Monoclonal antibody, antibody combination containing monoclonal antibody and application of monoclonal antibody in detection of pertussis toxin

Abstract

The invention discloses a monoclonal antibody, an antibody composition containing the monoclonal antibody and application of the monoclonal antibody to detection of pertussis toxin. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR1, an HCDR2 and an HCDR3 which are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 which are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The monoclonal antibody can be used as a capture antibody in ELISA sandwich detection, and can be applied to quantitative detection and quality control of pertussis toxin (PT) in each link in pertussis vaccine production.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and specifically to monoclonal antibodies, antibody combinations containing the same, and their uses in detecting pertussis toxin, such as the double-antibody sandwich ELISA quantitative detection method for pertussis toxin (PT). Background Art

[0002] Whooping cough is an acute respiratory infectious disease with strong infectivity and is one of the major infectious diseases seriously threatening human health. Since the introduction of the whole-cell pertussis vaccine (wP) and the acellular pertussis vaccine (APV), the mortality and morbidity rates of whooping cough have decreased significantly. Nowadays, both vaccines are in use, but due to problems such as relatively large side effects and safety of the whole-cell pertussis vaccine, it has been gradually replaced by the acellular pertussis vaccine with fewer side effects and better immune effects.

[0003] Pertussis toxin (PT) is a major virulence factor produced by Bordetella pertussis of the genus Bordetella, with various biological activities. It is the main factor causing many clinical symptoms of whooping cough and is also the only antigen component in the acellular pertussis vaccine that is not controversial. After detoxification, it can be used as an important and effective component of the acellular pertussis vaccine.

[0004] Currently, electrophoresis, Kjeldahl method, and Lowry method are mostly used to detect the contents of various components of whooping cough. Electrophoresis can only perform semi-quantitative analysis, while the Kjeldahl method and the Lowry method can only quantify total proteins and are not specific. Therefore, it is necessary to establish an accurate and specific method for detecting pertussis toxin (PT) antigen for quality control in the production of acellular pertussis vaccines. There are records in the prior art of detecting PT using ELISA kits, such as CN117736317A, CN108254556A, etc. However, for the implementation of the double-antibody sandwich ELISA method in these two patents, the paired antibodies used are monoclonal antibody - polyclonal antibody (i.e., the capture antibody is the mouse monoclonal antibody described in the patent, and the detection antibody is rabbit polyclonal antibody), and the specificity and sensitivity during the detection process need to be further improved.

[0005] Therefore, there is an urgent need in the art to develop a new method for detecting PT. Summary of the Invention

[0006] Based on this, it is necessary to provide at least one monoclonal antibody, an antibody combination containing the same, and their uses in detecting pertussis toxin.

[0007] In the first aspect of the present application, a monoclonal antibody specifically binding to pertussis toxin is provided, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.

[0008] In the second aspect of the present application, an antibody combination is provided, which comprises the monoclonal antibody as described in the first aspect as a capture antibody.

[0009] In the third aspect of the present application, a nucleic acid is provided, which encodes the monoclonal antibody as described in the first aspect, or the antibody combination as described in the second aspect.

[0010] In the fourth aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid as described in the third aspect.

[0011] In the fifth aspect of the present application, a host cell is provided, which expresses the monoclonal antibody as described in the first aspect, or the antibody combination as described in the second aspect.

[0012] In the sixth aspect of the present application, a kit for detecting pertussis toxin is provided, which comprises the monoclonal antibody as described in the first aspect, or the antibody combination as described in the second aspect.

[0013] In the seventh aspect of the present application, the use of the monoclonal antibody as described in the first aspect or the antibody combination as described in the second aspect in the preparation of a reagent or kit for detecting pertussis toxin is provided.

[0014] In the eighth aspect of the present application, a method for detecting pertussis toxin for non-diagnostic purposes is provided, which comprises the following steps:

[0015] Coating: Dilute the capture antibody with a coating solution and add it to an enzyme-linked immunosorbent assay (ELISA) plate for coating, then wash and remove the excess residual liquid;

[0016] Blocking: Add a blocking solution to the ELISA plate, then wash and remove the excess residual liquid;

[0017] Incubating the sample: Add the sample to be tested to the ELISA plate, after reaction, wash and remove the excess residual liquid;

[0018] Incubating the enzyme-labeled antibody: Add the detection antibody labeled with a biological marker or a chemical marker to the ELISA plate, after reaction, wash and remove the excess residual liquid;

[0019] Color development: Add a chromogenic solution for incubation;

[0020] Termination: Add the termination solution to terminate the reaction;

[0021] Detection: Read the OD on an enzyme-linked immunosorbent assay (ELISA) reader 405 value.

[0022] Use of the monoclonal antibody as described in the first aspect, the antibody as described in the second aspect, or the kit as described in the sixth aspect of the present application in the quality detection of a vaccine containing pertussis toxin antigen.

[0023] A specific monoclonal antibody was screened using the hybridoma technology. It can be used as a capture antibody in ELISA sandwich detection, and a paired detection antibody was also screened. These two antibodies specifically bind to different antigenic epitopes of pertussis toxin (PT) and do not interfere with each other. The established double-antibody sandwich ELISA method using these antibodies has excellent sensitivity, stability, and reproducibility, and can be applied to the quantitative detection and quality control of pertussis toxin (PT) in all aspects of diphtheria, pertussis, and tetanus (DPT) vaccine production. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments and implementation manners of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or implementation manners. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in the description of the present application.

[0025] Figure 1 This is the gel electrophoresis diagram of monoclonal antibody 48H7 in an embodiment of the present application.

[0026] Figure 2 This is the gel electrophoresis diagram of monoclonal antibody 45H5 in an embodiment of the present application.

[0027] Figure 3 This is the screening of the capture antibody concentration in an embodiment of the present application (relationship diagram between the capture antibody concentration and the OD 405 value when the antigen concentration is 250 ng / ml).

[0028] Figure 4 This is the screening of the detection antibody concentration in an embodiment of the present application (relationship diagram between the detection antibody concentration and the OD 405 value when the antigen concentration is 31 ng / ml).

[0029] Figure 5 This is the standard curve in an embodiment of the present application.

[0030] Figure 6This is a superimposed graph of standard curves for PT quantitative detection at different reaction times in one embodiment of the present application.

[0031] Figure 7 This is a superimposed graph of standard curves for PT quantitative detection at different reaction temperatures in one embodiment of the present application.

[0032] Figure 8 This is a superimposed graph of standard curves for PT quantitative detection at different post-termination standing times in one embodiment of the present application. Detailed implementation manners

[0033] For ease of understanding the present application, the present application will be described more comprehensively below with reference to the relevant attached drawings. Preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] In the present application, unless otherwise stated, "one or more" means any one of the listed items or any combination of the listed items. Similarly, in other cases where "one or more" and the like are used to mean "one or more", the same understanding shall be made unless otherwise stated.

[0036] The "combinations thereof", "any combinations thereof", "any combination manners thereof", etc. used in the present application include all suitable combination manners of any two or more of the listed items.

[0037] In the present application, "suitable combination manners", "suitable manners", "any suitable manners", etc., the "suitable" is subject to being able to implement the technical solution of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application.

[0038] In the present application, "further", "even further", "especially", "for example", "such as", "example", "exemplification", etc. are used for descriptive purposes, indicating that there is an association in the covered content between the different technical solutions before and after, but it should not be understood as a limitation on the previous technical solution, nor should it be understood as a limitation on the protection scope of the present application. In the present application, unless otherwise stated, A (such as B) means that B is a non-limiting example in A, and it can be understood that A is not limited to B.

[0039] In this application, "optionally", "optional", and "option" mean that it is optional, that is, it refers to any one of two alternative options of "yes" or "no". If the term "optional" appears multiple times in a technical solution, without special instructions, and without contradictions or mutual restrictions, each "optional" is independent. Unless otherwise specified, descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or may not include".

[0040] The terms "comprising", "containing", and "including" used in this application are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0041] In this application, in a technical feature or technical solution described in an open language, it includes a closed technical feature or technical solution composed of the listed content, and also includes an open technical feature or technical solution containing the listed content.

[0042] In this application, the exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may cover, but are not limited to, the following meanings: These solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0043] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within the numerical interval is considered continuous, and includes the two numerical endpoints of the numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical values" in the numerical interval can be any quantitative values, such as numbers, percentages, ratios, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0045] In this application, when a method process involves multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order limit, and it can be executed in an order other than the described one. Moreover, any one step can include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be executed at the same time, but can be executed at different times, and their execution order does not necessarily need to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0046] One aspect of this application provides a monoclonal antibody that specifically binds to pertussis toxin, which comprises a heavy chain variable region and a light chain variable region.

[0047] Unless otherwise specified, the terms "specifically bind", "selectively bind", "selectively binds", and "specifically binds" in this application refer to the binding of an antibody to an epitope on a pre-determined antigen (such as pertussis toxin).

[0048] Unless otherwise specified, the term "monoclonal antibody" or "mAb" or "monoclonal antibody composition" in this application refers to an antibody molecule product composed of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0049] In some embodiments, the heavy chain variable region of the monoclonal antibody comprises HCDR1, HCDR2, and HCDR3 with sequences shown in SEQ ID NO:1 (GYSITSDYA), SEQ ID NO:2 (IGYSGNT), and SEQ ID NO:3 (ARGSNLSYDYDGFAY) respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with sequences shown in SEQ ID NO:4 (ENIHNF), SEQ ID NO:5 (NAK), and SEQ ID NO:6 (QHFWSTPLT) respectively.

[0050] The monoclonal antibody of the present application may have the above CDRs or a derivative fragment of the above CDRs. The derivative fragment is formed by no more than 6-site amino acid substitutions ("conservative modification" or "conservative substitution or replacement") relative to its corresponding CDR, and retains the biological activity consistent with its corresponding complementarity-determining region. For example, the derivative fragment may have substitutions at 1, 2, 3, 4, 5, or 6 sites relative to its corresponding complementarity-determining region. It may be that one amino acid is replaced by another amino acid, or one amino acid is replaced by multiple (such as 2) amino acids.

[0051] Among the CDRs provided in the present application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, or 3 amino acids in the amino acid sequence of the antibody of the present application with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0052] Table A

[0053] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0054] "Conservative modification" or "conservative substitution or replacement" means replacing an amino acid in a protein with other amino acids having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.), so that changes can be frequently made without changing the biological activity of the protein. Those skilled in the art know that generally, a single amino acid substitution in a non-essential region of a polypeptide basically does not change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). In addition, substitutions of amino acids with similar structures or functions are unlikely to destroy the biological activity.

[0055] In some embodiments, the heavy chain variable region comprises an amino acid fragment having a sequence as shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid fragment having a sequence as shown in SEQ ID NO:8.

[0056] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGFIGYSGNTAYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGSNLSYDYDGFAYWGQGTLVTVSA(SEQ ID NO:7)

[0057] DIQMTQSPVSLSASVGETVTITCRASENIHNFLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPLTFGAGTKLELK(SEQ ID NO:8)

[0058] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 or has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity with the amino acid sequence of SEQ ID NO:7.

[0059] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 or has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity with the amino acid sequence of SEQ ID NO:8.

[0060] "Amino acid sequence ""identity"" means the percentage of amino acid residues in a first sequence that are identical with the amino acid residues in a second sequence when the amino acid sequences are aligned (introducing gaps if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, the alignment can be achieved in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequences being compared.

[0061] In some embodiments, the above monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid fragment having a sequence as shown in SEQ ID NO:9, and the light chain comprising an amino acid fragment having a sequence as shown in SEQ ID NO:10.

[0062] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGFIGYSGNTAYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGSNLSYDYDGFAYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO:9)

[0063] DIQMTQSPVSLSASVGETVTITCRASENIHNFLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10)

[0064] Another aspect of the present application provides an antibody combination comprising the monoclonal antibody as described above as a capture antibody.

[0065] Without wishing to be bound by any theory, it is believed that the functions of the capture antibody include at least immobilizing on the surface of a solid-phase carrier (such as an ELISA plate) for specifically "capturing" the target antigen in the sample to be tested.

[0066] In some embodiments, the antibody combination further comprises a detection antibody. Exemplarily, the detection antibody is a monoclonal antibody.

[0067] It should be understood that the detection antibody can bind to the captured antigen and can be used for signal detection and amplification.

[0068] Without wishing to be bound by any theory, it is believed that compared with the pairing of mouse monoclonal antibody - rabbit polyclonal antibody in the current technology, the paired monoclonal antibodies often have better specificity and sensitivity during application (such as the implementation of sandwich ELISA).

[0069] Exemplarily, the detection antibody comprises a heavy chain variable region and a light chain variable region.

[0070] In some embodiments, the heavy chain variable region of the detection antibody comprises HCDR1, HCDR2, and HCDR3 with sequences shown in SEQ ID NO:11 (GYSFTGYT), SEQ ID NO:12 (INPYNGGT), and SEQ ID NO:13 (VRADGYYWYFDV) respectively, and the light chain variable region of the detection antibody comprises LCDR1, LCDR2, and LCDR3 with sequences shown in SEQ ID NO:14 (QSLLYSSNQKNY), SEQ ID NO:15 (WAS), and SEQ ID NO:16 (QQYYTYT) respectively.

[0071] In some embodiments, the heavy chain variable region of the detection antibody comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 17, and the light chain variable region of the detection antibody comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 18.

[0072] EVQLQQSGPELVKPGDSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYNQKFK GKATLTVDRSSSTAYMEVLSLTSEDSAVYYCVRADGYYWYFDVWGAGTTVTVSS(SEQ ID NO:17)

[0073] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYTFGGGTKLEIK(SEQ ID NO:18)

[0074] In some embodiments, the amino acid sequence of the heavy chain variable region of the detection antibody is as shown in SEQ ID NO: 17 or has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to the amino acid sequence of SEQ ID NO: 17.

[0075] In some embodiments, the amino acid sequence of the light chain variable region of the detection antibody is as shown in SEQ ID NO: 18 or has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to the amino acid sequence of SEQ ID NO: 18.

[0076] In some embodiments, the detection antibody comprises a heavy chain and a light chain. Exemplarily, the heavy chain comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 19, and the light chain comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 20.

[0077] EVQLQQSGPELVKPGDSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYNQKFKGKATLTVDRSSSTAYMEVLSLTSEDSAVYYCVRADGYYWYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQID NO:19)

[0078] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIAKSFNRNEC(SEQ ID NO:20)

[0079] Another aspect of the present application also provides a nucleic acid encoding the monoclonal antibody or antibody combination as described above.

[0080] Unless otherwise specified, the term "nucleic acid" in the present application may refer to a nucleic acid molecule, such as an isolated nucleic acid molecule. The nucleic acid molecule in the present application mainly refers to an isolated nucleic acid molecule. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" does not mean that these materials are completely absent or that water, buffer, or salt is absent.

[0081] Exemplarily, the nucleic acid encodes the heavy chain variable region of the above monoclonal antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain variable region of the above monoclonal antibody is as shown in SEQ ID NO:21, or comprises the sequence as shown in SEQ ID NO:21.

[0082] GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTTCATAGGCTACAGTGGTAACACTGCCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGCTCCAATCTTTCCTATGATTACGACGGTTTTGCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ IDNO:21)

[0083] Exemplarily, the nucleic acid encodes the light chain variable region of the above monoclonal antibody. In some embodiments, the sequence of the nucleic acid encoding the light chain variable region of the above monoclonal antibody is as shown in SEQ ID NO:23, or comprises the sequence as shown in SEQ ID NO:23.

[0084] GACATCCAGATGACTCAGTCTCCAGTCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAAAATATTCACAATTTTTTAGCGTGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:23)Exemplarily, the nucleic acid encodes the heavy chain of the above monoclonal antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain of the above monoclonal antibody is as shown in SEQ ID NO:22, or comprises the sequence as shown in SEQ ID NO:22.

[0085]

[0086] Exemplarily, the nucleic acid encodes the light chain of the above monoclonal antibody. In some embodiments, the sequence of the nucleic acid encoding the light chain of the above monoclonal antibody is as shown in SEQ ID NO:24, or comprises the sequence as shown in SEQ ID NO:24.

[0087] GACATCCAGATGACTCAGTCTCCAGTCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAAAATATTCACAATTTTTTAGCGTGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGA(SEQ ID NO:24)

[0088] Exemplarily, the nucleic acid encodes the heavy chain variable region of the above detection antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain variable region of the above detection antibody is as shown in SEQ ID NO:25, or comprises the sequence as shown in SEQ ID NO:25.

[0089] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGATTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGGTACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAGGTCATCCAGCACAGCCTACATGGAGGTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGAGCCGATGGTTACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:25)

[0090] Exemplarily, the nucleic acid encodes the light chain variable region of the above-mentioned detection antibody. In some embodiments, the sequence of the nucleic acid encoding the light chain variable region of the above-mentioned detection antibody is as shown in SEQ ID NO:27, or comprises the sequence as shown in SEQ ID NO:27.

[0091] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAACAATATTATACCTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO:27) Exemplarily, the nucleic acid encodes the heavy chain of the above-mentioned detection antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain of the above-mentioned detection antibody is as shown in SEQ IDNO:26, or comprises the sequence as shown in SEQ ID NO:26.

[0092]

[0093] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAACAATATTATACCTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGCCAAGAGCTTCAACAGGAATGAGTGTTGA(SEQ ID NO:28)Another aspect of the present application provides a recombinant expression vector comprising the nucleic acid as described above.

[0094] The term "vector", which may also be referred to as "nucleic acid construct", refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and thereby replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes effectively ligated thereto. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in the present application. Generally, expression vectors useful in recombinant DNA technology usually exist in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

[0095] In the present application, the above-mentioned recombinant expression vector may contain a nucleic acid encoding the heavy chain of the above-mentioned monoclonal antibody. In some embodiments, the nucleic acid further contains a sequence encoding a leader peptide of the heavy chain. Exemplarily, the leader peptide contains an amino acid fragment having a sequence as shown in SEQ ID NO: 29 (MRVLILLWLFTAFPGILS). Optionally, the nucleic acid encoding it contains a nucleic acid sequence as shown in SEQ ID NO: 30 (ATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCT).

[0096] In the present application, the above-mentioned recombinant expression vector may contain a nucleic acid encoding the light chain of the above-mentioned monoclonal antibody. In some embodiments, the nucleic acid further contains a sequence encoding a leader peptide of the light chain. Exemplarily, the leader peptide contains an amino acid fragment having a sequence as shown in SEQ ID NO: 31 (MSVLTQVLALLLLWLTGARC). Optionally, the nucleic acid encoding it contains a nucleic acid sequence as shown in SEQ ID NO: 32 (ATGAGTGTGCTCACTCAGGTCCTGGCGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT).

[0097] In the present application, the above-mentioned recombinant expression vector may contain a nucleic acid encoding the heavy chain of the above-mentioned detection antibody. In some embodiments, the nucleic acid further contains a sequence encoding the leader peptide of the heavy chain. Exemplarily, the leader peptide contains an amino acid fragment with a sequence as shown in SEQ ID NO: 33 (MGWSWIFLFLLSGTAGVHS). Optionally, the nucleic acid encoding it contains a nucleic acid sequence as shown in SEQ ID NO: 34 (ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT).

[0098] In the present application, the above-mentioned recombinant expression vector may contain a nucleic acid encoding the light chain of the above-mentioned detection antibody. In some embodiments, the nucleic acid further contains a sequence encoding the leader peptide of the light chain. Exemplarily, the leader peptide contains an amino acid fragment with a sequence as shown in SEQ ID NO: 35 (MDSQAQVLMLLLLWVSGTCG). Optionally, the nucleic acid encoding it contains a nucleic acid sequence as shown in SEQ ID NO: 36 (ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGG).

[0099] Another aspect of the present application provides a host cell that expresses the monoclonal antibody as described above, or the antibody combination as described above.

[0100] The term "cell", also referred to as "host cell", refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Bacteria that are easily transformable include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0101] Another aspect of the present application provides a method for preparing the above-mentioned host cell, including the step of introducing the above-mentioned or recombinant expression vector into a target cell.

[0102] In one example, the introduction is carried out by transfection.

[0103] The term "transfection" refers to the process of introducing nucleic acids into cells, such as non-human mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0104] Another aspect of the present application provides a method for producing the monoclonal antibody or antibody combination as described above, comprising: culturing a host cell, and isolating the monoclonal antibody or antibody combination from the resulting culture supernatant.

[0105] Another aspect of the present application provides a product for detecting pertussis toxin, which comprises the monoclonal antibody or antibody combination as described above.

[0106] In some embodiments, the product comprises the above antibody combination, wherein the detection antibody is biolabeled or chemically labeled.

[0107] In some embodiments, the detection antibody is enzyme-labeled, including but not limited to being labeled with horseradish peroxidase or alkaline phosphatase.

[0108] In some embodiments, the product further comprises one or more of a coating solution, a blocking solution, a dilution solution, a substrate buffer solution, a washing solution, a chromogenic solution, and a termination solution.

[0109] Among them, the coating solution is, for example, or includes a carbonate buffer solution.

[0110] The blocking solution is optionally a 1% w / v - 4% w / v BSA-PBST solution, such as a 1% w / v BSA-PBST solution, a 1.5% w / v BSA-PBST solution, a 2% w / v BSA-PBST solution, a 2.5% w / v BSA-PBST solution, a 3% w / v BSA-PBST solution, a 3.5% w / v BSA-PBST solution, a 4% w / v BSA-PBST solution, or a range or value between any two values. Exemplarily, the meaning of a 1% w / v BSA-PBST solution is 1 g of BSA (Bovine Serum Albumin) dissolved in 100 ml of PBST solution (phosphate buffer solution).

[0111] Without wishing to be bound by any theory, it has been found that when the concentration of BSA-PBST in the blocking solution is less than 1% w / v, the blank value of ELISA detection may be too high; when it is greater than 4% w / v, the sensitivity of the ELISA detection method may decrease.

[0112] The diluent is optionally a 0.2% w / v to 1% w / v BSA-PBST solution, such as a 0.2% w / v BSA-PBST solution, a 0.3% w / v BSA-PBST solution, a 0.4% w / v BSA-PBST solution, a 0.5% w / v BSA-PBST solution, a 0.6% w / v BSA-PBST solution, a 0.7% w / v BSA-PBST solution, a 0.8% w / v BSA-PBST solution, a 0.9% w / v BSA-PBST solution, a 1% w / v BSA-PBST solution, etc.

[0113] Without wishing to be bound by any theory, it has been found that when the concentration of BSA-PBST in the diluent is less than 0.2% w / v, the blank value of ELISA detection may be too high; when it is greater than 1% w / v, the sensitivity of the ELISA detection method may decrease.

[0114] The above substrate buffer is optionally a citric acid solution, such as a citric acid solution with a concentration of 0.05 M.

[0115] The embodiments of the present application do not particularly limit the types of detection products, such as diagnostic reagents, test strips, test plates or reagent kits. In the detection product, the monoclonal antibody competes with the pertussis toxin in the sample to be detected.

[0116] When the term "compete" is used in the context of antigen-binding proteins that compete for the same epitope, it means competition between the antigen-binding proteins, which is determined by the following assay: in the assay, the antigen-binding protein to be detected (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such assays including: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assay, solid-phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA with I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct label RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Generally, the assay involves using a purified antigen that binds to a solid surface or cell bearing either the unlabeled test antigen-binding protein or the labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Generally, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope adjacent to the binding epitope of the reference antigen-binding protein that is sufficiently close such that the two epitopes sterically hinder binding. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Generally, when the competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more of the reference antigen-binding protein to the common antigen. In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97% or more.

[0117] Another aspect of the present application also provides the use of the monoclonal antibody as described above or the antibody combination as described above in the preparation of a product for detecting pertussis toxin.

[0118] Another aspect of the present application also provides a method for detecting pertussis toxin using the monoclonal antibody as described above or the antibody combination as described above in the preparation of a product for detecting pertussis toxin.

[0119] Exemplarily, the method is a sandwich ELISA quantitative detection method.

[0120] In some embodiments, the above method comprises the following steps:

[0121] Coating: Dilute the capture antibody with coating buffer and add it to the ELISA plate for coating, then wash and remove the excess residual liquid (such as by centrifugation);

[0122] Blocking: Add blocking buffer to the ELISA plate, then wash and remove the excess residual liquid (such as by centrifugation);

[0123] Incubating the sample: Add the sample to be tested to the ELISA plate, wash and remove the excess residual liquid (such as by centrifugation) after reaction;

[0124] Incubating the enzyme-labeled antibody: Add the detection antibody labeled with a biological or chemical label to the ELISA plate, wash and remove the excess residual liquid (such as by centrifugation) after reaction;

[0125] Color development: Add the chromogenic solution for incubation;

[0126] Termination: Add the termination solution to terminate the reaction;

[0127] Detection: Read the OD 405 value;

[0128] Wherein, the capture antibody is as defined above, and the detection antibody is as defined above.

[0129] Unless otherwise specified, the term "OD 405 " in the present application refers to the measured value of the optical density (OD) at a wavelength of 405 nm, and the concentration or activity of a specific substance in the sample is determined by this measured value. Exemplarily, some chromogenic reactions (such as the termination product after horseradish peroxidase HRP catalyzes ABTS chromogenesis) can be detected at 405 nm.

[0130] The above method can be for diagnostic purposes or non-diagnostic purposes.

[0131] In some embodiments, in the coating step, the concentration of the capture antibody in the ELISA plate is 0.5 μg / ml to 16 μg / ml. Exemplarily, for example, it is 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, or the range or value between any two values. In some embodiments, the concentration of the capture antibody is 2 μg / ml to 8 μg / ml.

[0132] In some embodiments, in the step of incubating the enzyme-labeled antibody, the concentration of the detection antibody in the ELISA plate is 0.05 μg / ml to 5 μg / ml; for example, 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.1 μg / ml, 1.2 μg / ml, 1.3 μg / ml, 1.4 μg / ml, 1.5 μg / ml, or the range or value between any two values.

[0133] In some embodiments, the concentration of the detection antibody in the ELISA plate is 0.8 μg / ml to 1.5 μg / ml.

[0134] Exemplarily, this method is implemented using the product (such as a kit) described above.

[0135] In some embodiments, this method includes the following steps:

[0136] (1) Coating: Dilute the monoclonal antibody 48H7 to 2 μg / ml with the coating solution, 100 μl / well, and place it at 2 - 8 °C for overnight coating;

[0137] (2) Washing the plate: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of the washing solution, and pat dry the residual liquid in the ELISA plate;

[0138] (3) Blocking: Add the blocking solution to the ELISA plate at 150 μl / well, and block at 37 °C for 2 h;

[0139] (4) Washing the plate: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of the washing solution, and pat dry the residual liquid in the ELISA plate;

[0140] (5) Incubating the samples: Set up standard wells, blank wells, and sample wells. Add different concentrations of standards to the standard wells in sequence, add the sample diluent to the blank well, and add the samples to be tested to the sample wells. Add the samples at 100 μl / well, and incubate at 37 °C for 1 h;

[0141] (6) Plate washing: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of the washing solution, and pat dry the residual liquid in the microplate reader;

[0142] (7) Incubate the enzyme-labeled antibody: Dilute the monoclonal antibody 45H5 labeled with HRP to 1 μg / ml with the diluent, add the sample at 100 μl / well, and incubate at 37 °C for 1 h;

[0143] (8) Plate washing: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of the washing solution, and pat dry the residual liquid in the microplate reader;

[0144] (9) Color development: Add the color developing solution to the microplate at 100 μl / well and develop color at 37 °C in the dark for 30 min;

[0145] (10) Termination: Add the termination solution to the microplate at 50 μl / well to terminate the reaction;

[0146] (11) Reading: Place the microplate in the microplate reader to measure the OD 405nm absorbance;

[0147] (12) Result processing: Subtract the OD value of the blank well from the average OD value of each standard to obtain the correction value. Use the OD 405nm value of the standard as the dependent variable Y, and use the standard concentration as the independent variable X to make a curve; Use the four-parameter Logistic mathematical model to fit the equation: Y = ((A - D) / (1 + (X / C)^B)) + D, and substitute the OD 405nm absorbance value of the sample into the formula to calculate the content of pertussis toxin in the sample; where A is the estimated value of the asymptote on the curve, D is the estimated value of the asymptote under the curve, B is the slope of the curve, and C is the dose corresponding to half of the maximum binding.

[0148] Another aspect of the present application provides the use of the monoclonal antibody, antibody combination, product (such as a kit) as described above in the quality detection of vaccines containing pertussis toxin antigen.

[0149] Exemplarily, the scenarios of quality detection include but are not limited to production quality control, laboratory testing, and quality sampling inspection, etc.

[0150] Exemplarily, the vaccine is a pertussis vaccine.

[0151] Some embodiments are provided below.

[0152] The implementation plan of the present application will be described in detail below in combination with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following embodiments, the guidance given in the present application shall be preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0153] Example 1 Preparation of Mouse Monoclonal Antibody Against Pertussis Toxin (PT)

[0154] 1.1 Preparation of Immunogen

[0155] The detoxified pertussis toxin after purification (concentration 200 μg / ml) was mixed with adjuvant in equal volume and emulsified evenly to form a water-in-oil state to prepare a vaccine for immunizing mice. The adjuvant used for the primary immunization was Freund's complete adjuvant, and the adjuvant used for the booster immunization was Freund's incomplete adjuvant.

[0156] 1.2 Immunization Strategy

[0157] The immunized animals were SPF-grade Balb / C mice, female, 4 - 6 weeks old. Each mouse was immunized subcutaneously 3 times, with 20 μg of immunogen per mouse each time, and the interval between each immunization was 2 weeks. After the third immunization, blood was collected from the cheek, the serum was separated, and the antibody titer was detected by the indirect ELISA method, and the antibody titer was required to reach above 1:800000.

[0158] 1.3 Cell Fusion

[0159] Mouse myeloma cells SP2 / 0 were prepared, and the cell state was adjusted to the logarithmic growth phase. The mouse with the highest ELISA titer was selected, and the mouse was boosted immunized intraperitoneally 3 days before fusion. After 3 days, the mouse spleen was taken, the spleen cells were collected after mechanical fragmentation, filtered through a 200-mesh sieve, and washed 3 times with PBS; the SP2 / 0 cells were collected and washed 3 times with PBS; after cell counting, they were mixed according to the quantity ratio of SP2 / 0: spleen cells = 1:2.5, and the PBS was discarded after centrifugation, and a cell fusion instrument was used for cell fusion. After the fusion was completed, the cells were centrifuged and then added to a complete medium containing HAT (hypoxanthine, aminopterin, and thymidine), and the cells were resuspended and mixed evenly and then plated in 96-well plates; a semi-medium change was performed 5 days after fusion.

[0160] 1.4 Screening of Hybridomas

[0161] The cell culture supernatant aspirated 7 days after fusion was detected by the indirect ELISA method, the positive cell wells were recorded, marked, and a semi-medium change was performed; the next day, the positive wells with replenished medium were rechecked, and the clone numbers with stable titers were recorded.

[0162] 1.5 Establishment of Stable Cell Lines

[0163] 1.5.1 First subcloning

[0164] Select specific positive cell wells from 1.4 for subcloning screening. Using the limited dilution method, in complete medium, seed the cells at a density of 1 cell per well in a 96-well plate. Observe under the microscope 7 days later, mark the monoclonal wells, and perform ELISA detection the next day. Discard the clone numbers that turn negative. For the positive clones, select the wells with vigorous growth for a single medium replenishment;

[0165] 1.5.2 Second subcloning

[0166] Select specific positive cell wells from the previous step for subcloning screening. Using the limited dilution method, in complete medium, seed the cells at a density of 1 cell per well in a 96-well plate; Observe under the microscope 7 days later, mark the monoclonal wells, and perform ELISA detection the next day. When the positive rate of ELISA detection reaches 100%, select the wells with vigorous growth for expansion culture and cryopreservation for storage.

[0167] 1.6 Antibody subtype identification

[0168] Use the indirect ELISA method to detect the cell culture supernatant of the stable cell line. The secondary antibody is the goat anti-mouse heavy chain subtype-specific secondary antibody labeled with HRP. At the same time, set a positive control (goat anti-mouse IgG(H+L) labeled with HRP) and a negative control (antibody diluent). The antibody subtype of the mouse anti-pertussis toxin (PT) monoclonal antibody 48H7 is IgG2b; the antibody subtype of the mouse anti-pertussis toxin (PT) monoclonal antibody 45H5 is IgG1.

[0169] 1.7 Ascites preparation and purification

[0170] Inject the stable hybridoma cells into the peritoneal cavity of mice, 1×10 6 cells / mouse. Sacrifice the mice 10 days later when the peritoneal cavity of the mice is significantly swollen, and collect the ascites. After centrifugation of the ascites, filter it through a 0.22μm pore size filter membrane, and then purify it using Protein G. The purified antibody is exchanged and concentrated with an ultrafiltration tube, and finally exchanged to PBS and sterile filtered through a 0.22μm pore size filter membrane in a laminar flow hood, and stored at -20°C for later use.

[0171] 1.8 Detection of titer, purity and concentration of the purified antibody

[0172] 1.8.1 Antibody titer detection

[0173] The purified antibodies were detected by indirect ELISA. The antibody titers of mouse anti - pertussis toxin (PT) monoclonal antibody 48H7 and mouse anti - pertussis toxin (PT) monoclonal antibody 45H5 were both > 1:1000000. The purity of the antibodies was detected by SDS - PAGE, and the antibody purity reached over 95%. Moreover, detected by Nano - 500, the concentrations of mouse anti - pertussis toxin (PT) monoclonal antibody 48H7 and 45H5 were both 2 mg / ml.

[0174] 1.8.2 Concentration determination

[0175] The concentrations of the antibodies were detected using Nano - 500. The concentration of mouse anti - pertussis toxin (PT) monoclonal antibody 48H7 was 2 mg / ml; the concentration of mouse anti - pertussis toxin (PT) monoclonal antibody 45H5 was 2 mg / ml.

[0176] 1.9 Purification, HRP labeling and titer detection of antibodies

[0177] The purified monoclonal antibodies were freeze - dried and then conjugated with HRP. The HRP - conjugated antibodies were detected by indirect ELISA. The antibody titers of HRP - monoclonal antibody 48H7 and HRP - monoclonal antibody 45H5 were both > 1:100000.

[0178] Full - length sequencing of monoclonal antibody genes from hybridoma cells

[0179] (1) Total RNA was extracted from lysed hybridoma cells;

[0180] (2) RNA was reverse - transcribed into cDNA using RACE technology;

[0181] (3) Full - length heavy and light chains were obtained by PCR amplification;

[0182] (4) The target fragments were ligated to vectors using ligase, and the ligation products were transformed into competent Escherichia coli cells. Then, single colonies were picked for sequencing;

[0183] (5) Analysis and annotation of the sequencing results were performed.

[0184] The heavy chain amino acid sequence of the monoclonal antibody 48H7 is as shown in SEQ ID NO:9 (the encoding nucleic acid sequence is as shown in SEQ ID NO:22), and the light chain amino acid sequence is as shown in SEQ ID NO:10 (the encoding nucleic acid sequence is as shown in SEQ ID NO:24); the heavy chain variable region amino acid sequence is as shown in SEQ ID NO:7 (the encoding nucleic acid sequence is as shown in SEQ ID NO:21), and the light chain variable region amino acid sequence is as shown in SEQ ID NO:8 (the encoding nucleic acid sequence is as shown in SEQ ID NO:23). According to the Chothia definition, the amino acid sequences of its HCDR1-HCDR3 are respectively as shown in SEQ ID NO:1-3, and the amino acid sequences of its LCDR1-LCDR3 are respectively as shown in SEQ ID NO:4-6.

[0185] The heavy chain amino acid sequence of the monoclonal antibody 45H5 is as shown in SEQ ID NO:19 (the encoding nucleic acid sequence is as shown in SEQ ID NO:26), and the light chain amino acid sequence is as shown in SEQ ID NO:20 (the encoding nucleic acid sequence is as shown in SEQ ID NO:28). The heavy chain variable region amino acid sequence is as shown in SEQ ID NO:17 (the encoding nucleic acid sequence is as shown in SEQ ID NO:25), and the light chain variable region amino acid sequence is as shown in SEQ ID NO:18 (the encoding nucleic acid sequence is as shown in SEQ ID NO:27). According to the Chothia definition, the amino acid sequences of its HCDR1-HCDR3 are respectively as shown in SEQ ID NO:11-13, and the amino acid sequences of its LCDR1-LCDR3 are respectively as shown in SEQ ID NO:14-16.

[0186] Example 2 Establishment and verification of a double antibody sandwich ELISA quantitative detection method for pertussis toxin (PT)

[0187] 2.1 Materials and instruments: Monoclonal antibody 48H7, HRP-labeled monoclonal antibody 45H5, pertussis toxin (PT) antigen reference product, coating solution, washing solution, blocking solution, dilution solution, substrate buffer solution, chromogenic solution and termination solution; enzyme-linked immunosorbent assay (ELISA) plate; constant temperature incubator, microplate reader, plate washer.

[0188] 2.2 Solution preparation

[0189] 2.2.1 Coating solution (0.05M carbonate buffer, pH 9.6): Accurately weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, and dissolve them in 1.0 L of purified water.

[0190] 2.2.2 10×PBS (0.1 M Phosphate Buffered Saline): Accurately weigh 80.0 g of sodium chloride, 2.0 g of potassium dihydrogen phosphate, 14.3 g of disodium hydrogen phosphate dihydrate (28.8 g of disodium hydrogen phosphate dodecahydrate), and 2.0 g of potassium chloride. Stir and dissolve in 1.0 L of purified water. Dilute 10-fold with purified water before use.

[0191] 2.2.3 Washing Solution (PBST Solution): Accurately measure 1 L of 1×PBS solution, add 500 μl of Tween 20, and stir until completely dissolved. Set aside for use.

[0192] 2.2.4 Blocking Solution: Accurately weigh 4.0 g of BSA and dissolve it in 100 ml of PBST solution.

[0193] 2.2.5 Dilution Solution: Accurately weigh 1.0 g of BSA and dissolve it in 100 ml of PBST solution.

[0194] 2.2.6 Substrate Buffer (0.05 M Citric Acid Solution): Accurately weigh 10.51 g of citric acid monohydrate (9.607 g of anhydrous citric acid) and dissolve it in 1 L of pure water. Adjust the pH to 4.0 with 400 g / L NaOH solution.

[0195] 2.2.7 Chromogenic Solution (0.05% ABTS): Shortly before use, accurately weigh 0.01 g of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and dissolve it in 20 ml of 0.05 M citric acid solution. Immediately add 5 μl of 30% H2O2 solution before use.

[0196] 2.2.8 Stop Solution (1% SDS): Accurately weigh 1.0 g of SDS and dissolve it in 100 ml of pure water.

[0197] 2.3 Establishment of a Quantitative Detection Method for Pertussis Toxin (PT) by Double Antibody Sandwich ELISA

[0198] 2.3.1 Determination of Capture Antibody and Detection Antibody

[0199] Capture antibody concentration: 1 μg / ml; Sandwich antigen concentration: starting from 1000 ng / ml, diluted serially by 3-fold, a total of 7 gradients, and a blank control was set up simultaneously; Detection antibody concentration: 0.1 μg / ml. Perform ELISA detection. The results showed that when the capture antibody was 48H7 and the detection antibody was HRP-45H5, the optical absorption value of the standard product was relatively high and the blank value was relatively low (high P / N value), which was a better pairing (see Table 1 for reference).

[0200] Table 1 Screening of Antibody Pairings

[0201]

[0202] 2.3.2 Capture Antibody Concentration Confirmation

[0203] The capture antibody concentration is 0.5 μg / ml to 16 μg / ml; the sandwich antigen concentration starts from 1000 ng / ml and is diluted 2-fold in a serial dilution, with a total of 7 gradients. At the same time, a blank control is set up; the detection antibody concentration is 0.1 μg / ml. ELISA detection is carried out. The results show that when the coating concentration is 0.5 - 1 μg / ml, as the coating concentration increases, the OD 405 value shows an obvious upward trend; when the coating concentration is 1 - 16 μg / ml, the OD 405 values are not much different and basically tend to be stable. The capture antibody coating concentration is determined to be 2 μg / ml (see Figure 3 ).

[0204] 2.3.3 Detection Antibody Concentration Confirmation

[0205] The capture antibody concentration is 2 μg / ml; the sandwich antigen concentration starts from 1000 ng / ml and is diluted 2-fold in a serial dilution, with a total of 7 gradients. At the same time, a blank control is set up; the detection antibody concentration is 0.05 μg / ml to 5 μg / ml. ELISA detection is carried out. The results show that when the detection antibody concentration is 0.05 - 1 μg / ml, as the detection antibody concentration increases, the OD value shows an obvious upward trend; when the detection antibody concentration is 1 - 5 μg / ml, the OD 405 values are not much different and basically tend to be stable. The detection antibody concentration is determined to be 1 μg / ml (see Figure 4 ).

[0206] 2.4 Verification of the Double-Antibody Sandwich ELISA Quantitative Detection Method for Pertussis Toxin (PT)

[0207] 2.4.1 Linearity and Range

[0208] The concentration of the standard product and the optical absorption value are fitted by four parameters (4-P) to prepare a standard curve, and the correlation coefficient R 2 of the standard curve is verified. The experiment is repeated 6 times. (See the standard curve fitting diagram in Figure 5 ) Calculate the coefficient of variability (CV) of the optical absorption values at each point of the standard curve, and at the same time determine the detection range. The results show that the correlation coefficients R 2 of the 6 standard curves are all ≥ 0.999, and the CV values of the 6 detection results at each concentration are all within 20%. The detection range is 40 ng / ml - 0.625 ng / ml (see Table 2).

[0209] Table 2 Linear Range

[0210]

[0211] 2.4.2 Specificity

[0212] ELISA tests were performed using the remaining components of DTaP protein and their mixed solutions (FHA, PRN, DT, TT) with known concentrations to verify whether there was cross-reactivity with the remaining components of DTaP. The results showed that when the remaining four components of the DTaP vaccine were detected in the quantitative detection system for the antigen content of pertussis toxin (PT), even at a concentration as high as 25 μg / ml, the detected value remained negative, indicating no cross-reactivity with this detection system and good specificity of the system (see Table 3).

[0213] Table 3 Specificity Verification

[0214]

[0215]

[0216] 2.4.3 Accuracy

[0217] The pertussis intermediate samples with known antigen content were detected 3 times repeatedly, and the recovery rate was calculated according to the formula [Recovery rate (%) = Measured value / Theoretical value × 100%]. (Taking the Lowry detected value as the theoretical value). The results showed that the PT detected values of each intermediate sample (high, medium, and low antigen samples) were close to the theoretical values, and the recovery rate was between 80% - 120%, indicating good accuracy of the system (see Table 4).

[0218] Table 4 Accuracy Verification

[0219]

[0220] 2.4.4 Precision

[0221] 2.4.4.1 Repeatability

[0222] Three samples with different antigen contents were detected 3 times repeatedly by the same experimenter, and the coefficient of variation (CV) of the 3 detection results of each sample was calculated. The results showed that the CV of the three detected values of the high, medium, and low antigen samples by the same experimenter was within 20%, indicating good repeatability of the system (see Table 5).

[0223] Table 5 Repeatability Verification

[0224]

[0225] 2.4.4.2 Intermediate Precision

[0226] The same antigen sample was repeatedly detected 3 times by 3 different experimental personnel, and the coefficient of variability (CV) of the 9 test results was calculated. The results showed that when 3 testers performed 3 repeated experiments on the same sample, the CV of the detected value of the sample was within 20%, indicating good intermediate precision of the system (see Table 6).

[0227] Table 6 Verification of Intermediate Precision

[0228]

[0229] 2.4.5 Durability

[0230] 2.4.5.1 Reaction Time

[0231] Under the reaction time (±10 min reaction time, ±5 min color development time), ELISA tests were performed on high, medium, and low concentration antigen samples, and the coefficient of variability (CV) of the test results of each sample at different reaction times was calculated. The results showed that when high, medium, and low antigen samples were detected by the same experimental personnel at three reaction times, the CV was within 20%, and the recovery rate was between 80% and 120%, indicating good durability of the system (see Table 7). At the three reaction times, the optical absorption values of each concentration of the standard curve had little difference, indicating good durability of the system (see Figure 6 ).

[0232] Table 7 Verification of Durability at Reaction Time

[0233]

[0234] 2.4.5.2 Reaction Temperature

[0235] Under the reaction temperature (±2 °C), ELISA tests were performed on high, medium, and low concentration antigen samples, and the coefficient of variability (CV) of the test results of each sample at different temperatures was calculated. The results showed that when high, medium, and low antigen samples were detected by the same experimental personnel at three reaction temperatures, the CV was within 20%, and the recovery rate was between 80% and 120%, indicating good durability of the system (see Table 8). At the three reaction temperatures, the optical absorption values of each concentration of the standard curve had little difference, indicating good durability of the system (see Figure 7 ).

[0236] Table 8 Verification of Durability at Reaction Temperature

[0237]

[0238] 2.4.5.3 Stability of the Solution after Termination

[0239] At 0 h, 15 min, 30 min, 1 h, 2 h, and 4 h after termination, ELISA was performed on high, medium, and low concentration antigen samples, and the coefficient of variability (CV) of the test results of each sample at different time periods after termination was calculated. The results showed that at 4 h after termination, the OD of the solution 405 showed no obvious change, and the solution had good stability. For high, medium, and low concentration antigen samples, the detected value CV was within 20%, and the system had good durability (see Table 9). Under six reading nodes, the optical absorption values of each concentration of the standard curve showed little difference, and the system had good durability (see Figure 8 ).

[0240] Table 9 Verification of solution stability after termination

[0241]

[0242] 2.5 Specific operation of double antibody sandwich ELISA quantitative detection method for pertussis toxin (PT)

[0243] In summary, the optimal coating concentration of the monoclonal antibody 48H7 is 200 ng / well, and the optimal coating condition is coating overnight at 2 - 8 °C; the enzyme-linked immunosorbent assay (ELISA) plate uses a PBST solution containing 4% BSA as the blocking solution, and the blocking condition is blocking for 2 h at 37 °C; the pertussis toxin (PT) antigen reference is diluted to 40 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, and 0.625 ng / ml with the diluent; the detection antibody HRP-45H5 has an optimal working concentration of 1 μg / ml. The specific operation of the double antibody sandwich ELISA quantitative detection method for pertussis toxin (PT) is as follows:

[0244] (1) Coating: Dilute the monoclonal antibody 48H7 to 2 μg / ml with the coating solution, 100 μl / well, and place it at 2 - 8 °C for coating overnight;

[0245] (2) Plate washing: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of the washing solution, and pat dry the residual liquid in the ELISA plate;

[0246] (3) Blocking: Add the blocking solution to the ELISA plate at 150 μl / well and block at 37 °C for 2 h;

[0247] (4) Plate washing: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of the washing solution, and pat dry the residual liquid in the ELISA plate;

[0248] (5) Incubate the samples: Set up standard wells, blank wells, and sample wells. Add standards with different concentrations to the standard wells in sequence, add sample diluent to the blank well, and add the sample to be tested to the sample wells. Add the samples at 100 μl / well and incubate at 37 °C for 1 h;

[0249] (6) Wash the plate: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of washing solution, and pat dry the residual liquid in the ELISA plate;

[0250] (7) Incubate the enzyme-labeled antibody: Dilute the monoclonal antibody 45H5 labeled with HRP to 1 μg / ml with diluent, add the samples at 100 μl / well, and incubate at 37 °C for 1 h;

[0251] (8) Wash the plate: Discard the liquid in the wells, wash the plate 3 times with 300 μl / well of washing solution, and pat dry the residual liquid in the ELISA plate;

[0252] (9) Develop color: Add the color-developing solution to the ELISA plate at 100 μl / well and develop color in the dark at 37 °C for 30 min;

[0253] (10) Terminate: Add the termination solution to the ELISA plate at 50 μl / well to terminate the reaction;

[0254] (11) Read the value: Put the ELISA plate into the ELISA reader to measure the OD 405nm absorbance;

[0255] (12) Result processing: Subtract the OD value of the blank well from the average OD value of each standard as the correction value. Use the OD 405nm value of the standard as the dependent variable Y, and use the standard concentration as the independent variable X to make a curve; Use the four-parameter Logistic mathematical model to fit the equation: Y = ((A - D) / (1 + (X / C)^B)) + D. Substitute the OD 405nm absorbance value of the sample into the formula to calculate the content of pertussis toxin in the sample.

[0256] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0257] The above-described embodiments merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A monoclonal antibody that specifically binds to pertussis toxin, characterized in that, It comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

2. The monoclonal antibody according to claim 1, wherein The heavy chain variable region comprises an amino acid fragment with a sequence shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid fragment with a sequence shown in SEQ ID NO:8; Optionally, the monoclonal antibody comprises a heavy chain and a light chain. The heavy chain comprises an amino acid fragment with a sequence shown in SEQ ID NO:9, and the light chain comprises an amino acid fragment with a sequence shown in SEQ ID NO:

10.

3. An antibody combination, characterized in that, It comprises the monoclonal antibody as claimed in claim 1 or 2 as a capture antibody; optionally, it further comprises a detection antibody; Wherein, the detection antibody: Optionally, it comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with sequences shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16 respectively; the heavy chain variable region optionally comprises an amino acid fragment with a sequence shown in SEQ ID NO:17, and the light chain variable region optionally comprises an amino acid fragment with a sequence shown in SEQ ID NO:18; Further optionally, it comprises a heavy chain and a light chain. The heavy chain comprises an amino acid fragment with a sequence shown in SEQ ID NO:19, and the light chain comprises an amino acid fragment with a sequence shown in SEQ ID NO:

20.

4. A nucleic acid, characterized in that, It encodes the monoclonal antibody as claimed in claim 1 or 2, or the antibody combination as claimed in claim 3; Optionally, the nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:

21. Further optionally, the nucleic acid sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO:22; Optionally, the nucleic acid sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

23. Further optionally, the nucleic acid sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO:24; Optionally, the nucleic acid sequence encoding the heavy chain variable region of the detection antibody is shown in SEQ ID NO:

25. Further optionally, the nucleic acid sequence encoding the heavy chain of the detection antibody is shown in SEQ ID NO:26; Optionally, the nucleic acid sequence encoding the variable region of the light chain of the detection antibody is as shown in SEQ ID NO:

27. Further optionally, the nucleic acid sequence encoding the light chain of the detection antibody is as shown in SEQ ID NO:

28.

5. Recombinant expression vector, characterized in that, It comprises the nucleic acid as described in claim 4.

6. A host cell, characterized in that, It expresses the monoclonal antibody as described in claim 1 or 2, or the antibody combination as described in claim 3; Optionally, the host cell is transformed with the recombinant expression vector as described in claim 5; Optionally, the host cell comprises a non-human mammalian cell.

7. A kit for detecting pertussis toxin, characterized in that, It comprises the monoclonal antibody as described in claim 1 or 2, or the antibody combination as described in claim 3; Optionally, the kit comprises the antibody combination, wherein the detection antibody is biolabeled or chemically labeled; the detection antibody is optionally enzyme-labeled, and further optionally labeled with horseradish peroxidase or alkaline phosphatase; Optionally, the kit further comprises one or more of coating solution, blocking solution, diluent, substrate buffer, washing solution, chromogenic solution and termination solution; wherein, the coating solution is optionally carbonate buffer solution; the blocking solution is optionally 1% w / v - 4% w / v BSA-PBST solution; the diluent is optionally 0.2% w / v - 1% w / v BSA-PBST solution; the substrate buffer is optionally citric acid solution.

8. Use of the monoclonal antibody as described in claim 1 or 2 or the antibody combination as described in claim 3 in the preparation of a reagent or kit for detecting pertussis toxin.

9. A method for detecting pertussis toxin for non-diagnostic purposes, characterized in that, It comprises the following steps: Coating: Dilute the capture antibody with the coating solution and add it to the ELISA plate for coating, then wash and remove the excess residual liquid; Blocking: Add the blocking solution to the ELISA plate, then wash and remove the excess residual liquid; Incubating the sample: Add the sample to be tested to the ELISA plate, wash after reaction and remove the excess residual liquid; Incubating the enzyme-labeled antibody: Add the detection antibody which is biolabeled or chemically labeled to the ELISA plate, wash after reaction and remove the excess residual liquid; Chromogenic reaction: Add the chromogenic solution for incubation; Termination: Add the termination solution to terminate the reaction; Detection: Read the OD value on the microplate reader; 405 value Wherein, the capture antibody and the detection antibody are as defined in claim 3; Optionally, in the coating step, the concentration of the capture antibody in the ELISA plate is 0.5 μg / ml - 16 μg / ml, optionally 2 μg / ml - 8 μg / ml; Optionally, in the step of incubating the enzyme-labeled antibody, the concentration of the detection antibody in the ELISA plate is 0.05 μg / ml - 5 μg / ml, optionally 0.8 μg / ml - 1.5 μg / ml; Optionally, the method is carried out using the kit as described in claim 7.

10. Application of the monoclonal antibody as described in claim 1 or 2, the antibody combination as described in claim 3 or the kit as described in claim 7 in the quality detection of a vaccine containing pertussis toxin antigen; Optionally, the vaccine is a pertussis vaccine; Optionally, the quality detection includes one or more of production quality control, laboratory detection and quality sampling inspection.

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