0077] It should be noted that certain terms used in the specification and claims are defined as follows. The following definitions are provided for the terms used herein.

[0078] Scientific and technical terms used in this specification generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless otherwise specifically defined herein. In case of conflict between the definitions and the explanations in the specification and the claims, the definitions in the claims shall control.

[0079] In general, the terms used in the specification have the following meanings.

[0080] In the present specification, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can arise during production of the monoclonal antibody preparation, such variants being present in minor amounts. Such monoclonal antibody preparations are typically produced by hybridoma methods, recombinant DNA methods, phage-display methods, and transgenic animals, and are inclusive of antibodies of intact molecules as well as fragments thereof, including but not limited to Fab and F(ab')2 fragments, produced by in vitro cleavage of the intact molecule, that bind to the same epitope as the intact monoclonal antibody from which they were derived. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and use of transgenic animals containing all or part of the human immunoglobulin loci, as described herein and as described generally in U.S. Patent No. 6,150,584.

[0081] In the present specification, CDR is an abbreviation of complement determine region, also known as "hypervariable region". LCDR is an abbreviation of light chain hypervariable region, and HCDR is an abbreviation of heavy chain hypervariable region. Generally, the heavy chain and the light chain of an antibody each have three CDRs, which together form the antigen-binding site of Ig. At this site, the antibody can form a precise complement with the antigenic determinant in the spatial structure. Hereinafter, the two monoclonal antibodies claimed in the present application are used as "capture antibody" and "anti-antibody" in the analysis technique to specifically bind to the anti-human TSLP monoclonal antibody, that is, to depend on the specific binding ability of the CDR region defined herein to the specific antigenic determinant on the anti-human TSLP monoclonal antibody.

[0082] In the present specification, CVR is an abbreviation of "variable domain", which is defined in relation to "constant domain". The variable domain of the heavy chain and the light chain each includes three "complement determining regions" described above.

[0083] In the present specification, human TSLP refers to a secreted protein derived from a human. Upon stimulation by allergens, viruses, and the like, the release of hTSLP is at the top of the inflammatory cascade and is upstream of other targets in signal transduction, playing a central role in regulating type II immune responses. hTSLP forms a ternary complex with the thymic stromal lymphopoietin 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 immunoregulatory effects. Monoclonal antibody 6 of the anti-anti-TSLP monoclonal antibody of the present application is a neutralizing antibody that specifically recognizes the anti-human TSLP monoclonal antibody, and monoclonal antibody 11 of the anti-anti-TSLP monoclonal antibody is a non-neutralizing antibody that specifically recognizes the anti-human TSLP monoclonal antibody, both of which can specifically bind to the anti-human TSLP monoclonal antibody and have different binding epitopes.

[0084] In the present specification, "identity" in the context of amino acid sequences refers to the number of identical residues in two sequences when aligned for maximum correspondence. There are many different algorithms known in the art that can be used to measure the percent amino acid identity (i.e., the Basic Local Alignment Tool or BLAST®). Unless otherwise indicated, the default parameters of a particular program or algorithm are used.

[0085] In the present specification, "anti-human TSLP monoclonal antibody" means a monoclonal antibody which is capable of binding to human TSLP with sufficient affinity so that the monoclonal antibody can be used for identifying / detecting human TSLP, and also can be used as an antibody drug, for example, as a drug for treating an autoimmune disease. Therefore, the sandwich ELISA method established hereinafter with the kit of the present application can be used as an analytical means for pharmacokinetic study of such an antibody drug.

[0086] In the present specification, "affinity" means the strength of the noncovalent 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" used in the present specification means 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 represented by the equilibrium dissociation constant (Kd) of the complex X-Y. Affinity can be measured by common methods known in the art. D ) represents. Affinity can be measured by common methods known in the art.

[0087] In the present specification, "enzyme-linked immunosorbent assay (ELISA)" means a detection method in which free target proteins and target proteins bound to a solid support are combined using the property that antibody molecules can specifically bind to antigen molecules, and are qualitatively or quantitatively analyzed using a special marker. The principle is that antigens or antibodies can be physically adsorbed to a solid surface and maintain their immunological activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immunological activity or enzymatic activity; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of the immune reaction can be determined by color reaction after the addition of a substrate, and the color reaction is proportional to the amount of the antigen or antibody of interest in the sample. Depending on the substance to be detected and the conditions for detection, various types of detection methods can be designed, and the sandwich method is the most commonly used method for detecting antigens.

[0088] In the present specification, "standard curve" means a linear relationship between the content of a sample to be measured and the measurable value (e.g., absorbance value) of the analysis method, in order to achieve quantitative analysis of the sample to be measured by means thereof.

[0089] The present application relates to monoclonal antibody 6 of anti-anti-human TSLP monoclonal antibody and monoclonal antibody 11 of anti-anti-human TSLP monoclonal antibody. Among them, the monoclonal antibody 6 of anti-anti-human TSLP monoclonal antibody comprises: a) antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has the amino acid sequence of sequence SEQ ID NO. 1: STYWIC or has at least 90% identity with SEQ ID NO. 1, CDR-H2 has the amino acid sequence of sequence SEQ ID NO. 2: CIYAGFTGNTYYASWVNG or has at least 90% identity with SEQ ID NO. 2, CDR-H3 has the amino acid sequence of sequence SEQ ID NO. 3: NAHDDSGEYIPHNF or has at least 90% identity with SEQ ID NO. 3; b) antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the amino acid sequence of sequence SEQ ID NO. 4: QASEDIYSNLA or has at least 90% identity with SEQ ID NO. 4, CDR-L2 has the amino acid sequence of sequence SEQ ID NO. 5: RASTLAS or has at least 90% identity with SEQ ID NO. 5, CDR-L3 has the amino acid sequence of sequence SEQ ID NO. 6: LGVASYISVDGYNA or has at least 90% identity with SEQ ID NO. 6.

[0090] In a specific embodiment, the above-mentioned antibody comprises antibody heavy chain variable region HCVR and antibody light chain variable region LCVR, wherein: HCVR has the amino acid sequence of sequence SEQ ID NO. 7: QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSS or has at least 90% identity with SEQ ID NO. 7; LCVR has the amino acid sequence of sequence SEQ ID NO. 8: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRAS TLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNA FGGGTEVVVK or has at least 90% identity with SEQ ID NO. 8.

[0091] In one embodiment, the antibody comprises a heavy chain and a light chain, wherein:

[0092] the heavy chain amino acid sequence is the amino acid sequence of SEQ ID NO. 9: QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or at least 90% identity to SEQ ID NO. 9; and the light chain amino acid sequence is the amino acid sequence of SEQ ID NO. 10: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or at least 90% identity to SEQ ID NO. 10.

[0093] The monoclonal antibody 11 of the anti-human TSLP 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 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, 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 complementarity determining region comprising: CDR-L1, CDR-L2, 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, CDR-L2 has an amino acid sequence of SEQ ID NO. 15: DASDLAS or has at least 90% identity with SEQ ID NO. 15, CDR-L3 has an amino acid sequence of SEQ ID NO. 16: QQGYTSYNVENA or has at least 90% identity with SEQ ID NO. 16.

[0094] In one 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: QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPGTLVTVSS or has at least 90% identity with SEQ ID NO. 17; LCVR has an amino acid sequence of SEQ ID NO. 18: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVK or has at least 90% identity with SEQ ID NO. 18.

[0095] In one specific embodiment, the above-mentioned antibody comprises a heavy chain and a light chain, wherein:

[0096] the heavy chain amino acid sequence is the amino acid sequence of SEQ ID NO. 19: QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMSWVRQAPGKGLEWIGYISYGGTTDYASWAKGRFTISKTSSTTVDLKMTSLTPSDTATYFCARGYGDGFNPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or at least 90% identity to SEQ ID NO. 19; the light chain amino acid sequence is the amino acid sequence of SEQ ID NO. 20: TYDMTQTPASVEVAVGGTVTIKCQASQSISTKLAWYQQKPGQPPELLIYDASDLASGVPSRFRGRGSGTEFTLTISSVQCDDAATYYCQQGYTSYNVENAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or at least 90% identity to SEQ ID NO. 20.

[0097] In the present 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.

[0098] 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.

[0099] In one specific embodiment, the capture antibody is monoclonal antibody 6 of the anti-anti-human TSLP monoclonal antibody described above; the second antibody is monoclonal antibody 11 of the anti-anti-human TSLP monoclonal antibody described above; and the test antibody is QX008N, which comprises heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the CDR-H1 of the test antibody QX008N has an amino acid sequence of SEQ ID NO. 21: SYYMS, the CDR-H2 has an amino acid sequence of SEQ ID NO. 22: FISYGGSAYHATWAQG, the CDR-H3 has an amino acid sequence of SEQ ID NO. 23: EFRSMTYGAEWGI; the CDR-L1 has an amino acid sequence of SEQ ID NO. 24: QASESIYDTLA, the CDR-L2 has an amino acid sequence of SEQ ID NO. 25: SASSLAS, and the CDR-L3 has an amino acid sequence of SEQ ID NO. 26: QQGYTMPDVDKNP.

[0100] In one specific embodiment, the capture antibody is monoclonal antibody 6 of the anti-anti-human TSLP monoclonal antibody described above; the second antibody is monoclonal antibody 11 of the anti-anti-human TSLP monoclonal antibody described above, which is coupled with Biotin protein and purified by a desalting column to obtain a biotin-labeled second antibody; and the test antibody is QX008N, which comprises heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the CDR-H1 of the test antibody QX008N has an amino acid sequence of SEQ ID NO. 21: SYYMS, the CDR-H2 has an amino acid sequence of SEQ ID NO. 22: FISYGGSAYHATWAQG, the CDR-H3 has an amino acid sequence of SEQ ID NO. 23: EFRSMTYGAEWGI; the CDR-L1 has an amino acid sequence of SEQ ID NO. 24: QASESIYDTLA, the CDR-L2 has an amino acid sequence of SEQ ID NO. 25: SASSLAS, and the CDR-L3 has an amino acid sequence of SEQ ID NO. 26: QQGYTMPDVDKNP.

[0101] In one embodiment, the kit comprises a capture antibody and a biotin-labeled secondary antibody, wherein the capture antibody is monoclonal antibody 6 against anti-human TSLP monoclonal antibody; the secondary antibody is monoclonal antibody 11 against anti-human TSLP monoclonal antibody, which is coupled with Biotin protein and purified by desalting column to obtain biotin-labeled secondary antibody. The antibody to be tested is QX008N, which comprises an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 27 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 28.

[0102] SEQ ID NO. 27:

[0103] EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWGQGTLVTVSS

[0104] SEQ ID NO. 28:

[0105] AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVEIK

[0106] The capture antibody is monoclonal antibody 6 against anti-human TSLP monoclonal antibody, which comprises an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 7 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 8.

[0107] The secondary antibody is monoclonal antibody 11 against anti-human TSLP monoclonal antibody, which comprises an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 17 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 18.

[0108] In one embodiment, the kit comprises a capture antibody and a biotin-labeled secondary antibody, wherein the capture antibody is monoclonal antibody 6 against anti-human TSLP monoclonal antibody, which comprises an amino acid sequence of a heavy chain as shown in SEQ ID NO. 9 and an amino acid sequence of a light chain as shown in SEQ ID NO. 10. The antibody to be tested is QX008N, which comprises an amino acid sequence of a heavy chain as shown in SEQ ID NO. 29 and an amino acid sequence of a light chain as shown in SEQ ID NO. 30.

[0109] SEQ ID NO. 29:

[0110] EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0111] SEQ ID NO. 30:

[0112] AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0113] The second antibody is monoclonal antibody 11 against anti-human TSLP monoclonal antibody, which is coupled with Biotin protein and purified by desalting column to obtain biotin-labeled second antibody; it comprises the amino acid sequence of heavy chain as shown in SEQ ID NO. 19; and the amino acid sequence of light chain as shown in SEQ ID NO. 20.

[0114] In one specific embodiment, the capture antibody is immobilized on a solid support, and the antibody to be detected is bound to the capture antibody.

[0115] In one specific embodiment, the kit further comprises: horseradish peroxidase-labeled streptavidin (SA-HRP), washing solution, substrate developing solution, blocking solution, dilution solution and termination solution.

[0116] In one specific embodiment, the solid support is an enzyme-labeled plate, preferably a 96-well enzyme-labeled plate, and specifically a transparent flat-bottom non-sterile 96-well enzyme-labeled plate from Thermo. The substrate developing solution is 3,3',5,5'-tetramethylbenzidine (TMB) developing solution; the washing solution is a mixture of PBS solution and Tween 20 (V:V=100:0.05); the dilution / blocking solution is a mixed solution of PBS solution, BSA powder, Tween 20 and Proclin 300 (V:W:V:V=200:1:0.1:0.1); and the termination solution is a mixture of water and phosphoric acid (V:V=4:1).

[0117] In one specific embodiment, the ELISA kit comprises:

[0118] 1) solid support: enzyme-labeled plate, 1 piece;

[0119] 2) capture antibody: monoclonal antibody 6 of anti-anti-human TSLP monoclonal antibody;

[0120] 3) standard / quality control of antibody to be detected QX008N;

[0121] 4) secondary antibody: biotin-labeled secondary antibody, which is monoclonal antibody 11 of anti-anti-human TSLP monoclonal antibody;

[0122] 5) detection antibody: horseradish peroxidase-labeled streptavidin (SA-HRP);

[0123] 6) substrate: 3,3',5,5'-tetramethylbenzidine (TMB), used after mixing equal volumes of A and B;

[0124] 7) capture antibody coating dilution solution: phosphate buffer (1x PBS);

[0125] 8) washing solution: a mixture of PBS solution and Tween 20 (V:V=100:0.05);

[0126] 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);

[0127] 10) Stop solution: mixture of water and phosphoric acid (V:V = 4:1).

[0128] In one embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit. Preferably, the kit is a modified sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0129] The present application also relates to a method for quantitatively detecting the content of anti-human TSLP monoclonal antibody, wherein a modified sandwich enzyme-linked immunosorbent assay is used to perform ELISA detection using a capture antibody and a biotin-labeled secondary antibody.

[0130] In one embodiment, the capture antibody is monoclonal antibody 6 of the anti-human TSLP monoclonal antibody described above. In one embodiment, the secondary antibody is monoclonal antibody 11 of the anti-human TSLP monoclonal antibody described above. In one embodiment, the antibody to be detected is QX008N, which comprises heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the CDR-H1 of the antibody to be detected QX008N has an amino acid sequence of SEQ ID NO. 21: SYYMS, the CDR-H2 has an amino acid sequence of SEQ ID NO. 22: FISYGGSAYHATWAQG, the CDR-H3 has an amino acid sequence of SEQ ID NO. 23: EFRSMTYGAEWGI; the CDR-L1 has an amino acid sequence of SEQ ID NO. 24: QASESIYDTLA, the CDR-L2 has an amino acid sequence of SEQ ID NO. 25: SASSLAS, and the CDR-L3 has an amino acid sequence of SEQ ID NO. 26: QQGYTMPDVDKNP.

[0131] In one embodiment, monoclonal antibody 6 of the anti-human TSLP monoclonal antibody is used as a capture antibody to bind the antibody to be detected (such as QX008N), and biotin-labeled secondary antibody is used to bind the antibody to be detected, wherein the secondary antibody is monoclonal antibody 11 of the anti-human TSLP monoclonal antibody, and a modified sandwich enzyme-linked immunosorbent assay is used to perform ELISA detection, and the method comprises the following steps: fixing the capture antibody to a solid-phase carrier and coating and blocking; adding the antibody to be detected for incubation; then adding the biotin-labeled secondary antibody for incubation; adding diluted horseradish peroxidase-labeled streptavidin (SA-HRP) for incubation; adding substrate developing solution, incubating in the dark, then adding stop solution, measuring the OD value, and preparing a standard curve; and substituting the OD value of the sample to be detected into the equation to calculate the content of the anti-human TSLP monoclonal antibody in the sample to be detected.

[0132] In one embodiment, the ELISA kit described herein can be used to quantitatively detect the content of anti-human TSLP monoclonal antibody in a sample, comprising the following steps:

[0133] (1) Coating: dilute the capture antibody to 1 μg / ml coating working solution with 1xPBS, then add 100 μl / well to the enzyme-labeled plate (96-well Thermo enzyme-labeled plate), and coat at 2-8°C overnight;

[0134] (2) Blocking: discard the coating working solution in the enzyme-labeled plate, wash the plate 3 times with washing solution (phosphate buffer solution (PBS) containing 0.05% Tween 20), then add blocking solution (phosphate buffer solution (PBS) containing 0.5% BSA powder, 0.05% Tween 20 and 0.05% Proclin 300) at 200 μl / well, and block at room temperature for about 2 hours;

[0135] (3) Sample addition: pretreat the standard and quality control with diluent at a minimum dilution ratio (MRD) of 20 times; discard the blocking solution in the enzyme-labeled plate, wash the plate 3 times with washing solution, then add the pretreated standard and quality control to the enzyme-labeled plate at 100 μl / well, and incubate at room temperature for 2 hours; wherein the standard and quality control are both QX008N;

[0136] (4) Addition of biotin-labeled secondary antibody: discard the standard and quality control in the enzyme-labeled plate, wash the plate 3 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 enzyme-labeled plate, and incubate at room temperature for about 1 hour in the dark;

[0137] (5) Addition of detection antibody: discard the biotin-labeled secondary antibody in the enzyme-labeled plate, wash the plate 3 times with washing solution, then dilute horseradish peroxidase-labeled streptavidin (SA-HRP) to a detection antibody working solution of 33.3 ng / ml with diluent, and add 100 μl / well to the enzyme-labeled plate, and incubate at room temperature for about 1 hour in the dark;

[0138] (6) Color development: mix A and B liquids of substrate (3,3',5,5'-tetramethylbenzidine (TMB)) in equal volumes as required; discard the detection antibody working solution in the enzyme-labeled plate, wash the plate 3 times with washing solution, then add 100 μl / well of substrate, and develop color at room temperature for 10-15 minutes in the dark;

[0139] (7) Termination and reading: add termination solution to the enzyme-labeled plate at a dosage of 100 μl / well to terminate the reaction, then use an enzyme-labeled instrument to measure the OD value of each well of the enzyme-labeled plate at a wavelength of 450 nm, fit a standard curve according to the OD value of the standard, and substitute the OD value of the quality control sample into the equation to calculate the back-calculation concentration and recovery rate of the quality control sample.

[0140] Examples

[0141] Preparation and purification of the capture antibody and the second antibody in Example 1

[0142] Rabbits were immunized with the (F(ab)2) of the anti-human TSLP monoclonal antibody as an antigen, and neutralizing active and / or non-neutralizing active rabbit monoclonal antibodies capable of binding to the anti-human TSLP monoclonal antibody and not cross-reacting with other isotype human IgG were screened by B cell cloning technology (i.e., the capture antibody and the second antibody were obtained). Sequencing was performed using conventional methods in the art, and the CDR sequences of the capture antibody were SEQ ID NO. 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 NO. 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.

[0143] Biotinylation of the second antibody in Example 2

[0144] 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. The second antibody was mixed with Sulfo-NHS-LC-biotin and incubated, and then purified through a desalting column to remove unbound biotin to obtain a biotinylated second antibody. The CDR sequences of the second antibody were SEQ ID NO. 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. Biotin-labeled antibodies facilitate detection using streptavidin, with higher sensitivity and specificity.

[0145] Performance identification of the capture antibody and the second antibody in Example 3

[0146] 1. Identification of binding activity

[0147] The anti-human TSLP monoclonal antibody disclosed in CN113683694B and human IgG were used as target proteins to coat the enzyme-labeled plate (purchased from Thermo) respectively. After blocking, the gradient-diluted rabbit monoclonal antibody prepared in Example 1 was added and incubated at room temperature for 2 hours. After washing the plate, horseradish peroxidase-labeled goat anti-rabbit IgG antibody was added and incubated at room temperature for 1 hour. After the final washing of the plate, color development was performed, and the absorbance at OD 450 Figure 1A Figure 1B Figure 1A is a result graph of the binding of the rabbit monoclonal antibody prepared in Example 1 to the anti-human TSLP monoclonal antibody, Figure 1B is a result graph of the binding of the rabbit monoclonal antibody prepared in Example 1 to human IgG.

[0148] As can be seen from Figure 1A Figure 1B , the rabbit monoclonal antibody has good binding activity to the anti-human TSLP monoclonal antibody and does not cross-react with the same type of IgG.

[0149] 2. Neutralization activity identification

[0150] The enzyme-labeled plate was coated with human TSLP as an antigen, and after blocking, 100 ng / ml biotin-labeled anti-human TSLP monoclonal antibody disclosed in CN113683694B and a gradient-diluted mixture of the rabbit monoclonal antibody 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. After the final washing of the plate, color development was performed, and the absorbance at OD 450 Figure 2 Figure 2 is a result graph of the blocking of the rabbit monoclonal antibody prepared in Example 1 to the binding of the anti-human TSLP monoclonal antibody to human TSLP.

[0151] As can be seen from Figure 2 , the monoclonal antibody 6 against the anti-human TSLP monoclonal antibody blocks the binding of the anti-human TSLP monoclonal antibody to its target human TSLP, and has neutralization activity; the monoclonal antibody 11 against the anti-human TSLP monoclonal antibody does not block the binding of the anti-human TSLP monoclonal antibody to its target human TSLP, and thus does not have neutralization activity.

[0152] Example 4 Identification of the binding epitope of the capture antibody and the secondary antibody

[0153] ​​​​​​The rabbit monoclonal antibody prepared in Example 1 was used as a coating reagent to coat an enzyme-labeled plate (purchased from Thermo) overnight, and then the plate was washed and blocked. At the same time, a fixed concentration of biotin-labeled anti-human TSLP monoclonal antibody and gradient-diluted rabbit monoclonal antibody prepared in Example 1 were added into the enzyme-labeled plate, and incubated in the dark for 2 hours. After washing the plate, horseradish peroxidase-labeled streptavidin (SA-HRP) was added, and incubated at room temperature for 1 hour. Finally, after washing the plate, color development was performed, and the absorbance at OD 450 Figure 3A Figure 3B Figure 3A is a graph showing the results of competition of monoclonal antibody 6 against monoclonal antibody 11 of anti-human TSLP monoclonal antibody for binding to anti-human TSLP monoclonal antibody; Figure 3B is a graph showing the results of competition of monoclonal antibody 11 against monoclonal antibody 6 of anti-human TSLP monoclonal antibody for binding to anti-human TSLP monoclonal antibody.

[0154] As can be seen from Figure 3A Figure 3B , monoclonal antibody 6 of anti-human TSLP monoclonal antibody and monoclonal antibody 11 of anti-human TSLP monoclonal antibody do not cross the binding epitope of anti-human TSLP monoclonal antibody.

[0155] Example 5 uses the "antibody pair" prepared in Example 1 to construct an ELISA kit

[0156] The capture antibody is monoclonal antibody 6 of anti-human TSLP monoclonal antibody and is immobilized on an enzyme-labeled plate; the second antibody is monoclonal antibody 11 of anti-human TSLP monoclonal antibody and is biotinylated; the antibody to be tested can be bound to the coated capture antibody and the biotinylated second antibody added later; 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 a detection antibody, and the ELISA kit is constructed using the method shown in Figure 4

[0157] Experimental process:

[0158] (1) Coating: Dilute monoclonal antibody 6 of anti-human TSLP monoclonal antibody to 1 μg / ml of coating working solution with 1xPBS, and then add 100 μl / well to a 96-well enzyme-labeled plate (purchased from Thermo) at 2-8°C for coating overnight;

[0159] ​​​​​(2) Blocking: Discard the coating working solution in the enzyme-labeled plate, wash the plate 3 times with washing solution (phosphate buffer solution (PBS) containing 0.05% Tween 20), then add blocking solution at a dosage of 200 μl / well, and block at room temperature for about 2 hours;

[0160] (3) Sample addition: uniformly pretreat the standard and quality control samples by diluting 20 times with diluent for MRD, discard the blocking solution in the enzyme-labeled plate, wash the plate 3 times with washing solution, then add the pretreated standard and quality control sample working solution to the enzyme-labeled plate at a dosage of 100 μl / well, and incubate at room temperature for 2 hours; wherein the standard and quality control samples are the antibody QX008N to be tested;

[0161] (4) Addition of secondary antibody: discard the standard and quality control sample working solution in the enzyme-labeled plate, wash the plate 3 times with washing solution, dilute the monoclonal antibody 11 of the "secondary antibody" anti-human TSLP monoclonal antibody prepared in Example 1 to a secondary antibody working solution of 20 ng / ml with diluent, then add to the enzyme-labeled plate at a dosage of 100 μl / well, and incubate at room temperature for about 1 hour in the dark;

[0162] (5) Addition of detection antibody: discard the secondary antibody working solution in the enzyme-labeled plate, wash the plate 3 times with washing solution, dilute horseradish peroxidase-labeled streptavidin (SA-HRP) to a detection antibody working solution of 33.3 ng / ml with diluent, then add to the enzyme-labeled plate at a dosage of 100 μl / well, and incubate at room temperature for about 1 hour in the dark;

[0163] (6) Color development: mix A and B liquids of the substrate (3,3',5,5'-tetramethylbenzidine (TMB)) in equal volumes as needed; discard the detection antibody working solution in the enzyme-labeled plate, wash the plate 3 times with washing solution, then add the substrate at a dosage of 100 μl / well, and develop color at room temperature in the dark for 10-15 minutes;

[0164] (7) Termination and reading: add the termination solution to the enzyme-labeled plate at a dosage of 100 μl / well to terminate the reaction, then use an enzyme-labeled instrument to measure the OD values of the wells in the enzyme-labeled plate at a wavelength of 450 nm, fit a standard curve according to the OD values of the standard, and calculate the back-calculated concentration and recovery rate of the quality control sample by substituting the OD value of the quality control sample into the equation.

[0165] Among them, the reagent information is:

[0166] Phosphate buffer solution (1×PBS): 23.48 g of PBS powder is dissolved in 2000 ml of ultrapure water;

[0167] Washing solution: add 500 μl of Tween 20 to 1,000 ml of 1×PBS;

[0168] Diluent / blocking solution: 2.5 g of BSA powder was weighed, dissolved in 500 ml of 1xPBS, 250 μl of Tween20 and 250 μl of Proclin 300 were added, and mixed thoroughly;

[0169] TMB developing solution: equal volumes of A and B were taken, mixed thoroughly after returning to room temperature, and used immediately, avoiding light, and prepared immediately before use;

[0170] Termination solution: 100 ml of H3PO4 was added to 400 ml of ultrapure water;

[0171] Example 6 Establishment of an enzyme-linked immunoassay method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum

[0172] 1. Establishment of a standard curve

[0173] The anti-human TSLP monoclonal antibody was diluted with human mixed serum (10 healthy individual blank serum, mixed in equal volume) to 3.91, 7.81, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 ng / ml, and enzyme-linked immunoassay experiments were 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 abscissa, and the OD value was taken as the ordinate to draw a four-parameter curve, as shown in 450 Figure 5 The recovery rate was calculated, and the results are shown in Table 1.

[0174] Table 1: Recovery rate statistics of each concentration point in the establishment of the method standard curve

[0175]

[0176] Curve equation: Fit: y = (0.046-3.421) / (1+(x / 208.7)^1.238)+3.421

[0177] Note: *marked as anchor point, recovery rate not required

[0178] As can be seen from Figure 5 , the anti-human TSLP monoclonal antibody concentration in the range of 7.81-1000 ng / ml has a good recovery rate.

[0179] As can be seen from Table 1, the concentration of the enzyme-linked immunoassay method established in the present application is 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 not required).

[0180] ​2. Methodology study

[0181] 2.1 Quantitative range

[0182] At least 3 independent batches of standard curves were evaluated by the established ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum, and the results are shown in Table 2. As can be seen from Table 2, except for the anchor point, at the LLOQ and ULOQ concentration levels: the inter-batch accuracy RE% was in the range of -19.4% to 5.6%, and the precision CV% was in the range of 1.0% to 21.6%. 1000 ng / ml and 10 ng / ml can be used as the upper and lower limits of quantification of the method.

[0183] Table 2: Quantitative range study of the method

[0184]

[0185] 2.2 Accuracy and precision

[0186] At least 3 independent batches of quality control samples (including LLOQ, LQC, MQC, HQC, ULOQ) were evaluated by the established ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum, and the results are shown in Table 3.

[0187] As can be seen from Table 3, at the LLOQ and ULOQ concentration levels: the inter-batch accuracy RE% was in the range of -17.3% to -3.0%, and the precision CV% was in the range of 7.6% to 11.8%. At the QC concentration levels: the inter-batch accuracy RE% was in the range of -5.9% to -1.3%, and the precision CV% was in the range of 8.1% to 11.0%.

[0188] Table 3: Accuracy and precision study of the method

[0189]

[0190]

[0191] 2.3 Selectivity

[0192] At least 10 blank matrices from healthy individuals were added with LLOQ concentration level of anti-human TSLP monoclonal antibody, and the results are shown in Table 4.

[0193] As can be seen from Table 4, the response values of blank samples from 12 individuals were lower than the 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%, meeting the selectivity passing of at least 8 / 10 individuals.

[0194] Table 4: Method selectivity study

[0195]

[0196] 2.4 Specificity

[0197] The established ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum was investigated by adding gradient concentrations of interferents (including human TSLP and anti-anti-human TSLP monoclonal antibody polyclonal antibody) at the concentration levels of blank sample, LLOQ and ULOQ, and the results are shown in Table 5.

[0198] As can be seen from Table 5, at the ULOQ concentration, 1 μg / ml of anti-anti-human TSLP monoclonal antibody polyclonal antibody and 1 ng / ml of human TSLP target antigen can be tolerated; at the LLOQ concentration, 0.1 μg / ml of anti-anti-human TSLP monoclonal antibody polyclonal antibody and 100 pg / ml of human TSLP target antigen can be tolerated.

[0199] Table 5: Method specificity study

[0200]

[0201] Example 7: Content determination of anti-human TSLP monoclonal antibody in serum using the ELISA kit constructed in Example 5 and the method established in Example 6

[0202] The anti-human TSLP monoclonal antibody sample to be tested was pretreated by 20-fold dilution with diluent, and then diluted by an appropriate multiple with 5% serum diluent (the diluent contains 5% human mixed serum) to fall within the quantitative range of the calibration curve; the content of anti-human TSLP monoclonal antibody in serum was determined using the ELISA kit constructed in Example 5 and the method established in Example 6, which can accurately determine the content of anti-human TSLP monoclonal antibody in serum. In summary, the ELISA method for quantitatively detecting the content of anti-human TSLP monoclonal antibody in serum provided by the present application is a method for forming a solid phase carrier by coating an enzyme-labeled plate with a capture antibody, combining the anti-human TSLP monoclonal antibody in the biological sample to be tested with the capture antibody, 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 strong specificity, the method established in the present application has strong specificity, high sensitivity, good precision and accuracy.

[0203] The above descriptions are only the preferred embodiments of the present application, not intended to limit the present application in any manner. Any modification, equivalent replacement, and improvement made by any person with the technical contents disclosed by the present application shall be within the protection scope of the present 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: The sequence of CDR-H1 is shown as the amino acid sequence of SEQ ID NO. 1: STYWIC. The sequence of CDR-H2 is shown in the amino acid sequence of SEQ ID NO. 2: CIYAGFTGNTYYASWVNG. The sequence of CDR-H3 is shown as the amino acid sequence of SEQ ID NO. 3: NAHDDSGEYIPHNF; b) Antibody light chain complementarity-determining regions, which include: CDR-L1, CDR-L2, and CDR-L3, wherein: The sequence of CDR-L1 is shown in the amino acid sequence of SEQ ID NO. 4: QASEDIYSNLA. The sequence of CDR-L2 is shown as the amino acid sequence of SEQ ID NO. 5: RASTLAS. The sequence of CDR-L3 is shown as the 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: The sequence of HCVR is shown in SEQ ID NO. 7: The amino acid sequence of QQLEQSGGGAEGGLVKPGGSLELCCKASGFALSSTYWICWVRQAPGKGLEWIGCIYAGFTGNTYYASWVNGRFTLSRDIDQSTGCLQMNSLTAADTAMYYCSRNAHDDSGEYIPHNFWGQGTLVTVSS is shown below; The sequence of LCVR is shown in SEQ ID NO. 8: The amino acid sequence of ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVK is shown.

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 the amino acid sequence of SEQ ID NO. 10: ALVMTQTPSPVSAAVGGTVTIKCQASEDIYSNLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGLQCDDAATYYCLGVASYISVDGYNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

4. A kit for detecting monoclonal antibodies against thymic stromal lymphopoietin, wherein, The kit includes a capture antibody and a biotin-labeled second antibody, wherein the capture antibody is a monoclonal antibody against 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 antibody to be tested 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: The sequences of the test antibodies are as follows: CDR-H1 as shown in SEQ ID NO. 21: SYYMS; CDR-H2 as shown in SEQ ID NO. 22: FISYGGSAYHATWAQG; CDR-H3 as shown in SEQ ID NO. 23: EFRSMTYGAEWGI; CDR-L1 as shown in SEQ ID NO. 24: QASESIYDTLA; CDR-L2 as shown in SEQ ID NO. 25: SASSLAS; and CDR-L3 as shown in SEQ ID NO. 26: QQGYTMPDVDKNP.

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 (SA-HRP), washing buffer, 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 capture antibody is a monoclonal antibody against 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 (SA-HRP); 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 antibody to be tested comprises 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: The sequences of CDR-H1 are shown in SEQ ID NO. 21: SYYMS; the sequences of CDR-H2 are shown in SEQ ID NO. 22: FISYGGSAYHATWAQG; the sequences of CDR-H3 are shown in SEQ ID NO. 23: EFRSMTYGAEWGI; the sequences of CDR-L1 are shown in SEQ ID NO. 24: QASESIYDTLA; the sequences of CDR-L2 are shown in SEQ ID NO. 25: SASSLAS; and the sequences of CDR-L3 are shown in SEQ ID NO. 26: QQGYTMPDVDKNP.

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