Monoclonal antibody aiming at bordetella pertussis antibody as well as preparation method and application thereof

Abstract

The invention discloses a monoclonal antibody aiming at a bordetella pertussis antibody as well as a preparation method and application of the monoclonal antibody. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of which the sequences are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of which the sequences 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 bordetella pertussis quality control antibody, is applied to the clinical diagnosis of pertussis, and provides reference for the clinical diagnosis of pertussis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to monoclonal antibodies against Bordetella pertussis antibodies, and preparation method and application thereof. BACKGROUND

[0002] Whooping cough is caused by Bordetella pertussis, and in recent years it has shown a global resurgence trend, especially in infants and young adults / adults who have not completed the immunization program. Its typical symptoms include paroxysmal convulsive cough, and the course can last for several months, and in severe cases it can lead to pneumonia, encephalopathy and even death, posing a constant threat to public health. Early diagnosis of whooping cough is difficult, and the symptoms are easily confused with colds, and the culture of Bordetella pertussis has a long cycle and low positive rate. The current diagnosis relies on nucleic acid detection (PCR), serological detection (such as PT-IgG antibody) and pathogen isolation. However, the detection methods, reagent sensitivity and specificity of different laboratories differ greatly, and there is an urgent need for quality control antibodies as standard products to ensure the comparability and accuracy of test results, and to provide timely reference and guidance for clinical rapid diagnosis and treatment.

[0003] After natural infection of Bordetella pertussis, antibodies related to pertussis antigens (PT, FHA, PRN, FIM and whole microorganisms) can appear, and Bordetella pertussis can express multiple virulence factors, among which pertussis toxin (Pertussis toxin, PT) is a unique virulence factor. Determining the concentration of PT-IgG antibody in serum is the goal of serological detection, which generally uses ELISA or multiplex immunoassay (MIA) method. For patients with a cough course of less than 2 weeks, the serological positive rate may be low due to the relatively short time of suffering from whooping cough, and not enough antibodies have been formed. However, if the patient has been coughing for 2-3 weeks or more, the ECDC recommends that the most effective way to diagnose is to detect the level of anti-PT-IgG by serological method, especially for older children, adolescents and adults who often visit the doctor later. Choose pertussis toxin (PT) as the immunogen, develop human chimeric antibodies as the quality control antibody for Bordetella pertussis, and provide methods and references for the diagnosis of whooping cough in clinic.

[0004] In the existing methods, suspected and / or clinical diagnosis can be made according to the patient's epidemiological history, clinical manifestations and blood routine results, which requires higher clinical experience of doctors, is not easy to distinguish in clinic, and needs pathogenic or immunological examination for identification, but the defects are that the work experience of medical staff will affect the diagnosis of the disease, and delay the treatment of the disease; or, Bordet-Gengou agar medium (hereinafter referred to as B-G medium) containing commercialized Bordetella pertussis selective additive or appropriate concentration of cefalexin and Regan-Lowe charcoal agar medium (hereinafter referred to as R-L medium) can be selected, and after sampling, the partition streak method is used for inoculating the B-G medium or R-L medium plate containing the selective additive. After the partition streak inoculation, 35°C~37°C culture is carried out, and continuous observation is carried out for 3 days~7 days, if there is no any bacterial growth trace, negative can be reported on the 7th day, if there is bacterial growth evidence, continuous culture and observation can be carried out for 14 days, but the defect of this method is that the clinical diagnosis is carried out through the culture of Bordetella pertussis, which takes a long time and cannot treat the disease in time and effectively, increasing the burden of the patient. SUMMARY

[0005] Therefore, the present application provides at least one monoclonal antibody against Bordetella pertussis, a preparation method and application thereof.

[0006] In a first aspect of the present application, a monoclonal antibody against Bordetella pertussis 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, the sequences of which are respectively shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the sequences of which are respectively shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0007] In a second aspect of the present application, a nucleic acid molecule is provided, which comprises nucleic acid fragment 1 and nucleic acid fragment 2 encoding the monoclonal antibody of the first aspect; wherein the nucleic acid fragment 1 comprises a nucleic acid fragment encoding the heavy chain variable region, and the nucleic acid fragment 2 comprises a nucleic acid fragment encoding the light chain variable region.

[0008] In a third aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid molecule of the second aspect.

[0009] In a fourth aspect of the present application, a host cell is provided, which expresses the monoclonal antibody of the first aspect.

[0010] In a fifth aspect of the present application, a method for preparing the monoclonal antibody of the first aspect is provided, comprising:

[0011] culturing the host cell of the fourth aspect to obtain a culture;

[0012] isolating the monoclonal antibody from the culture.

[0013] In a sixth aspect of the present application, a kit for quantitatively detecting Bordetella pertussis antibodies in a sample is provided, comprising the monoclonal antibody of the first aspect.

[0014] In a seventh aspect of the present application, the use of the monoclonal antibody of the first aspect in the preparation of a reagent or kit for detecting Bordetella pertussis antibodies is provided.

[0015] In an eighth aspect of the present application, a method for using the kit of the seventh aspect is provided, comprising the steps of:

[0016] mixing and incubating the monoclonal antibody in the kit with a sample;

[0017] reading the signal.

[0018] In one aspect of the present application, Balb / c mice are immunized with a pertussis toxin antigen, and their spleens are used to prepare a single-cell suspension. Memory B cells specific to the pertussis toxin are sorted, and the antibody genes are obtained through reverse transcription PCR of the single B cells. Through activity screening of the obtained antibody genes, a pertussis toxin antibody with good specificity and high sensitivity is obtained, which is used as a Bordetella pertussis quality control antibody and applied in the clinical diagnosis of pertussis, thereby providing a reference for the clinical diagnosis of pertussis. BRIEF DESCRIPTION OF DRAWINGS

[0019] 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 in the embodiment or example description will be briefly introduced below. 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 labor. 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.

[0020] Figures 1A to 1E The schematic diagrams of flow sorting specific B cells in an embodiment of the present application are shown respectively.

[0021] Figure 2 The antibody light chain expression vector diagram in an embodiment of the present application is shown.

[0022] Figure 3Figure 1 shows a map of an antibody heavy chain expression vector in an embodiment of the present application. DETAILED DESCRIPTION

[0023] For the purposes of this application, the application will now be described in more detail with reference to the enclosed drawings. The figures show preferred embodiments of the application. However, the application can be implemented in many different ways and is not limited to the embodiments described herein. Rather, the embodiments are provided so that the disclosure of the application will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

[0024] 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 herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0025] 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.

[0026] As used herein, "combinations thereof", "any combination thereof", "any combination" and the like include all suitable combinations of any two or more of the listed items.

[0027] In the present application, "suitable combinations", "suitable ways", "any suitable way" and the like, "suitable" means that the technical solutions of the present application can be implemented, the technical problems of the present application can be solved, and the intended technical effects of the present application can be achieved.

[0028] In the present application, "further", "even further", "in particular", "for example", "such as", "for instance", "for example" and the like are used for descriptive purposes and indicate that the preceding and following technical solutions have a correlation in terms of coverage, but should not be understood as a limitation on the preceding technical solution, nor as a limitation on 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.

[0029] In the present application, "optionally", "optional", "option" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "optional" in a technical solution, if there is no special description, and there is no contradiction or mutual restriction relationship, each "optional" is independent. If there is no other description, the present application is described as "optionally includes", "optionally contains" and the like. For example, "optionally includes" means "may include or not include".

[0030] The terms "contain", "include" and "comprise" used in the present application are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members or features. Members or features, such as materials or components, structures, elements, instruments, etc.; Non-limiting examples of members or features also include actions, conditions, timing, states, etc.

[0031] 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.

[0032] In the present application, the exemplary description involving "in some embodiments (or examples)", "in one embodiment (or example)" and the like can cover but is not limited to the following meanings: these schemes can be combined with other schemes in a suitable manner to form new technical solutions.

[0033] In the present application, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth" and the like are only for description purposes, 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" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0034] In the present application, when a numerical interval (i.e., a numerical range) is involved, the distribution of the optional numbers 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 each number between the two numerical endpoints, unless otherwise specified. When a numerical interval refers only to integers in the numerical interval, including both endpoint integers and each integer between the two endpoints, it is equivalent to directly listing each integer, unless otherwise specified. When multiple numerical ranges are provided to describe a feature or a characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein are to be interpreted to include any and all sub-ranges therein. The "numbers" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is intended to broadly include numerical interval types such as percentage interval, ratio interval, and the like.

[0035] Unless otherwise specified, the English full name of the term "OD value" in the present application is optical density, which refers to the absorbance of a certain substance at a certain specific wavelength.

[0036] In the present application, unless otherwise specified, the execution of the steps involved in the method flow does not have strict order restrictions, and the steps can be executed in other orders described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily be executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0037] A quality control monoclonal antibody for detecting Bordetella pertussis antibodies is disclosed in the present application, and the antibody is used for the development of an in vitro diagnostic kit, which is applied to the quantitative detection of human Bordetella pertussis antibodies in clinical samples.

[0038] After the human body is infected, PT-IgG can be detected in the serum 2-3 weeks or more. By detecting the content of PT-IgG in the serum, the effect of detecting Bordetella pertussis infection can be achieved. The Balb / c mice are immunized with pertussis toxin antigen, and the spleen is taken to prepare a single cell suspension. The memory B cells specific to pertussis toxin are sorted, and the antibody gene is obtained by reverse transcription-PCR (RT-PCR) of single B cells. Through the activity screening of the obtained antibody gene, a pertussis toxin antibody with good specificity and high sensitivity is obtained, which is used as a quality control antibody for Bordetella pertussis and applied in the clinical diagnosis of pertussis, providing a reference for the clinical diagnosis of pertussis.

[0039] Using single B-cell antibody discovery technology, the sequence of antibodies against pertussis toxin was obtained, compared with traditional hybridoma technology, the development cycle was shortened to 3 months (including the time of immunization); a large number of antibody sequences can be obtained by identification.

[0040] Unless otherwise specified, the term "monoclonal antibody" or "monoclonal" 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.

[0041] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the sequences set forth in SEQ ID NO: 1 (WIE), SEQ ID NO: 2 (EILPGSDKINYNEKFTG) and SEQ ID NO: 3 (GGDGYAMDY), respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the sequences set forth in SEQ ID NO: 4 (RASQSIGTNIH), SEQ ID NO: 5 (YTSESIS) and SEQ ID NO: 6 (QQSNIWPTT), respectively.

[0042] 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 modification" or "conservative substitution or replacement") of the corresponding CDR, and 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 of the corresponding complementarity determining region 1, 2, 3, 4, 5 or 6, which can be a substitution of one amino acid with another amino acid, or a substitution of one amino acid with multiple (e.g., 2) amino acids.

[0043] In the CDRs provided in the present application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, 3 amino acids with amino acids of similar or similar properties compared with the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0044] Table A

[0045]

[0046] "Conservative modifications" or "conservative substitutions or replacements" refer to the replacement of an amino acid in a protein with another amino acid having 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, substitutions of amino acids with similar structures or functions are not likely to disrupt biological activity.

[0047] In some embodiments, the heavy chain variable region comprises an amino acid fragment having a sequence as set forth in SEQ ID NO: 7 (QVHLQQSGAEVMKPGASVKISCKAIGFSRYWIEWVKQRPGHGLEWIGEILPGSDKINYNEKFTGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGGDGYAMDYWGQGTSVTVSS) and the light chain variable region comprises an amino acid fragment having a sequence as set forth in SEQ ID NO: 8 (DILLTQSPAILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYTSESISGIPSRFSGSGSGTDFTLSINSVESEDIAVYYCQQSNIWPTTFGAGTKLELK).

[0048] In some embodiments, the amino acid sequence of the heavy chain variable region described above is as set forth 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 to SEQ ID NO: 7.

[0049] In some embodiments, the amino acid sequence of the light chain variable region described above is as set forth 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 to SEQ ID NO: 8.

[0050] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence when aligning amino acid sequences (introducing gaps where necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0051] In some embodiments, the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid fragment with the sequence shown in SEQ ID NO: 9, and the light chain comprising an amino acid fragment with the sequence shown in SEQ ID NO: 10.

[0052] In some embodiments, the amino acid sequence of the heavy chain described above is as shown in SEQ ID NO: 9 or the amino acid sequence 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 SEQ ID NO: 9.

[0053] In some embodiments, the amino acid sequence of the light chain described above is as shown in SEQ ID NO: 10 or the amino acid sequence 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 SEQ ID NO: 10.

[0054] Another aspect of this application provides a nucleic acid molecule that encodes a monoclonal antibody as described above.

[0055] In some embodiments, the nucleic acid molecule comprises nucleic acid fragment 1 and nucleic acid fragment 2.

[0056] In some embodiments, nucleic acid fragment 1 comprises a nucleic acid fragment encoding a heavy strand variable region, and nucleic acid fragment 2 comprises a nucleic acid fragment encoding a light strand variable region.

[0057] In some embodiments, nucleic acid fragment 1 comprises a nucleic acid fragment encoding a heavy chain, and nucleic acid fragment 2 comprises a nucleic acid fragment encoding a light chain.

[0058] In some embodiments, the nucleic acid molecule is a combination product comprising a nucleic acid molecule 1 and a nucleic acid molecule 2, each comprising nucleic acid segment 1 and nucleic acid segment 2, respectively.

[0059] Unless otherwise indicated, the nucleic acid molecules in the present application can be isolated nucleic acid molecules. The nucleic acid molecules of the present application refer primarily to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cellular debris and growth media. Generally, the term "isolated" is not intended to mean completely free from these materials or free from water, buffers, or salts.

[0060] Another aspect of the present application provides a recombinant expression vector comprising the nucleic acid molecule as described above.

[0061] 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.

[0062] In some embodiments, the vector is a pCMV vector.

[0063] Another aspect of the present application provides a host cell expressing the monoclonal antibody as described above.

[0064] The term "cell" or "host cell" refers to a cell into which an 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, 293T cells, or HEK293F cells.

[0065] Another aspect of the present application provides a method of producing the above-mentioned host cell, comprising the step of introducing the above-mentioned or recombinant expression vector into a target cell.

[0066] In one example, the introduction is by transfection.

[0067] 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.

[0068] Another aspect of the present application provides a method of producing the above-mentioned monoclonal antibody, comprising:

[0069] culturing the above-mentioned host cell to obtain a culture;

[0070] isolating the monoclonal antibody from the culture.

[0071] The method of isolating the monoclonal antibody is exemplified by, for example, affinity chromatography and ion exchange chromatography.

[0072] Another aspect of the present application provides a kit for quantitatively detecting Bordetella pertussis antibodies in a sample, comprising the monoclonal antibody as described above.

[0073] Illustratively, the sample can be blood, serum, plasma, etc.

[0074] In some embodiments, the kit further comprises a positive control and / or a negative control.

[0075] In some embodiments, the kit further comprises an antibody diluent.

[0076] Another aspect of the present application also provides use of the monoclonal antibody as described above in the preparation of a reagent or kit for detecting Bordetella pertussis antibodies.

[0077] Another aspect of the present application provides a method for using the kit as described above, comprising the following steps:

[0078] mixing / incubating the monoclonal antibody in the kit with a sample;

[0079] reading the signal.

[0080] Some embodiments are provided below.

[0081] Embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. In the following examples, the experimental methods without specified conditions are preferred to refer to the guidance given in the present 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.

[0082] Example 1

[0083] 1. Mouse immunization and spleen B cell enrichment

[0084] 8-12 week old Balb / c mice were immunized with pertussis toxin antigen, using Freund's adjuvant emulsification with antigen and subcutaneous immunization, and the tail blood of the mice was detected by ELISA indirect method until the titer of the tail blood reached 10 5The mice were immunized for 3 days, and then stopped immunization. The mice were euthanized, and the spleen was removed using dissection tools. The connective tissue remaining in the spleen was removed as much as possible, and the spleen was placed in a 40 μm polyester sterile cell filter, immersed in FACS buffer, and placed in a culture dish on ice. The spleen was cut into 3 mm pieces using scissors, and the spleen tissue was ground using a 10 mL syringe piston rubber head to prepare a single cell suspension. The spleen cells passing through the 40 μm polyester sterile cell filter were transferred to a 15 mL conical tube, and the final volume was adjusted to 15 mL with cold FB. The cells were counted, and the cell density was 1.11x10E7 / mL, and the cell viability was 82.13%. 0.2 mL of the cell precipitate was added to 1 mL of red blood cell lysis solution, mixed gently, and lysed for 1-2 minutes. 15-20 mL of PBS, HBSS, normal saline, or serum-free culture medium was added, mixed, and centrifuged at 400-500g for 5 minutes. The red supernatant was discarded. The memory B cells in the mouse spleen were enriched by a commercial kit, and then resuspended in 1xPBS solution.

[0085] 2. Mouse-specific B cell markers and sorting

[0086] After the enrichment of mouse spleen memory B cells is completed, the fluorescently labeled pertussis toxin antigen and mouse memory B cell surface marker fluorescent antibodies are used for labeling. The B cell density obtained by enrichment is adjusted to 1x10E7 / mL using 5 mL of FACS buffer, 5 μL of CD16 / CD32 Monoclonal Antibody is added to the B cell suspension, the dilution concentration is 1 / 1000, and the working concentration is 0.5 μg / mL, and 4°C incubation for 30 min. After the Fc fragment blocking is completed, centrifuge at 300 x g for 5 min, and resuspend the cell density to 1x10E7 / mL using FACS buffer. Specific B cell labeling is performed, goat anti-mouse IgG1 gamma chain (Cy5 ®) working concentration dilution 1 / 500, CD45R (B220) Monoclonal Antibody (RA3-6B2), eFluor™ 660 working concentration dilution 1 / 500, CD38 Monoclonal Antibody (90), PE-eFluor™ 610 working concentration dilution 1 / 400, IgM Monoclonal Antibody (II / 41), eFluor™ 450 working concentration dilution 1 / 400, Ag-488 working concentration dilution 1 / 500; the above fluorescently labeled antibodies are added at the same time, respectively, and incubated at 4°C in the dark for 30 min. The fluorescently labeled memory B cells are sorted by flow cytometry instrument, and the specific memory B cells are sorted into 96-well PCR plates. The flow cytometry sorting settings are shown in Figures 1A to 1E .

[0087] 3. Reverse transcription to synthesize cDNA and antibody gene to obtain an expression vector

[0088] 96-well plates are used for reverse transcription to synthesize cDNA, and then cDNA is used as a template for PCR amplification of cDNA. Primers specific to the variable region of mouse antibodies are designed, and the PCR amplification product of cDNA is used as a template for PCR amplification to obtain antibody gene PCR products. Nucleic acid gel electrophoresis is used to detect PCR products, and the amplified products of antibody light chains and heavy chains are selected for sequencing. The sequencing results are analyzed, and finally 88 antibodies are obtained. Primers specific to the variable region of antibodies are designed, and the necessary sequences for seamless cloning are added. The sequencing sample is used as a template for PCR reaction, gel electrophoresis and gel recovery to obtain the target fragment. The target fragment is recovered by gel recovery kit, the concentration of the recovered product is determined and labeled. The PCR product and double-digested pCMV vector are connected by seamless cloning kit, and the plasmid is extracted by transformation and amplification for sequencing. The correct construction of the expression vector is confirmed, and the transient expression vector is shown in Figure 2 and Figure 3 .

[0089] 4. Antibody plasmid extraction, transfection, expression and activity screening

[0090] 293T cells were seeded in 96-well plates 12h before transfection, and PEI was used for transfection, with a total of 1 μg of DNA per well, and the ratio of light chain to heavy chain was 2:1. The cell supernatant was collected 72h after transfection, and ELISA was used to screen the supernatant with antibody activity. The ELISA results are shown in Table 1. Through ELISA screening, 88 antibodies were screened, and 79 antibodies were confirmed to have activity.

[0091] Table 1. ELISA detection results of supernatant 72h after transfection

[0092]

[0093] 5. Preparation of recombinant antibodies and detection of pertussis toxin antibody specificity and quality control performance

[0094] The antibody with an OD value greater than 3.0 in the above experiment was extracted to obtain light and heavy chain plasmids. PEI was used to transfect mammalian cells HEK293F, and the cell density was 2.0x10 6 / mL, and the cell volume was 400mL. The expression product was purified by Protein A affinity chromatography and ion exchange chromatography to obtain pertussis toxin antibodies. The obtained antibodies were diluted to 1.0mg / mL, and ELISA was used for detection. Pertussis filamentous hemagglutinin (FHA), tetanus toxoid (TTd) and human serum albumin (HSA) were coated on a 96-well plate (1ug / mL, pH9.6 carbonate buffer) at the same time, and pertussis toxin antigen was used as a positive control. PT-31-IgG and PT-68-IgG were added at a dilution of 1000 times, incubated at 37°C for 1 hour, and then HRP-labeled anti-human IM secondary antibody was used for color development (TMB substrate), and the values of A450 and A630 were read. The ELISA detection results are shown in Table 2. PT-31-IgG and PT-68-IgG have high specificity for recombinant and natural PT antigens. The purified antibodies were diluted to 1.0mg / mL, and the performance of the antibodies was tested using a calibration mode, and the test results are shown in Table 3. The luminescence value and the concentration of the pertussis toxin antibodies have a linear relationship under the condition of gradient dilution, and the back-calculated concentration calculated according to the dilution factor meets the use requirements.

[0095] Table 2. Specificity detection of pertussis toxin antibodies

[0096]

[0097] Table 3. Performance detection of pertussis toxin antibodies

[0098]

[0099] Table 2 shows that PT antibody diluted 1000-fold from 1.0 mg / mL stock solution, the reactivity to different antigens was detected by ELISA, only in the well plate coated with PT antigen, signal was detected, which proved that PT antibody has high specificity.

[0100] Table 3 shows that the concentration point after dilution 5000-fold from 1.0 mg / mL stock solution (i.e. 0.2 μg / mL) still has measurable luminescence value and concentration value. It can be seen that the sensitivity of the pertussis toxin antibody can be as low as 0.2 μg / mL or even lower. In addition, within the dilution range of 100-fold to 5000-fold, the luminescence signal of the antibody and the concentration showed a good linear relationship, indicating that the detection system is stable, which proves the reliability of the antibody concentration prediction after dilution, which meets the quality control requirements.

[0101] 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, however, as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present disclosure.

[0102] The above-described embodiments only express several implementation manners of the present application, the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of variations and improvements can be made, which are all within the scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims, the description and drawings can be used to explain the content of the claims.

Claims

1. A monoclonal antibody against Bordetella pertussis, characterized in that, It contains variable regions of heavy chains and variable regions of light chains; The heavy chain variable region includes sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region includes sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

2. The monoclonal antibody as described in claim 1, characterized in that, The heavy chain variable region comprises an amino acid fragment with the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid fragment with the sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody as described in claim 1 or 2, characterized in that, The monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid fragment with the sequence shown in SEQ ID NO: 9, and the light chain comprising an amino acid fragment with the sequence shown in SEQ ID NO:

10.

4. A nucleic acid molecule, characterized in that, It comprises nucleic acid fragment 1 and nucleic acid fragment 2 encoding the monoclonal antibody as described in any one of claims 1 to 3; wherein, nucleic acid fragment 1 comprises a nucleic acid fragment encoding the heavy chain variable region, and nucleic acid fragment 2 comprises a nucleic acid fragment encoding the light chain variable region; Optionally, the nucleic acid molecule is a combination product and comprises nucleic acid molecule 1 and nucleic acid molecule 2, which respectively contain nucleic acid fragment 1 and nucleic acid fragment 2.

5. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It expresses the monoclonal antibody as described in any one of claims 1 to 3; Optionally, the host cell comprises the nucleic acid molecule as described in claim 4 and / or the recombinant expression vector as described in claim 5; Optionally, the host cell includes non-human mammalian cells; for example, 293T cells.

7. The method for preparing the monoclonal antibody according to any one of claims 1 to 3, characterized in that, It includes: Culture the host cells as described in claim 6 to obtain a culture; The monoclonal antibody was isolated from the culture.

8. A kit for the quantitative detection of Bordetella pertussis antibodies in samples, characterized in that, It comprises the monoclonal antibody as described in any one of claims 1 to 3; Optionally, the sample may include one or more of blood, serum, and plasma.

9. Use of the monoclonal antibody as described in any one of claims 1 to 3 in the preparation of reagents or kits for detecting antibodies against Bordetella pertussis.

10. The method of using the reagent kit as described in claim 7 or 8, characterized in that, It includes the following steps: Mix the monoclonal antibody from the kit with the sample and incubate. Read the signal.

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