CN115975021B

CN115975021B - Coxsackie virus A10 monoclonal antibody and application thereof

Info

Publication number: CN115975021B
Application number: CN202211451179.3A
Authority: CN
Inventors: 刘庆伟; 王晓黎; 阮丽珠; 杨义; 边金; 张玺; 程飞
Original assignee: Huasong Shanghai Biomedical Technology Co ltd
Current assignee: Huasong Shanghai Biomedical Technology Co ltd
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2024-08-16
Anticipated expiration: 2042-11-18
Also published as: CN115975021A

Abstract

The application relates to the field of antibodies, in particular to a coxsackievirus A10 monoclonal antibody and application thereof. The A10 monoclonal antibody comprises one or more of the following technical characteristics: the heavy chain variable region comprises CDR-H1 with an amino acid sequence shown as SEQ ID No. 1; the heavy chain variable region comprises CDR-H2 with an amino acid sequence shown as SEQ ID No. 2; the heavy chain variable region comprises CDR-H3 with an amino acid sequence shown as SEQ ID No. 3; the light chain variable region comprises CDR-L1 with an amino acid sequence shown as SEQ ID No. 4; the light chain variable region comprises CDR-L2 with an amino acid sequence shown as SEQ ID No. 5; the light chain variable region comprises CDR-L3 of the amino acid sequence shown in SEQ ID No. 6. Through screening detection, a monoclonal antibody which can specifically bind to A10 and has neutralization activity is obtained: 10H 11. The 10H11 can be used for developing antiviral drugs of CV A10, and can be used as a high-efficiency sensitive detection reagent in clinical diagnosis of A10 infection, an A10 vaccine screening and qualitative and quantitative detection and detection reagent of vaccine antigens, and an antibody reference for evaluating the activity of the A10 vaccine.

Description

Coxsackie virus A10 monoclonal antibody and application thereof

Technical Field

The application relates to the field of antibodies, in particular to a coxsackievirus A10 monoclonal antibody and application thereof.

Background

Hand-foot-mouth disease (Hand foot mouth disease, HFMD) is a common infectious disease of infants and infants caused by different enteroviruses, and clinical symptoms of the infectious disease are common in hand, foot, mouth rash and herpes; most hand-foot-and-mouth disease has lighter illness and self-limiting, but a small number of patients have rapid illness progress, and serious central nervous system disease complications are accompanied, so that the patients die from heart-lung failure, and the life health of children is seriously threatened in view of wide HFMD transmission range and serious hazard, and the patients are classified as the type-III infectious disease in 2008 in China. The causative agent of hand-foot-and-mouth disease is enterovirus type a (EV a), mainly comprising enterovirus type 71 (EV a 71) coxsackievirus (coxsackievirus, CV) (CV a16, CV A6, and CV a 10), wherein EV a71 and CV a16 are the main causative agents causing HFMD. However, in recent years, researches show that the hand-foot-and-mouth disease cases caused by CV A10 and CV A6 infection show an ascending trend, and CV A10 is gradually transformed into dominant pathogens of HFMD in partial areas of China.

Clinical manifestations of hand-foot-and-mouth disease caused by different virus infections are slightly different. After CV A10 infection, only mild and self-limiting disease symptoms such as fever, canker sore, rash or herpes on hands and feet and the like are caused, and serious hand-foot-mouth disease complications such as nail fall-off, herpangina, neuromyelitis, meningitis and the like are also caused, or death is caused. Currently, monovalent EV A71 inactivated vaccines independently developed in China are marketed, but cannot prevent HFMD caused by other enteroviruses such as CV A10 and the like. There is no preventive vaccine or specific therapeutic drug on the market for CV A10, so the preparation of a high-efficiency, low-cost and low-side-effect passive immune preparation becomes a research hot spot. In vitro antiviral neutralizing activity and in vivo protection of monoclonal antibodies against viral challenge many experiments have been obtained to demonstrate that neutralizing antibody drugs can prevent and treat viral infections in vivo to protect animals from viral challenge. The method has important significance for promoting the treatment and research of severe and dead cases caused by hand-foot-mouth disease viruses in China, and has extremely important value for further advanced clinical research, epidemiological development and passive treatment.

Disclosure of Invention

In view of the above-described shortcomings of the prior art, the present study prepared neutralizing monoclonal antibodies that specifically recognized CV A10 using CV A10 VLPs as immunogens. The 10H11 monoclonal antibody can be used for sensitively detecting and analyzing CV A10 through methods such as Elisa, SDS-PAGE, western blot, neutralization experiments and the like, has stronger neutralization activity, and has great potential in the diagnosis of virus infection and the research of vaccine development.

To achieve the above and other related objects, a first aspect of the present application provides a coxsackievirus a10 monoclonal antibody, the a10 monoclonal antibody comprising one or more of the following technical features:

The heavy chain variable region comprises CDR-H1 with the amino acid sequence shown in SEQ ID No. 1: GFTFTEYS;

the heavy chain variable region comprises CDR-H2 having the amino acid sequence shown in SEQ ID No. 2: IRNKANGYRT;

the heavy chain variable region comprises CDR-H3 having the amino acid sequence shown in SEQ ID No. 3: AREISGIHYFGYGFAY;

the light chain variable region comprises CDR-L1 with the amino acid sequence shown in SEQ ID No. 4: QTIVHSSGNTY;

the light chain variable region comprises CDR-L2 having the amino acid sequence shown in SEQ ID No. 5: RVS;

the light chain variable region comprises CDR-L3 with the amino acid sequence shown in SEQ ID No. 6: FQGSHVPWT.

In any embodiment of the application, the heavy chain variable region and the light chain variable region further comprise framework regions FR1-FR4.

In any embodiment of the application, the amino acid sequence of FR1 of the heavy chain variable region comprises the sequence set forth in SEQ ID No: 7: EVKLVESGGGLVQPGGSLRLSCTTS;

The amino acid sequence of FR2 of the heavy chain variable region comprises the sequence shown in SEQ ID No. 8: MSWVRQPPGKALEWLGF;

The amino acid sequence of FR3 of the heavy chain variable region comprises the sequence shown in SEQ ID No. 9: EYNPSMEGRFTISRDNSQSTLYLQMNTLSTEDSATYYC;

The amino acid sequence of FR4 of the heavy chain variable region comprises the sequence shown in SEQ ID No. 10: WGQGTLVTVSA.

In any embodiment of the application, the amino acid sequence of FR1 of the light chain variable region comprises the sequence set forth in SEQ ID No: 11: DVLMTQTPLSLPVSLGDQASISCRSS;

the amino acid sequence of FR2 of the light chain variable region comprises the sequence shown in SEQ ID No. 12: LEWYLQKPGQSPKVLIY;

The amino acid sequence of FR3 of the light chain variable region comprises the sequence shown in SEQ ID No. 13: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC;

The amino acid sequence of FR4 of the light chain variable region comprises the sequence shown in SEQ ID No. 14: FGGGTKLEIK.

In any embodiment of the application, the heavy chain further comprises a signal peptide; the heavy chain signal peptide comprises an amino acid sequence shown in SEQ ID No. 15: MKLWLNWIFLVTLLNGIQC.

In any embodiment of the application, the light chain further comprises a signal peptide; the light chain signal peptide comprises an amino acid sequence shown as SEQ ID No. 16: MKLPVRLLVLMFWIPASSS.

In any embodiment of the application, the amino acid sequence of the heavy chain comprises the sequence shown in SEQ ID No. 17;

And/or the amino acid sequence of the light chain comprises the sequence shown in SEQ ID No. 18.

In a second aspect, the application provides a nucleotide molecule encoding said monoclonal antibody.

In a third aspect the application provides a construct comprising said nucleotide molecule.

In a fourth aspect the application provides an expression system comprising said construct or genome incorporating said nucleotide molecule.

In a fifth aspect, the application provides the use of said monoclonal antibody or said nucleotide molecule or said construct or said expression system in the preparation of a virus detection product, in the preparation of a product for the prevention or treatment of a virus infection, or for screening and detection of a virus vaccine or for evaluation of vaccine activity.

In any embodiment of the application, the virus is an enterovirus, preferably coxsackievirus a10; more preferably, the monoclonal antibody is used for preparing a hand-foot-and-mouth disease detection product, a hand-foot-and-mouth disease prevention or treatment product, or an antibody reference product for screening hand-foot-and-mouth disease vaccines, qualitative and quantitative detection and verification of vaccine antigens or evaluation of vaccine activity.

Compared with the prior art, the application has the beneficial effects that:

1. the monoclonal antibody provided by the application can specifically recognize the A10 virus-like particle, but cannot recognize the denatured virus-like particle, which suggests that the epitope recognized by the monoclonal antibody may be a conformational epitope.

2. The monoclonal antibody provided by the application has a minimum detection limit of 15.625ng on A10 virus-like particles, and is a powerful and effective detection tool.

3. The monoclonal antibody provided by the application has the neutralization concentration of 3.12 mug/ml, shows strong neutralization activity, can be used for developing antiviral drugs of CV A10, can be used as a high-efficiency and sensitive detection reagent in clinical diagnosis of A10 infection, and can also be used as an antibody reference for evaluating vaccine activity.

Drawings

FIG. 1 shows a schematic diagram of SDS-PAGE analysis of monoclonal antibodies.

FIG. 2 shows a schematic diagram of a monoclonal antibody specificity assay.

FIG. 3 shows a schematic diagram of Western blot analysis of monoclonal antibodies.

FIG. 4 shows a schematic diagram of sandwich ELISA assay monoclonal antibodies.

FIG. 5 shows a schematic diagram of sequence validation of 10H11 mab.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application will be further described with reference to examples. It is to be understood that the examples are provided for the purpose of illustrating the application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventor of the present application has found a coxsackievirus A10 monoclonal antibody and application thereof through a great deal of research and study, and completed the present application on the basis.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') ₂ fragments; antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules; or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.

Monoclonal antibodies can be produced by several known techniques, such as phage technology, bacterial, yeast or ribosome display, as well as classical methods exemplified by antibodies derived from hybridomas. Monoclonal antibodies can be obtained by various methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by hybridoma methods, or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries.

In one aspect, the application provides a coxsackievirus A10 monoclonal antibody, wherein the A10 monoclonal antibody comprises one or more of the following technical characteristics:

the heavy chain variable region comprises CDR-H1 with an amino acid sequence shown as SEQ ID No. 1;

the heavy chain variable region comprises CDR-H2 with an amino acid sequence shown as SEQ ID No. 2;

The heavy chain variable region comprises CDR-H3 with an amino acid sequence shown as SEQ ID No. 3;

the light chain variable region comprises CDR-L1 with an amino acid sequence shown as SEQ ID No. 4;

The light chain variable region comprises CDR-L2 with an amino acid sequence shown as SEQ ID No. 5;

the light chain variable region comprises CDR-L3 of the amino acid sequence shown in SEQ ID No. 6.

CDRs (complementarity determining regions, complementarity determining region) generally refer to regions of an antibody that can be spatially complementary to an epitope. The variability in antibodies is typically not uniformly distributed throughout the variable regions of the antibodies, the heavy and light chain variable regions of a monoclonal antibody typically have 3 hypervariable regions (hypervariable region, HVR) which are typically complementary to an epitope in spatial structure, so that the hypervariable regions are also known as complementarity determining regions (complementarity determining region, CDRs), i.e., the heavy chain variable regions typically comprise three complementarity determining regions, i.e., CDR-H1, CDR-H2, and CDR-H3, and the light chain variable regions typically comprise three complementarity determining regions, i.e., CDR-L1, CDR-L2, and CDR-L3. The CDR sequence of the monoclonal antibody No. 10H11 provided by the application is shown in table 1.

TABLE 1 10H11 monoclonal antibody CDR sequences

In the coxsackievirus A10 monoclonal antibody provided by the application, the heavy chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of the FR1 comprises a sequence shown as SEQ ID No. 7. The amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No. 8. The amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No. 9. The amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 10. The sequence of the framework region of the heavy chain variable region of the monoclonal antibody No. 10H11 provided by the application is shown in table 2.

TABLE 2 10H11 heavy chain variable region framework region sequences

In the coxsackievirus A10 monoclonal antibody provided by the application, the light chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of the FR1 comprises a sequence shown as SEQ ID No. 11. The amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No. 12. The amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No. 13. The amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 14. The sequence of the framework region of the light chain variable region of the monoclonal antibody 10H11 provided by the application is shown in table 3.

TABLE 3 10H11 monoclonal antibody light chain variable region framework region sequences

In the coxsackievirus A10 monoclonal antibody provided by the application, the heavy chain also comprises a signal peptide. The heavy chain signal peptide comprises a sequence shown as SEQ ID No. 15. The light chain further comprises a signal peptide. The light chain signal peptide comprises a sequence shown as SEQ ID No. 16. The signal peptide sequence of the monoclonal antibody No. 10H11 provided by the application is shown in Table 4.

TABLE 4 monoclonal antibody Signal peptide sequences

	Amino acid sequence	SEQ ID NO：
			Heavy chain signal peptide	MKLWLNWIFLVTLLNGIQC	15
Light chain signal peptide	MKLPVRLLVLMFWIPASSS	16

The application provides a coxsackievirus A10 monoclonal antibody, wherein the amino acid sequence of the heavy chain comprises a sequence shown as SEQ ID No. 17. The amino acid sequence of the light chain comprises a sequence shown as SEQ ID No. 18. The sequence is as follows:

in another aspect, the application provides a nucleotide molecule encoding said monoclonal antibody. The full-length nucleotide sequence of the monoclonal antibody of the present application or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

The heavy chain nucleotide sequence of the monoclonal antibody is shown as SEQ ID No. 19 (wherein, the single underlined part is a signal peptide sequence, the italic part is a variable region sequence, and the dotted line is a constant region sequence). The sequence is as follows:

The light chain nucleotide sequence of the monoclonal antibody is shown as SEQ ID No. 20 (wherein, the single underlined part is a signal peptide sequence, the italic part is a variable region sequence, and the dotted line is a constant region sequence). The sequence is as follows:

in another aspect, the application provides a construct comprising said nucleotide molecule. The construct may be constructed from the nucleotide molecules described above inserted into the multiple cloning site of an expression vector. The construct may be transformed, transduced or transfected into a host cell to allow expression of the genetic material elements carried thereby within the host cell. The construct is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC), phages such as lambda phage or M13 phage, animal viruses, and the like. The viral vector comprises: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-vacuolated viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist in its entry into the cell, including, but not limited to, viral particles, liposomes, or protein shells.

In another aspect, the application provides an expression system comprising said construct or genome incorporating said nucleotide molecule. The expression system may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells such as mammalian cells, etc. The expression system includes a number of cell types such as prokaryotic cells like E.coli or Bacillus subtilis, fungal cells like yeast cells or Aspergillus, insect cells like S2 Drosophila cells or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

In another aspect, the application provides the use of said monoclonal antibody or said nucleotide molecule or said construct or said expression system in the preparation of a virus detection product, in the preparation of a product for the prevention or treatment of a virus infection, or in the screening and detection of a virus vaccine or in the evaluation of vaccine activity.

In the use of the application, the virus detection product may be, for example, a vaccine composition. The vaccine composition may be monovalent (containing only one virus-like particle) or multivalent (containing multiple virus-like particles). The vaccine composition can be prepared into various conventional dosage forms, such as: injection, granule, tablet, pill, suppository, capsule, suspension, spray, etc. The vaccine composition may be administered directly to an individual using known methods for the virus-like particles of the application. These vaccines are typically administered by the same route of administration as conventional vaccines and/or by a route that mimics pathogen infection. The route of administration of the vaccine composition includes: intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral routes of administration. The routes of administration may be combined, if desired, or adjusted according to the disease condition. The vaccine composition may be administered in a single dose or in multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.

In the use of the present application, the virus is enterovirus, preferably coxsackievirus a10; more preferably, the monoclonal antibody is used for preparing a hand-foot-and-mouth disease detection product, a hand-foot-and-mouth disease prevention or treatment product, or an antibody reference product for screening hand-foot-and-mouth disease vaccines, qualitative and quantitative detection and verification of vaccine antigens or evaluation of vaccine activity.

In vaccine screening, to ensure that the antigen detected is an active epitope antigen, a highly sensitive, highly specific and highly neutralizing antibody is required, which in one embodiment of the application can be minimally detected, for example, by a sandwich Elisa assay. The specificity can be detected by, for example, western blot experiments to detect the specific binding of the monoclonal antibody to the antigen. The neutralization activity can be determined, for example, by neutralization experiments. Qualitative and quantitative detection of the vaccine antigens can be obtained by a sandwich Elisa experiment, for example.

The application is further illustrated by the following examples, which are not intended to limit the scope of the application.

Information on the reagents described below is shown in Table 5.

TABLE 5 reagent information

Name of the name	Manufacturer/preparation method	Goods number
			Mouse secondary antibody	SIGMA	A0168-1ml
5' RACE kit	Invitrogen	18374058

EXAMPLE 1 selection of hybridoma cells secreting Coxsackie A10-specific antibody

CV a10 virus-like particles (VLPs) were prepared by expression using the pichia expression system (see patent CN 114836443). 5 μg CV A10 VLP was mixed with 500 μg aluminum adjuvant and 25 μg oligonucleotide adjuvant (CPg), and the mixed formulation was immunized intraperitoneally for 6 week female Balb/c mice four times with 2 weeks between immunizations. 2 weeks after the last immunization, 5CV of A10 VLPs were injected into the tail vein for booster immunization.

After 3 days of mouse tail vein boosting, hybridoma cells were prepared by fusing mouse spleen cells with myeloma cells SP2/0 using PEG 1450. After 9 days of hybridoma cell culture, antibodies specifically secreted against a10 VLPs were screened using an enzyme-linked immunosorbent assay and neutralization assay. The ELISA test steps are as follows: CV A10 VLPs were coated on 96-well ELISA plates (200 ng/well) and incubated overnight at 4 ℃; after blocking with PBST containing 5% skimmed milk, 50. Mu.l of hybridoma culture solution was added to each well and incubated at 37℃for 2 hours; then, the incubation was performed with HRP-labeled secondary antibody for 1 hour, and finally, a color reaction was performed to read the absorbance at OD450 nm. The neutralization test steps are as follows: after 50. Mu.l of the hybridoma culture broth was thoroughly mixed with 100TCID 50/50. Mu.l of enterovirus A10 (see patent CN 114836443), the mixture was placed in a 5% CO ₂ incubator and incubated at 37℃for 2 hours. RD cell working fluid (1.5X105/ml) is suspended and added to the 96-well plate after incubation, 100. Mu.l/well, and cultured in a 5% CO ₂ incubator at 35℃for 7 days. CPE was observed.

Finally, monoclonal antibodies numbered 5F10, 8H11 and 10H11 were selected based on their binding capacity and neutralizing activity to a10 VLP, wherein 10H11 had both better binding capacity and neutralizing activity, and the identification information is shown in table 6.

TABLE 6 identification of monoclonal antibody secreting hybridoma cell lines

Hybridoma cell line numbering	Heavy chain	Light chain	Binding ability to a10 VLP	Neutralization Activity ^#
					5F10	IgG1	kappa	+++	-
8H11	IgG2a	kappa	+++	-
					10H11	IgG2b	kappa	+++	+

The samples used for the analysis were 50. Mu.l of hybridoma culture cell supernatants.

*,+：OD450＞0.15；++：OD450＞0.3；+++：OD450＞0.5。

#, +: Has neutralizing activity; -, no neutralization activity

EXAMPLE 2 specificity analysis of anti-A10 mab

Antibody purification: female Balb/c mice were intraperitoneally injected with 500. Mu.l liquid paraffin oil, and 80 ten thousand hybridoma cells per mouse intraperitoneally after one week. After 1-2 weeks, collecting ascites with needle, centrifuging at 4000rpm for 10min, removing upper layer oil and lower layer precipitate, and purifying clarified ascites with antibody. According to the specification, the ascites fluid was purified using iProtein G Purose Fast Flow affinity column (thousand pure organisms) to obtain purified antibodies.

ELISA method: coating 96-well ELISA plates (200 ng/well) with A6 or A10 or A16 or A71 VLPs, and incubating overnight at 4 ℃; adding 5% skimmed milk PBST, sealing at 37deg.C for 1 hr; adding detection monoclonal antibody, and incubating for 2 hours at 37 ℃; HRP-labeled secondary antibody was then added and incubated for 1 hour, and finally absorbance OD450nm was read.

Protein experiment: the protein samples were mixed with a polypropylene gel electrophoresis (SDS-PAGE) loading buffer, boiled for 5min, and the protein samples were separated by a 12% polyacrylamide gel. Protein bands were visualized by coomassie blue staining or proteins were transferred to PVDF membranes for western blot analysis. Monoclonal antibody concentration 1ug/ml, rabbit anti-VP 0 polyclonal antibody 1:1000 was diluted for use, followed by incubation with HPR-labeled secondary antibody, and finally recorded with a luminescence image analyzer.

The results show that: SDS-PAGE identifies the purity and integrity of the A10 mab purified from ascites. As shown in FIG. 1, each of the monoclonal antibodies 5F10, 8H11 and 10H11 shows two major bands, which are about 55kDa and 25kDa in size, respectively, corresponding to heavy and light chains, respectively. Reactivity of the monoclonal antibodies with different antigens, including CV A6 VLP, CV A10VLP, CV A16VLP and EV A71 VLP, was examined by ELISA (see patent CN114836444 for preparation). As shown in fig. 2, each of the three mabs was able to specifically recognize CV a10VLP, but was unable to recognize CV A6 VLP, CV a16VLP, and EV a71 VLP. Finally, binding of the mab to CV A6 VLP, CV a10VLP, CV a16VLP and EV a71 VLP was analyzed by Western blot, and as shown in fig. 3, none of the three mabs recognized the denatured CV A6 VLP, CV a10VLP, CV a16VLP and EV a71 VLP, suggesting that the three mabs recognized epitopes may all be conformational epitopes.

EXAMPLE 3 Sandwich ELISA detection of Coxsackie virus A10 Virus-like particles

Sandwich ELISA: rabbit anti-CV A10 VLP polyclonal antisera were 1:8000 diluted and coated on 96 ELISA plates (100 μl/well) and incubated overnight at 4deg.C; after blocking for 1 hour at 37℃with PBST containing 5% skim milk, CV A10 VLP was added and incubated for 2 hours at 37 ℃; then, the antibody of example 2 (1.2 ng/. Mu.l) was added and incubated at 37℃for 2 hours; incubation was then performed with HPR-labeled murine secondary antibody, and finally absorbance OD450nm was read.

The minimal detection limit of the primary antibody against the diseased CV A10 VLPs was determined by sandwich ELISA (positive when OD450 > 0.15). As shown in FIG. 4, each of the 5F10, 8H11 and 10H11 mabs sensitively detected coxsackievirus A10 VLPs with a minimum detection of 15.625ng/ml, suggesting that it can be used for diagnosis of A10 infection.

Example 4 neutralization Activity assay

Adding the antibody of the example 2 into a 96-well dilution plate containing 2% FBS DMEM, blowing and mixing uniformly, carrying out 2-time gradient dilution downwards, carrying out 8 gradient dilutions altogether, adding 50 μl of the antibody with the gradient concentration of 5000ng/50 μl to 39ng/50 μl into the 96-well culture plate, and setting 2 multiple wells for each dilution; 100TCID50/50 μl Coxsackie virus A10 was taken. The working solution is added into the positive antibody diluted by the corresponding 96-well plate, and after being fully and evenly mixed, the positive antibody is put into a 5% CO ₂ incubator for incubation for 2 hours at 37 ℃. RD cell working fluid (1.5X10 ⁵/ml) was suspended in 96-well plates after incubation, 100. Mu.l/well, 5% CO ₂ incubator at 35℃for 7 days. CPE was observed.

The neutralization activities of the 5F10, 8H11 and 10H11 monoclonal antibodies are detected through a neutralization test, and the result shows that neither the 5F10 nor the 8H11 has the neutralization activity, but the 10H11 has stronger potential neutralization activity on the A10, wherein the neutralization concentration is 3.12 mug/ml, which suggests that the 10H11 can be used for the development of antiviral drugs of the Coxsackie virus A10.

EXAMPLE 5 Gene sequence analysis of 10H11 monoclonal antibody

The cells of the hybridoma cell line of example 1 were subjected to extraction of total RNA with Trizol reagent, and heavy and light chain full-length genes were amplified according to the 5' RACE kit instructions.

The obtained heavy chain nucleotide sequence of the 10H11 monoclonal antibody is shown as SEQ ID No. 19, and the light chain nucleotide sequence is shown as SEQ ID No. 20.

Further analysis of the 10H11 mab heavy chain variable region and light chain variable region sequences, the 10H11 mab heavy chain variable region amino acids were as follows (underlined as heavy chain CDR regions):

(SEQ ID No:21)。

The heavy chain variable region described above belongs to the IGHV7 subgroup.

The 10H11 mab light chain variable region amino acids are as follows (underlined as heavy chain CDR regions):

the light chain variable region belongs to IGKV1 subgroup.

EXAMPLE 6 recombinant expression and identification of monoclonal antibody genes

The coding sequences of the heavy chain and the light chain are respectively inserted between pcDNA3.3 (thermo) EcoRI and XhoI multiple cloning sites in a homologous recombination mode, constructs pcDNA3.3-10H11-H and pcDNA3.3-10H11-L are constructed, pcDNA3.3-10H11-H and pcDNA3.3-10H11-L are co-transfected into 293T cells by a liposome method, culture supernatants are collected for analysis after 3d, and the expression of antibodies in the culture supernatants is determined by an enzyme-linked immunosorbent assay method.

As shown in fig. 5, cell supernatants expressing the 10H11 mab sequences had high binding signals to a10 VLPs and the OD450nm gradually decreased with increasing dilution; while the supernatant of control cells not transfected with the relevant plasmid did not bind a signal, the result indicated that the amplified and expressed sequence was indeed the gene for 10H11 mab.

The aim of this study is to prepare a neutralizing monoclonal antibody that specifically binds to CV A10 VLP, so as to further develop a kit for diagnosing CV A10, and at the same time, can be used for the development of therapeutic monoclonal antibody drugs. Monoclonal antibodies 5F10, 8H11 and 10H11 were successfully obtained by using the hybridoma method, elisa and Werstern blot suggest that the antibodies specifically recognize CV A10 by binding with conformational epitopes, and sandwich Elisa results show that the lowest detection limit of three monoclonal antibodies to VLPs is 15.625ng/ml, suggesting that the antibodies can be used for development and application of a detection kit of CV A10. In addition, the neutralization test shows that the 10H11 monoclonal antibody has stronger neutralization activity on CV A10, and the neutralization concentration is 3.12ug/ml.

In conclusion, the CV A10 monoclonal antibody 10H11 obtained by the research has stronger specific recognition capability and neutralization activity, can be used as an effective material for developing a diagnosis method, and is beneficial to the clinical diagnosis of A10, the research of disease treatment mechanism, vaccine and drug research and development. The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A coxsackievirus a10 monoclonal antibody, comprising the following technical features:

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region and the light chain variable region further comprise framework regions FR1, FR2, FR3, FR4.

3. The monoclonal antibody of claim 2, wherein the monoclonal antibody,

The amino acid sequence of FR1 of said heavy chain variable region comprises the sequence shown in SEQ ID No. 7: EVKLVESGGGLVQPGGSLRLSCTTS;

The amino acid sequence of FR4 of the heavy chain variable region comprises the sequence shown in SEQ ID No. 10: WGQGTLVTVSA;

and/or, the amino acid sequence of FR1 of the light chain variable region comprises the sequence set forth in SEQ ID No: 11: DVLMTQTPLSLPVSLGDQASISCRSS;

4. The monoclonal antibody of claim 1, wherein the heavy chain further comprises a signal peptide; the amino acid sequence of the heavy chain signal peptide is shown as SEQ ID No. 15: MKLWLNWIFLVTLLNGIQC;

And/or, the light chain further comprises a signal peptide; the amino acid sequence of the signal peptide of the light chain is shown as SEQ ID No. 16: MKLPVRLLVLMFWIPASSS.

5. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain comprises the sequence set forth in SEQ ID No. 17;

6. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-5.

7. A construct comprising the nucleic acid molecule of claim 6.

8. An expression system comprising the construct of claim 7 or the nucleic acid molecule of claim 6 integrated with an exogenous source in the genome.

9. Use of a monoclonal antibody according to any one of claims 1 to 5 or a nucleic acid molecule according to claim 6 or a construct according to claim 7 or an expression system according to claim 8 for the preparation of a virus detection product, for the preparation of a product for the prophylaxis or treatment of a viral infection, for the screening and detection of a viral vaccine or for the evaluation of the activity of a viral vaccine, said virus being coxsackievirus a10.

10. The use according to claim 9, wherein the monoclonal antibody is used for preparing a hand-foot-and-mouth disease detection product, a hand-foot-and-mouth disease prevention or treatment product, or an antibody reference product for vaccine screening and qualitative and quantitative detection of vaccine antigens or vaccine activity evaluation, and the vaccine is a hand-foot-and-mouth disease vaccine.