Monoclonal antibody, antibody combination containing monoclonal antibody and application of monoclonal antibody in detection of filamentous hemagglutinin

Abstract

The invention discloses a monoclonal antibody, an antibody composition containing the monoclonal antibody and application of the monoclonal antibody to detection of filamentous hemagglutinin. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, amino acid sequences of HCDR1-HCDR3 of the heavy chain variable region are respectively shown as SEQ ID NO: 1-3, and amino acid sequences of LCDR1-LCDR3 of the light chain variable region are respectively shown as SEQ ID NO: 4-6. The monoclonal antibody can be used as a capture antibody in ELISA sandwich detection, and a detection antibody capable of being paired with the monoclonal antibody can be screened.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to monoclonal antibodies, antibody combinations containing the same, and their use in detecting filamentous hemagglutinin. Background Art

[0002] Whooping cough (whooping cough) is an acute respiratory infection with high contagion, a major threat to 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 significantly decreased. Today, both vaccines are in use, but due to significant side effects and safety concerns, the wP vaccine is being gradually replaced by the acellular vaccine, which has fewer side effects and a better immune response.

[0003] Filamentous hemagglutinin (FHA) is a hemagglutinin secreted by Bordetella pertussis. It has hemagglutinating activity and plays a key role in the adhesion of B. pertussis to respiratory epithelial cells. It is a key virulence factor in B. pertussis pathogenicity and the primary protective antigen of B. pertussis. Therefore, FHA is a key component of acellular pertussis vaccines.

[0004] Currently, the content of pertussis components is mostly determined using electrophoresis, Kjeldahl nitrogen determination, and Lowry's method. Electrophoresis provides only semi-quantitative analysis, while Kjeldahl nitrogen determination and Lowry's method can only quantify total protein, lacking specificity. Therefore, there is a need to establish an accurate and specific method for detecting filamentous hemagglutinin (FHA) antigen for quality control in acellular pertussis vaccine production. ELISA kits for FHA detection are already documented in the prior art, such as CN111004322B. However, the implementation of this double-antibody sandwich ELISA utilizes a monoclonal antibody-polyclonal antibody pairing (i.e., the capture antibody is the mouse monoclonal antibody described in the patent, and the detection antibody is a rabbit polyclonal antibody), which could further enhance the specificity and sensitivity of the detection process.

[0005] Therefore, there is an urgent need to develop new methods for detecting FHA in this field. Summary of the Invention

[0006] Based on this, it is necessary to provide at least a monoclonal antibody, an antibody combination containing the monoclonal antibody, and use thereof in detecting filamentous hemagglutinin.

[0007] In a first aspect of the present application, a monoclonal antibody specifically binding to filamentous hemagglutinin is provided, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the sequences as set forth 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 having the sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

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

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

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

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

[0012] In a sixth aspect of the present application, a kit for detecting filamentous hemagglutinin is provided, comprising the monoclonal antibody as described in the first aspect, or the antibody combination as described in the second aspect.

[0013] In a seventh aspect of the present application, 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 a kit for detecting filamentous hemagglutinin is provided.

[0014] In an eighth aspect of the present application, a method for detecting filamentous hemagglutinin for non-diagnostic purposes is provided, comprising the steps of:

[0015] coating: after diluting the capture antibody with coating solution, adding to the enzyme-labeled plate, washing and spinning dry;

[0016] blocking: adding blocking solution to the enzyme-labeled plate, washing and spinning dry;

[0017] incubating sample: adding the sample to be detected to the enzyme-labeled plate, washing and spinning dry after reaction;

[0018] incubating enzyme-labeled antibody: adding the detection antibody labeled with a biological marker or a chemical marker to the enzyme-labeled plate, washing and spinning dry after reaction;

[0019] color development: incubating with color developing solution;

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

[0021] Detection: read OD values on a microplate reader 405

[0022] In a ninth aspect of the application, there is provided use of a monoclonal antibody according to the first aspect, an antibody according to the second aspect or a kit according to the sixth aspect in quality control of a vaccine comprising filamentous haemagglutinin antigen.

[0023] In the present application, a specific monoclonal antibody is screened by using hybridoma technology, which can be used as a capture antibody in a sandwich ELISA assay, and a detection antibody that can be paired therewith is screened. The two antibodies specifically bind to different antigen epitopes of filamentous haemagglutinin (FHA) and do not interfere with each other, and the sandwich ELISA method established using the antibodies has excellent sensitivity, stability and reproducibility, and can be applied to quantitative detection and quality control of filamentous haemagglutinin (FHA) at various stages in the production of DPT vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the description of the embodiments or examples will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings. It should be noted that the drawings are drawn in a simplified form and are only used to conveniently and clearly assist in the description of the present application.

[0025] Figure 1 Gel electrophoresis diagram of monoclonal antibody 16E11 in an embodiment of the present application.

[0026] Figure 2 Gel electrophoresis diagram of monoclonal antibody 29B9 in an embodiment of the present application.

[0027] Figure 3 Figure of relationship between capture antibody concentration and OD value when the antigen concentration is 20 ng / mL in the screening of capture antibody concentration in an embodiment of the present application. 405

[0028] Figure 4 Figure of relationship between detection antibody concentration and OD value when the antigen concentration is 10 ng / mL in the screening of detection antibody concentration in an embodiment of the present application. 405

[0029] Figure 5 Standard curve in an embodiment of the present application.

[0030] Figure 6 ​​​Standard curve superimposed plot for quantitative detection of FHA under different reaction times in one embodiment of the present application.

[0031] Figure 7 Standard curve superimposed plot for quantitative detection of FHA under different reaction temperatures in one embodiment of the present application.

[0032] Figure 8A and Figure 8B Standard curve superimposed plot for quantitative detection of FHA under different post-termination holding times in one embodiment of the present application. DETAILED DESCRIPTION

[0033] For the purpose of promoting an understanding of the present application, the present application will now be described in greater detail with reference to the relevant drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this description, the terms "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] In the present application, "one or more" means any one of the listed items or any combination of the listed items, unless otherwise specified. Similarly, "one or more" and the like, which otherwise indicates "one or more" in other ways, are also understood in the same way, unless otherwise specified.

[0036] In the present application, "further", "even further", "in particular", "for example", "such as", "for instance", "for example", and the like are used for the purpose of description and indicate that the technical solutions before and after have a correlation in terms of coverage, but should not be understood as a limitation of the previous technical solution, nor as a limitation of the scope of protection of the present application. In the present application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0037] In the present application, "optionally", "optional", "optionally" means that it can or can not be present, that is, it means that it is selected from any one of the two parallel schemes of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "option" is independent. Unless otherwise specified, "optionally includes", "optionally contains", and the like are described in the present application, for example, "optionally includes" means "may include or not include".

[0038] The terms "comprise", "contain", and "include", as used herein, are synonymous with each other, are inclusive or open-ended and do not exclude additional, unrecited members, or features. Members or features, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also like actions, conditions, timing, states, etc.

[0039] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0040] In the present application, the exemplary description involving "in some embodiments (or examples)", "in an embodiment (or example)", etc. can cover but is not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0041] In the present application, in "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for description purpose, and cannot be understood as indicating or implying relative importance or quantity, nor can it 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 description, and should be understood as not constituting a closed limitation on the quantity.

[0042] In the present application, with respect to a numerical interval (i.e. a numerical range), if no special instructions are given, the distribution of the optional values in the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. the minimum value and the maximum value) of the numerical interval, and every value between the two numerical endpoints. If no special instructions are given, when the numerical interval only points to the integers in the numerical interval, including the two endpoint integers of the numerical range and every integer between the two endpoints, it is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or property, these numerical ranges can be combined. In other words, unless otherwise indicated, the numerical ranges disclosed herein should be understood to include any and all sub-ranges therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is allowed to include numerical interval types such as percentage interval, ratio interval, and value interval in a broad sense.

[0043] In the present application, unless otherwise explicitly stated, the execution of the steps involved in the method flow does not have strict order restrictions, and can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily have to be executed at the same time, but can be executed at different times, and the execution order does not necessarily have to be sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0044] One aspect of the present application provides a monoclonal antibody specifically binding to filamentous hemagglutinin, comprising a heavy chain variable region and a light chain variable region.

[0045] Unless otherwise specified, the terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" in the present application refer to the binding of an antibody to an epitope on a predetermined antigen (e.g., filamentous hemagglutinin).

[0046] Unless otherwise specified, the term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" in the present application refers to an antibody molecule preparation of a single molecular composition. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0047] In some embodiments, the heavy chain variable region of the monoclonal antibody described above comprises HCDR1, HCDR2 and HCDR3 having sequences as shown in SEQ ID NO: 1 (GYSITTDYT), SEQ ID NO: 2 (IHYSGGA) and SEQ ID NO: 3 (TRSWYDYGNYYSMDY), respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having sequences as shown in SEQ ID NO: 4 (QDIYNY), SEQ ID NO: 5 (YTS) and SEQ ID NO: 6 (QQGITLPFT), respectively.

[0048] The monoclonal antibody of the present application can have the above-mentioned CDRs, or a derivative fragment of the above-mentioned CDRs. The derivative fragment is formed by no more than 6 amino acid substitutions ("conservative modifications" or "conservative substitutions or replacements") of the corresponding CDR, which retains the biological activity consistent with the corresponding complementarity determining region. For example, the derivative fragment has 1, 2, 3, 4, 5 or 6 substitutions relative to the corresponding complementarity determining region, which can be one amino acid replaced by another amino acid, or one amino acid replaced by multiple (e.g., 2) amino acids.

[0049] In the CDRs (Kabat numbering convention) provided herein, a derivative fragment (conservative variant) is a polypeptide having 1, 2, 3 amino acids replaced with an amino acid of similar or comparable properties as compared to the amino acid sequence of the antibody of the present application. Preferably, these conservative variant polypeptides are generated by amino acid replacement according to Table A.

[0050] Table A

[0051]

[0052] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid of similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. As is known to those skilled in the art, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). Additionally, substitution of structurally or functionally similar amino acids is not likely to disrupt biological activity.

[0053] In some embodiments, the heavy chain variable region comprises an amino acid fragment having the sequence of SEQ ID NO: 7 and the light chain variable region comprises an amino acid fragment having the sequence of SEQ ID NO: 8:

[0054] DVQLQESGPDLVKPSQSLSLTCTVTGYSITTDYTWHWIRHFPGNKLEWMGYIHYSGGANYNPSLKSRVSVTRDTSKNQFFLQLNSVTSEDTATYYCTRSWYDYGNYYSMDYWGQGTSVTVSS (SEQ ID NO: 7);

[0055] DIQMTQTTSSLSASLGDRVTISCRASQDIYNYLNWYQRRPDGTVKLLIYYTSELHSGVPSRFSGSGSGTDYSLTISNLEHEDIATYFCQQGITLPFTFGSGTKLEIK (SEQ ID NO: 8).

[0056] In some embodiments, the amino acid sequence of the heavy chain variable region described above is set forth in SEQ ID NO: 7 or the amino acid sequence is 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%) identical to SEQ ID NO: 7.

[0057] In some embodiments, the amino acid sequence of the light chain variable region described above is set forth in SEQ ID NO: 8 or the amino acid sequence is 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%) identical to SEQ ID NO: 8.

[0058] 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 sequences are aligned for maximum sequence comparison, e.g., gaps can be introduced in one or both of a pair of sequences to maximize the percentage of sequence identity. Alignment can be achieved using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0059] In some embodiments, the monoclonal antibody described above comprises a heavy chain comprising an amino acid fragment having a sequence set forth in SEQ ID NO: 9 and a light chain comprising an amino acid fragment having a sequence set forth in SEQ ID NO: 10.

[0060] Another aspect of the present application provides an antibody combination comprising a monoclonal antibody as described above as a capture antibody.

[0061] Without wishing to be bound by any theory, it is believed that the role of the capture antibody at least includes immobilization on the surface of a solid support (e.g., an enzyme-linked plate) for specifically "capturing" the target antigen in the sample to be tested.

[0062] In some embodiments, the antibody combination further comprises a detection antibody. Illustratively, the detection antibody is a monoclonal antibody.

[0063] It is understood that the detection antibody is capable of binding to the antigen that has been captured and can be used for detection and amplification of the signal.

[0064] Without wishing to be bound by any theory, it is believed that the paired antibodies tend to have better specificity and sensitivity in application (e.g. performance of double antibody sandwich ELISA) than the pairing of mouse monoclonal antibody-rabbit polyclonal antibody in the current technology.

[0065] The detection antibody illustratively comprises a heavy chain variable region and a light chain variable region.

[0066] In some embodiments, the heavy chain variable region of the detection antibody comprises HCDR1, HCDR2 and HCDR3 having sequences as set forth in SEQ ID NO: 11 (GYSFTDYT), SEQ ID NO: 12 (INPYNGDT) and SEQ ID NO: 13 (ARWFYYGTTALDF), respectively, and the light chain variable region of the detection antibody comprises LCDR1, LCDR2 and LCDR3 having sequences as set forth in SEQ ID NO: 14 (ESIYSY), SEQ ID NO: 15 (NAK) and SEQ ID NO: 16 (QHHYGVPFT), respectively.

[0067] In some embodiments, the heavy chain variable region of the detection antibody comprises an amino acid fragment having a sequence as set forth in SEQ ID NO: 17, and the light chain variable region of the detection antibody comprises an amino acid fragment having a sequence as set forth in SEQ ID NO: 18:

[0068] EVQLQQSGPALVKPGTSMKISCKASGYSFTDYTMNWVKQSHGKNLEWIGLINPYNGDTHYNQRFKDKATLTVDKSSSTAYMDLLSLTSEDSAVYYCARWFYYGTTALDFWGQGTSVTVSS (SEQ ID NO: 17);

[0069] DIQMTQSPASLSASVGETVTITCRVSESIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGVPFTFGSGTKLEIK (SEQ ID NO: 18).

[0070] In some embodiments, the heavy chain variable region of the detection antibody has an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that is 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%) identical to SEQ ID NO: 17.

[0071] In some embodiments, the light chain variable region of the detection antibody has an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that is 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%) identical to SEQ ID NO: 18.

[0072] In some embodiments, the detection antibody comprises a heavy chain and a light chain. Illustratively, the heavy chain comprises an amino acid fragment having a sequence as set forth in SEQ ID NO: 19 and the light chain comprises an amino acid fragment having a sequence as set forth in SEQ ID NO: 20.

[0073] Another aspect of the present application provides a nucleic acid encoding the monoclonal antibody or the antibody combination as described above.

[0074] Unless otherwise indicated, the term "nucleic acid" in the present application can be a nucleic acid molecule, e.g., an isolated nucleic acid molecule. The nucleic acid molecule of the present application, primarily refers to an isolated nucleic acid molecule. "Isolated" means that the molecule is substantially free of other biological molecules, e.g., nucleic acids, proteins, lipids, carbohydrates, or other materials, e.g., cellular debris and growth media. Typically, the term "isolated" is not intended to mean the complete absence of such materials or the absence of water, buffers, or salts.

[0075] Illustratively, the nucleic acid encodes the heavy chain variable region of the monoclonal antibody described above. In some embodiments, the nucleic acid encoding the heavy chain variable region of the monoclonal antibody described above has a sequence as set forth in SEQ ID NO: 21 or comprises a sequence as set forth in SEQ ID NO: 21:

[0076] GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCACTGATTATACCTGGCACTGGATCCGGCACTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATACACTACAGTGGTGGCGCTAACTACAACCCATCTCTCAAAAGTCGAGTCTCTGTCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTTCTGAGGACACAGCCACATATTACTGTACAAGATCTTGGTATGATTACGGGAATTACTATTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0077] In some embodiments, the nucleic acid encodes a heavy chain of the monoclonal antibody described above. In some embodiments, the sequence of the nucleic acid encoding a heavy chain of the monoclonal antibody described above is set forth in SEQ ID NO: 22, or comprises a sequence set forth in SEQ ID NO: 22.

[0078] GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTTACAATTATTTAAACTGGTATCAGCGGAGACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAGAATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCACGAAGATATTGCCACTTACTTTTGCCAACAGGGTATTACGCTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0079] In some embodiments, the nucleic acid encodes a heavy chain of the monoclonal antibody described above. In some embodiments, the sequence of the nucleic acid encoding a heavy chain of the monoclonal antibody described above is set forth in SEQ ID NO: 22, or comprises a sequence set forth in SEQ ID NO: 22.

[0080] Exemplarily, the nucleic acid encodes the heavy chain of the detection antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain of the detection antibody is set forth in SEQ ID NO: 25, or comprises the sequence set forth in SEQ ID NO: 25.

[0081] Exemplarily, the nucleic acid encodes the heavy chain of the detection antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain of the detection antibody is set forth in SEQ ID NO: 25, or comprises the sequence set forth in SEQ ID NO: 25.

[0082] GAGGTCCAGCTGCAACAGTCTGGACCTGCGCTGGTGAAGCCTGGAACTTCAATGAAGATTTCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGATACTCACTACAACCAGAGATTCAAGGACAAGGCCACATTAACTGTGGACAAGTCATCCAGCACAGCCTACATGGACCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATGGTTTTACTACGGTACTACTGCTTTGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0083] Exemplarily, the nucleic acid encodes the heavy chain of the detection antibody. In some embodiments, the sequence of the nucleic acid encoding the heavy chain of the detection antibody is set forth in SEQ ID NO: 25, or comprises the sequence set forth in SEQ ID NO: 25.

[0084] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGTAAGTGAGAGTATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAGTCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAAATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAGATAAAA.

[0085] In some embodiments, the nucleic acid encodes a heavy chain of the detection antibody described above. In some embodiments, the sequence of the nucleic acid encoding a heavy chain of the detection antibody described above is set forth in SEQ ID NO: 26, or comprises a sequence as set forth in SEQ ID NO: 26.

[0086] In some embodiments, the nucleic acid encodes a heavy chain of the detection antibody described above. In some embodiments, the sequence of the nucleic acid encoding a heavy chain of the detection antibody described above is set forth in SEQ ID NO: 26, or comprises a sequence as set forth in SEQ ID NO: 26.

[0087] Another aspect of the present application provides a recombinant expression vector comprising the nucleic acid as described above.

[0088] The term "vector," which can also be referred to as a "nucleic acid construct," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. 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, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell 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 be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA technologies are often in the form of plasmids. However, other forms of expression vectors are available (e.g., viral vectors such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0089] In the present application, the aforementioned recombinant expression vector can comprise a nucleic acid encoding the heavy chain of the aforementioned monoclonal antibody. In some embodiments, the nucleic acid further comprises a sequence encoding a leader peptide of the heavy chain. Illustratively, the leader peptide comprises an amino acid fragment with a sequence as set forth in SEQ ID NO: 29 (MRMLILLCLFTAFPGFLS).

[0090] In some embodiments, the nucleic acid encoding the same comprises a nucleic acid sequence with a sequence as set forth in SEQ ID NO: 30 (ATGAGAATGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTTTCCTGTCT).

[0091] In the present application, the aforementioned recombinant expression vector can comprise a nucleic acid encoding the light chain of the aforementioned monoclonal antibody. In some embodiments, the nucleic acid further comprises a sequence encoding a leader peptide of the light chain. Illustratively, the leader peptide comprises an amino acid fragment with a sequence as set forth in SEQ ID NO: 31 (MMSSAQFLGLLLLCFQGSRC).

[0092] In some embodiments, the nucleic acid encoding the same comprises a nucleic acid sequence with a sequence as set forth in SEQ ID NO: 32 (ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTTCCAGATGT).

[0093] In the present application, the aforementioned recombinant expression vector can comprise a nucleic acid encoding the heavy chain of the aforementioned detection antibody. In some embodiments, the nucleic acid further comprises a sequence encoding a leader peptide of the heavy chain. Illustratively, the leader peptide comprises an amino acid fragment with a sequence as set forth in SEQ ID NO: 33 (MGWSWIFLFLLSGTAGVHS).

[0094] In some embodiments, the nucleic acid encoding the same comprises a nucleic acid sequence with a sequence as set forth in SEQ ID NO: 34 (ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT).

[0095] In the present application, the aforementioned recombinant expression vector can comprise a nucleic acid encoding the light chain of the aforementioned detection antibody. In some embodiments, the nucleic acid further comprises a sequence encoding a leader peptide of the light chain. Illustratively, the leader peptide comprises an amino acid fragment with a sequence as set forth in SEQ ID NO: 35 (MSVPTQVLGLLLLWLTGARC).

[0096] In some embodiments, the nucleic acid encoding the same comprises a nucleic acid sequence as set forth in SEQ ID NO: 36 (ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT).

[0097] Another aspect of the present application provides a host cell expressing the monoclonal antibody as described above, or the antibody combination as described above.

[0098] The term "cell" or "host cell" refers to a cell into which a recombinant expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Bacteria that are readily transformed include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, 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.

[0099] Another aspect of the present application provides a method of preparing the host cell described above, comprising the step of introducing the above-described or recombinant expression vector into a target cell.

[0100] In one example, the introduction is by transfection.

[0101] 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, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., 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.

[0102] Another aspect of the present application provides a method for producing the monoclonal antibody or antibody combination as described above, comprising: culturing the host cell, and isolating the monoclonal antibody or antibody combination from the resulting culture supernatant.

[0103] Another aspect of the present application provides a product for detecting filamentous hemagglutinin, comprising the monoclonal antibody or antibody combination as described above.

[0104] In some embodiments, the product comprises the antibody combination as described above, wherein the detection antibody is biologically or chemically labeled.

[0105] In some embodiments, the detection antibody is enzymatically labeled, including but not limited to labeled with horseradish peroxidase or alkaline phosphatase.

[0106] In some embodiments, the product further comprises one or more of a coating solution, a blocking solution, a dilution solution, a substrate buffer, a washing solution, a developing solution, and a termination solution.

[0107] In some embodiments, the coating solution is or comprises a carbonate buffer.

[0108] In some embodiments, the coating solution is a carbonate buffer with a concentration of 0.05 M, pH 9.6.

[0109] In some embodiments, the blocking solution is a 1% w / v~4% w / v BSA-PBST solution, for example, 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 of the values. For example, a 1% w / v BSA-PBST solution means 1 g of BSA (Bovine Serum Albumin) is dissolved in 100 mL of PBST solution (phosphate buffer solution).

[0110] Without wishing to be bound by any theory, it is found that when the concentration of BSA-PBST in the blocking solution is less than 1% w / v, the blank value of the ELISA detection may be high; and when the concentration of BSA-PBST in the blocking solution is greater than 4% w / v, the sensitivity of the ELISA detection method may be reduced.

[0111] In some embodiments, the diluent is 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, and the like.

[0112] Without wishing to be bound by any theory, it is found that when the concentration of BSA-PBST in the diluent is less than 0.2% w / v, the blank value of the ELISA detection can be high; when it is greater than 1% w / v, the sensitivity of the ELISA detection method can be reduced.

[0113] The above substrate buffer is exemplarily a citric acid solution, such as a 0.05M citric acid solution.

[0114] The detection product is not particularly limited in the embodiments of the present application, and is exemplarily a diagnostic reagent, a test strip, a detection plate, or a kit. In the detection product, the detection antibody and the capture antibody compete to bind the filamentous hemagglutinin in the sample to be detected.

[0115] 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 filamentous hemagglutinin.

[0116] Another aspect of the present application also provides a method for detecting filamentous hemagglutinin using the monoclonal antibody as described above or the antibody combination as described above.

[0117] Exemplarily, the method is a double-antibody sandwich ELISA quantitative detection method.

[0118] In some embodiments, the above method comprises the following steps:

[0119] Coating: after diluting the capture antibody with the coating solution, the coating solution is added to the enzyme-labeled plate, and then washed and removed (e.g., centrifuged) to remove the excess residual liquid;

[0120] Blocking: the blocking solution is added to the enzyme-labeled plate, and then washed and removed (e.g., centrifuged) to remove the excess residual liquid;

[0121] Incubating the sample: the sample to be detected is added to the enzyme-labeled plate, and then washed and removed (e.g., centrifuged) to remove the excess residual liquid after reaction;

[0122] Incubate enzyme-labeled antibody: add the biologically or chemically labeled detection antibody to the enzyme-labeled plate, after the reaction, wash and remove the excess residue (e.g. spin dry);

[0123] Color development: add color developing solution for incubation;

[0124] Termination: add termination solution to terminate the reaction;

[0125] Detection: read OD on the enzyme-labeled instrument 405 Value;

[0126] wherein the capture antibody is as defined above, and the detection antibody is as defined above.

[0127] Unless otherwise specified, the term "OD 405 " in the present application refers to the measurement of optical density (Optical Density, OD) at 405 nm wavelength, by which the concentration or activity of a specific substance in a sample is determined. Exemplarily, part of the color development reaction (such as the termination product after HRP catalyzes ABTS color development) can be detected at 405 nm.

[0128] The above method can be for diagnostic purposes or non-diagnostic purposes.

[0129] In some embodiments, in the coating step, the concentration of the capture antibody in the enzyme-labeled plate is 2 μg / mL~8 μg / mL. Exemplarily, for example, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, or a range or value between any two values.

[0130] In some embodiments, the concentration of the detection antibody in the enzyme-labeled plate is 0.2 μg / mL~1.6 μg / mL; for example, 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, 1.0 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, or a range or value between any two values.

[0131] Exemplarily, the method is implemented using the product (e.g. kit) as described above.

[0132] In some embodiments, the method comprises the following steps:

[0133] (1) Coating: dilute the monoclonal antibody (capture antibody) to 2 μg / mL with coating solution, 100 μL / well, and place in 2-8°C for coating overnight;

[0134] (2) Washing plate: discard the liquid in the wells, wash the plate with washing solution at 300 μL / well for 3 times, and pat dry the residual liquid in the enzyme-labeled plate;

[0135] (3) Blocking: add blocking solution to the enzyme-labeled plate at 150 μL / well, and block at 37°C for 2 h;

[0136] (4) Washing plate: discard the liquid in the wells, wash the plate with washing solution at 300 μL / well for 3 times, and pat dry the residual liquid in the enzyme-labeled plate;

[0137] (5) Incubate sample: set standard wells, blank wells, and sample wells, add different concentrations of standards to the standard wells in order, add sample diluent to the blank wells, and add the sample to be tested to the sample wells, with 100 μL / well, and incubate at 37°C for 1 h;

[0138] (6) Washing plate: discard the liquid in the wells, wash the plate with washing solution at 300 μL / well for 3 times, and pat dry the residual liquid in the enzyme-labeled plate;

[0139] (7) Incubate enzyme-labeled antibody: dilute the HRP-labeled detection antibody to 0.2 μg / mL with diluent, add 100 μL / well, and incubate at 37°C for 1 h;

[0140] (8) Washing plate: discard the liquid in the wells, wash the plate with washing solution at 300 μL / well for 3 times, and pat dry the residual liquid in the enzyme-labeled plate;

[0141] (9) Color development: add color developing solution to the enzyme-labeled plate at 100 μL / well, develop color at 37°C for 30 min in the dark;

[0142] (10) Termination: add termination solution to the enzyme-labeled plate at 50 μL / well to terminate the reaction;

[0143] (11) Read value: place the enzyme-labeled plate into an enzyme-labeled instrument to measure OD 405nm absorbance;

[0144] (12) Result processing: subtract the OD value of the blank wells from the average OD value of each standard as the correction value, take the OD 405nm value of the standard as the dependent variable Y, and take the standard concentration as the independent variable X to make a curve; use a four-parameter Logisitic mathematical model to fit the equation: Y=((A-D) / (1+(X / C)^B))+D, and substitute the sample OD 405nm absorbance value into the formula to calculate the content of the filamentous hemagglutinin in the sample; wherein A is the asymptote value on the curve, D is the asymptote value under the curve, B is the slope of the curve, and C is the dose corresponding to half of the maximum binding.

[0145] In another aspect of the application, there is provided use of a monoclonal antibody, antibody combination, product (e.g. kit) as described above in quality testing of a vaccine containing filamentous hemagglutinin antigen.

[0146] By way of example, scenarios of quality testing include, but are not limited to, production quality control, laboratory testing, and quality sampling, etc.

[0147] By way of example, the vaccine is a pertussis vaccine.

[0148] Some embodiments are provided below.

[0149] Embodiments of the application will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. In the following examples, the experimental methods not specified are preferably referred to the guidance given in the application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0150] Example 1

[0151] 1. Preparation of mouse anti-filamentous hemagglutinin (FHA) monoclonal antibody

[0152] 1.1 Preparation of immunogen

[0153] The purified filamentous hemagglutinin (concentration 200 μg / mL) was mixed with an equal volume of adjuvant to emulsify uniformly, in a water-in-oil state, to prepare a vaccine for immunizing mice. The adjuvant used for the first immunization was Freund's complete adjuvant, and the adjuvant used for the booster immunization was Freund's incomplete adjuvant.

[0154] 1.2 Immunization strategy

[0155] The immunized animals were SPF Balb / C mice, female, 4-6 weeks old. Each mouse was subcutaneously immunized 3 times, 20 μg of immunogen per mouse per time, with an interval of 2 weeks between each immunization. After the third immunization, cheek blood was collected, serum was separated, and the antibody titer was detected by indirect ELISA method. The antibody titer reached more than 1:51200000.

[0156] 1.3 Cell fusion

[0157] Prepare mouse myeloma cells SP2 / 0, adjust the cell state to the logarithmic growth phase, select the mouse with the highest ELISA titer, and give the mouse a booster immunization 3 days before fusion. Take the mouse spleen 3 days later, collect the spleen cells after mechanical disruption, filter through a 200-mesh screen, and wash with PBS 3 times; collect SP2 / 0 cells, wash with PBS 3 times; after cell counting, mix at a ratio of SP2 / 0:spleen cells = 1:2.5, discard the PBS after centrifugation, and use a cell fusion instrument for cell fusion. After the fusion is completed, add complete medium containing HAT to the cells after centrifugation, resuspend the cells, and plate them in a 96-well plate; perform a half medium exchange 5 days after fusion.

[0158] 1.4 Hybridoma screening

[0159] Detect the cell culture supernatant taken 7 days after fusion by indirect ELISA, record the positive cell wells, label them, and perform a half medium exchange; the next day, recheck the positive wells that were replenished, and record the clone numbers with stable titers.

[0160] 1.5 Establishment of stable cell lines

[0161] 1.5.1 First subcloning

[0162] Select specific positive cell wells from 1.4 for subcloning screening, use the limiting dilution method, complete medium, and plate 1 cell per well in a 96-well plate. Observe under a microscope 7 days later, label the single clone wells, perform ELISA detection the next day, discard the clone numbers that have turned negative, and select the wells with vigorous growth for a replenishment;

[0163] 1.5.2 Second subcloning

[0164] Select specific positive cell wells from the previous step for subcloning screening, use the limiting dilution method, complete medium, and plate 1 cell per well in a 96-well plate; observe under a microscope 7 days later, label the single clone wells, perform ELISA detection the next day, and select the wells with vigorous growth for expansion culture and cryopreservation when the subcloning has a 100% positive rate in ELISA detection.

[0165] 1.6 Antibody subtype identification

[0166] Detect the cell culture supernatant of the stable cell line by indirect ELISA, with HRP-labeled goat anti-mouse heavy chain subtype-specific secondary antibody as the secondary antibody, and set up a positive control (HRP-labeled goat anti-mouse IgG (H+L)) and a negative control (antibody diluent). The antibody subtype of mouse anti-filamentous hemagglutinin (FHA) monoclonal antibody 16E11 is IgG1; the antibody subtype of mouse anti-filamentous hemagglutinin (FHA) monoclonal antibody 29B9 is IgG2b.

[0167] 1.7 Ascites preparation and purification

[0168] Stable hybridoma cells were injected into the abdominal cavity of mice at 1*10 6 cells per mouse, and the mice were sacrificed after the abdominal cavity was obviously swollen 10 days later. Ascites was collected. After centrifugation, the ascites was filtered through a 0.22 μm pore size filter, and then purified using Protein G. The purified antibody was concentrated using an ultrafiltration tube, and finally exchanged into PBS. The product was sterile filtered through a 0.22 μm pore size filter in an ultra-clean bench, and stored at -20°C for standby use.

[0169] 1.8 Detection of antibody titer, purity and concentration after purification

[0170] The purified antibody was detected by indirect ELISA. The antibody titer of mouse anti-filamentous hemagglutinin (FHA) monoclonal antibody 16E11 was 1:486000, and the antibody titer of mouse anti-filamentous hemagglutinin (FHA) monoclonal antibody 29B9 was 1:486000. The purity of the antibody was detected by SDS-PAGE (see Figure 1 and Figure 2 ), and the purity of the antibody reached more than 95%. The concentration of mouse anti-filamentous hemagglutinin (FHA) monoclonal antibodies 16E11 and 29B9 was 2 mg / mL as detected by Nano-500.

[0171] 1.9 HRP labeling and titer detection of purified antibody

[0172] The purified monoclonal antibody was freeze-dried, and then coupled with HRP. The HRP-labeled antibody was detected by indirect ELISA. The antibody titer of HRP-monoclonal antibody 16E11 was >1:100000, and the antibody titer of HRP-monoclonal antibody 29B9 was >1:100000.

[0173] 1.10 Full-length sequencing of hybridoma cell monoclonal antibody gene

[0174] (1) Total RNA was extracted by lysing the hybridoma cells;

[0175] (2) cDNA was synthesized by reverse transcription of RNA using RACE technology;

[0176] (3) The full length of heavy and light chains was obtained by PCR amplification;

[0177] (4) The target fragment was ligated to the vector using ligase, and the ligation product was transformed into E. coli competent cells, and then a single colony was picked for sequencing;

[0178] (5) The sequencing results were analyzed and annotated.

[0179] The heavy chain amino acid sequence of the monoclonal antibody 16E11 is shown in SEQ ID NO: 9, and the light chain amino acid sequence is shown in SEQ ID NO: 10; the heavy chain amino acid sequence of the monoclonal antibody 29B9 is shown in SEQ ID NO: 19, and the light chain amino acid sequence is shown in SEQ ID NO: 20.

[0180] 2. Establishment and verification of a quantitative detection method of a fibrillar hemagglutinin (FHA) double-antibody sandwich ELISA

[0181] 2.1 Materials and instruments: monoclonal antibody 16E11, HRP-labeled monoclonal antibody 29B9, fibrillar hemagglutinin (FHA) antigen reference, coating solution, washing solution, blocking solution, dilution solution, substrate buffer, color developing solution and termination solution; enzyme-labeled plate; constant temperature incubator, enzyme-labeled instrument and plate washing machine.

[0182] 2.2 Preparation of solutions

[0183] 2.2.1 Coating solution (0.05M carbonate buffer solution pH 9.6): accurately weigh 1.59g of sodium carbonate and 2.93g of sodium bicarbonate, and dissolve in 1.0L of purified water.

[0184] 2.2.2 10×PBS (0.1M phosphate buffer solution): accurately weigh 80.0g of sodium chloride, 2.0g of potassium dihydrogen phosphate, 14.3g of sodium hydrogen phosphate dihydrate (28.8g of sodium hydrogen phosphate dodecahydrate) and 2.0g of potassium chloride, and dissolve in 1.0L of purified water. Before use, dilute 10 times with purified water.

[0185] 2.2.3 Washing solution (PBST solution): accurately measure 1L of 1×PBS solution, add 500μL of Tween 20, and stir until completely dissolved for standby use.

[0186] 2.2.4 Blocking solution: accurately weigh 4.0g of BSA, and dissolve in 100mL of PBST solution.

[0187] 2.2.5 Dilution solution: accurately weigh 1.0g of BSA, and dissolve in 100mL of PBST solution.

[0188] 2.2.6 Substrate buffer solution (0.05M citric acid solution): accurately weigh 10.51g of citric acid monohydrate (9.607g of anhydrous citric acid), and dissolve in 1L of purified water. Adjust the pH to 4.0 with 400g / L NaOH solution.

[0189] 2.2.7 Color reagent (0.05% ABTS): Just before use, accurately weigh 0.01 g of 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and dissolve in 20 mL of 0.05 M citric acid solution. Immediately before use, add 5 μL of 30% H2O2 solution.

[0190] 2.2.8 Stop solution (1% SDS): Accurately weigh 1.0 g of SDS and dissolve in 100 mL of pure water.

[0191] 2.3 Establishment of a quantitative detection method of filamentous hemagglutinin (FHA) double-antibody sandwich ELISA

[0192] 2.3.1 Determination of capture antibody and detection antibody

[0193] The capture antibody concentration was 1 μg / mL, and the sandwich antigen concentration was 1 μg / mL, with a 3-fold dilution, for a total of 7 gradients, with a blank control. The detection antibody concentration was 0.1 μg / mL. ELISA detection was performed. The results showed that when the capture antibody was 16E11 and the detection antibody was HRP-29B9, the sample optical absorbance value was high and the blank value was low (high P / N value), which was a better pair (see Table 1).

[0194] Table 1 Antibody pair screening

[0195]

[0196] 2.3.2 Confirmation of buffer system

[0197] ELISA detection was performed using PBST solutions containing 10% FBS / 2% FBS, 2.5% skim milk / 2.5% skim milk, 4% BSA / 1% BSA, 1% BSA / 0.2% BSA, 4% sucrose + 1% BSA / 4% sucrose + 1% BSA as blocking / dilution solutions, respectively. The results showed that when 4% BSA-PBST / 1% BSA-PBST or 1% BSA-PBST / 0.2% BSA-PBST was used as the buffer solution, the sample optical absorbance value was high and the blank value was low (high P / N value), so the buffer system 4% BSA-PBST / 1% BSA-PBST was used in the subsequent experiments (see Table 2).

[0198] Table 2 Buffer system screening

[0199]

[0200] 2.3.3 Confirmation of capture antibody concentration

[0201] Capture antibody concentration 0.25 μg / mL ~ 8 μg / mL; sandwich antigen concentration 40 ng / mL, 2-fold dilution, a total of 7 gradients, while setting up a blank control; detection antibody concentration 0.1 μg / mL. ELISA detection was performed. The results showed that when the coating concentration was 0.25 μg / mL ~ 2 μg / mL, with the increase of the coating concentration, the OD 405 value showed a clear upward trend; when the coating concentration was 2 μg / mL ~ 8 μg / mL, the OD 405 value had little difference, and basically tended to be stable. The coating concentration was determined to be 2 μg / mL (see Figure 3 ).

[0202] 2.3.4 Detection antibody concentration confirmation

[0203] Capture antibody concentration 2 μg / mL; sandwich antigen concentration 20 ng / mL, 2-fold dilution, a total of 7 gradients, while setting up a blank control; detection antibody concentration 0.05 μg / mL ~ 1.6 μg / mL. ELISA detection was performed. The results showed that when the detection antibody concentration was 0.05 μg / mL ~ 0.2 μg / mL, with the increase of the detection antibody concentration, the OD value showed a clear upward trend; when the detection antibody concentration was ≥ 0.2 μg / mL, the OD still showed an upward trend, but the trend was relatively flat (see Figure 4 ). Considering that with the increase of the OD value (system detection limit decreases), accompanied by the increase of the sample dilution multiple, it is easy to cause errors; and when the detection antibody concentration is 0.2 μg / mL, the system sensitivity meets the current sample detection requirements, the detection antibody concentration is determined to be 0.2 μg / mL (see Figure 4 ).

[0204] 2.4 Verification of the double antibody sandwich ELISA quantitative detection method of filamentous hemagglutinin (FHA)

[0205] 2.4.1 Linearity and range

[0206] The concentration of the standard was fitted with the light absorption value by four parameters (4-P), a standard curve was prepared, the correlation coefficient R 2 of the standard curve was verified, and the experiment was repeated 6 times, the coefficient of variability (CV) of the light absorption value of each point of the standard curve was calculated, and the detection range was determined. (The standard curve fitting graph is shown in Figure 5 ) The results showed that the correlation coefficients R 2 of the 6 standard curves were all ≥ 0.999, and the CV values of the 6 detection results of each concentration were all within 20%. The detection range was 20 ng / mL ~ 0.313 ng / mL (see Table 3).

[0207] Table 3 Linear range

[0208]

[0209] 2.4.2 Specificity

[0210] The ELISA test was performed with known concentrations of DPT remaining component proteins and their mixed solutions (PT, PRN, DT, TT) to verify whether the system has cross-reactivity with DPT remaining component proteins. The results showed that the DPT vaccine remaining four component proteins were detected in the FHA antigen content quantitative detection system, and the detection was still negative when the concentration was as high as 25 μg / mL, and there was no cross-reactivity in the detection system, and the specificity of the system was good (see Table 4).

[0211] Table 4 Specificity verification

[0212]

[0213] 2.4.3 Accuracy

[0214] The known antigen content of pertussis intermediate samples was detected three times, and the recovery rate was calculated according to the formula [recovery rate (%) = measured value / theoretical value x 100%] (the lowry detection value was used as the theoretical value). The results showed that the FHA detection value of each intermediate sample (high, medium and low antigen sample) was close to the theoretical value, and the recovery rate was between 80% and 120%, and the accuracy of the system was good (see Table 5).

[0215] Table 5 Accuracy verification

[0216]

[0217] 2.4.4 Precision

[0218] 2.4.4.1 Reproducibility

[0219] 3 samples with different antigen contents were detected three times by the same experimenter, and the coefficient of variability (CV) of the three detection results of each sample was calculated. The results showed that the CV of the three detection values of high, medium and low antigen samples by the same experimenter was within 20%, and the reproducibility of the system was good (see Table 6).

[0220] Table 6 Reproducibility verification

[0221]

[0222] 2.4.4.2 Intermediate precision

[0223] The same antigen sample was detected by 3 different experimenters for 3 repeated tests, and the coefficient of variability (CV) of 9 test results was calculated. The results showed that the CV of sample detection values of 3 repeated experiments of the same sample by 3 testers was within 20%, and the intermediate precision of the system was good (see Table 7).

[0224] Table 7 Verification of intermediate precision

[0225]

[0226] 2.4.5 Durability

[0227] 2.4.5.1 Reaction time

[0228] Under the reaction time (±10 min reaction time, ±5 min color development time), ELISA detection was performed on high, medium and low concentration antigen samples, and the coefficient of variability (Coefficient of Variability, CV) of the detection results of each sample under different reaction times was calculated. The results showed that the light absorption values of the standard curve at different concentrations were different under the three reaction times (see Table 8); but the CV of the detection of high, medium and low antigen samples by the same experimenter under the three reaction times was within 20%, and the recovery rate was between 80% and 120%, and the durability of the system was good (see Table 8). Figure 6

[0229] Table 8 Verification of durability of reaction time

[0230]

[0231] 2.4.5.2 Reaction temperature

[0232] Under the reaction temperature (±2℃), ELISA detection was performed on high, medium and low concentration antigen samples, and the coefficient of variability (Coefficient of Variability, CV) of the detection results of each sample under different temperatures was calculated. The results showed that the CV of the detection of high, medium and low antigen samples by the same experimenter under the three reaction temperatures was within 20%, and the recovery rate was between 80% and 120%, and the durability of the system was good (see Table 9). The light absorption values of the standard curve at different concentrations were not much different under the three reaction temperatures, and the durability of the system was good (see Table 9). Figure 7

[0233] Table 9 Verification of durability of reaction temperature

[0234]

[0235] 2.4.5.3 Solution stability after termination ​​

[0236] The ELISA detection was performed on high, medium and low concentration antigen samples at 0h, 15min, 30min, 1h, 2h and 4h after termination, and the coefficient of variability (CV) of the detection results of each sample at different time periods after termination was calculated. The results showed that at 4h after termination, the OD 405 There was no obvious change, and the solution stability was good. The CV of the detection values of the high, medium and low concentration antigen samples was within 20%, and the system had good durability (see Table 10). The light absorption values of the standard curve at each concentration were not much different at the six reading nodes, and the system had good durability (see Table 11). Figure 8A and Figure 8B ).

[0237] Table 10 Stability verification of solution after termination

[0238]

[0239] 2.5 Filamentous hemagglutinin (FHA) double antibody sandwich ELISA quantitative detection method specific operation

[0240] In summary, the preferred coating concentration of the monoclonal antibody 16E11 is 200 ng / well, and the preferred coating condition is overnight coating at 2-8°C; the enzyme-labeled plate uses 4% BSA PBST solution as the blocking liquid, and the blocking condition is 37°C for 2h; the reference sample of the filamentous hemagglutinin (FHA) antigen is diluted to 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0.313 ng / mL using the diluent; the detection antibody HRP-29B9 has a preferred working concentration of 0.2 μg / mL. The specific operation of the filamentous hemagglutinin (FHA) double antibody sandwich ELISA quantitative detection method is as follows:

[0241] (1) Coating: Dilute the monoclonal antibody 16E11 to 2 μg / mL with the coating liquid, 100 μL / well, and place it in 2-8°C for overnight coating;

[0242] (2) Washing the plate: Discard the liquid in the wells, wash the plate with 300 μL / well of washing liquid for 3 times, and tap the residual liquid in the enzyme-labeled plate;

[0243] (3) Blocking: Add 150 μL / well of the enzyme-labeled plate with the blocking liquid, and block at 37°C for 2h;

[0244] (4) Washing the plate: Discard the liquid in the wells, wash the plate with 300 μL / well of washing liquid for 3 times, and tap the residual liquid in the enzyme-labeled plate;

[0245] (5) Incubate the sample: set standard holes, blank holes and sample holes, add different concentrations of standards to the standard holes in order, add sample diluent to the blank holes, and add the sample to be tested to the sample holes, add 100 μL / hole, incubate at 37°C for 1 h;

[0246] (6) Wash the plate: discard the liquid in the holes, wash the plate with 300 μL / hole of washing solution for 3 times, and pat dry the residual liquid in the enzyme-labeled plate;

[0247] (7) Incubate the enzyme-labeled antibody: dilute the HRP-labeled monoclonal antibody 29B9 to 0.2 μg / mL with diluent, add 100 μL / hole, incubate at 37°C for 1 h;

[0248] (8) Wash the plate: discard the liquid in the holes, wash the plate with 300 μL / hole of washing solution for 3 times, and pat dry the residual liquid in the enzyme-labeled plate;

[0249] (9) Color development: add 100 μL / hole of color developing solution to the enzyme-labeled plate, develop color at 37°C for 30 min;

[0250] (10) Termination: add 50 μL / hole of termination solution to the enzyme-labeled plate to terminate the reaction;

[0251] (11) Read the value: place the enzyme-labeled plate into an enzyme-labeled instrument to measure the OD 405nm absorbance;

[0252] (12) Result processing: subtract the OD value of the blank hole from the average OD value of each standard as the correction value, take the OD 405nm value of the standard as the dependent variable Y, and take the standard concentration as the independent variable X to make a curve; use a four-parameter Logisitic mathematical model to fit the equation: Y = ((A-D) / (1+(X / C)^B))+D, and substitute the sample OD 405nm absorbance value into the formula to calculate the content of the filamentous hemagglutinin in the sample; wherein A is the asymptote value of the curve, D is the asymptote value under the curve, B is the slope of the curve, and C is the corresponding dose when the maximum binding is half.

[0253] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not contradict, they should be considered as within the scope of the present disclosure.

[0254] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the patent scope of the application. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should 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 filamentous hemagglutinin, 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 whose sequences are 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 whose sequences are 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 as shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid fragment as 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 whose sequence is shown in SEQ ID NO: 9, and the light chain comprises an amino acid fragment whose sequence is shown in SEQ ID NO:

10.

3. An antibody combination, characterized in that It comprises the monoclonal antibody according to claim 1 or 2 as a capture antibody; Optionally, it further comprises a detection antibody; Optionally, the detection antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 whose sequences are 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 whose sequences are 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 whose sequence is shown in SEQ ID NO: 17, and the light chain variable region optionally comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 18; Further optionally, the detection antibody comprises a heavy chain and a light chain, the heavy chain comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 19, and the light chain comprises an amino acid fragment whose sequence is shown in SEQ ID NO:

20.

4. A nucleic acid, characterized in that It encodes the monoclonal antibody according to claim 1 or 2, or the antibody combination according to 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 light chain variable region of the detection antibody is shown in SEQ ID NO:

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

28.

5. A recombinant expression vector, characterized in that: It comprises the nucleic acid according to claim 4.

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

7. A kit for detecting filamentous hemagglutinin, characterized in that: It comprises the monoclonal antibody according to claim 1 or 2, or the antibody combination according to claim 3; Optionally, the kit comprises the antibody combination, wherein the detection antibody is biologically or chemically labeled; the detection antibody is optionally enzyme-labeled, and further optionally horseradish peroxidase or alkaline phosphatase-labeled; Optionally, the kit further comprises one or more of a coating solution, a blocking solution, a diluent, a substrate buffer, a washing solution, a developing solution, and a stop solution; wherein the coating solution is optionally a carbonate buffer; the blocking solution is optionally a 1% w / v~4% w / v BSA-PBST solution; the diluent is optionally a 0.2% w / v~1% w / v BSA-PBST solution; and the substrate buffer is optionally a citric acid solution.

8. Use of the monoclonal antibody according to claim 1 or 2 or the antibody combination according to claim 3 in the preparation of a reagent or kit for detecting filamentous hemagglutinin.

9. A method for detecting filamentous hemagglutinin for non-diagnostic purposes, characterized in that: It includes the following steps: Coating: Dilute the capture antibody with coating solution and add it to the ELISA plate for coating, then wash and remove excess liquid; Blocking: Add blocking solution to the ELISA plate, wash and remove excess residual solution; Incubate samples: Add the sample to be tested to the ELISA plate, wash after the reaction, and remove excess liquid; Incubate enzyme-labeled antibodies: Add biologically or chemically labeled detection antibodies to the ELISA plate, wash after the reaction, and remove excess liquid; Color development: add color development solution and incubate; Termination: Add stop solution to terminate the reaction; Detection: Read OD on a microplate reader 405 value; wherein the capture antibody and the detection antibody are each independently as defined in claim 3; Optionally, during the coating step, the concentration of the capture antibody in the ELISA plate is 2 μg / mL to 8 μg / mL; Optionally, in the enzyme-labeled antibody incubation step, the concentration of the detection antibody in the enzyme-labeled plate is 0.2 μg / mL to 1.6 μg / mL; Optionally, the method is performed using a kit as described in claim 7.

10. Use of the monoclonal antibody according to claim 1 or 2, the antibody according to claim 3, or the kit according to claim 7 in quality testing of vaccines containing filamentous hemagglutinin antigen; Optionally, the vaccine is a pertussis vaccine; Optionally, the quality inspection includes one or more of production quality control, laboratory testing and quality random inspection.

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