CN114316035A - General type foot-and-mouth disease virus structural protein antibody, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a general foot-and-mouth disease virus structural protein antibody, a preparation method and application thereof. The invention takes O-type foot-and-mouth disease virus 146s as immunogen to immunize Balb/c mice, and obtains two positive hybridoma cell lines which efficiently secrete monoclonal antibodies through cell fusion and screening, and obtains two mouse monoclonal antibodies m33 and m 77. Immunoblotting and indirect immunofluorescence assays showed that the monoclonal antibodies m33 and m77 had broad-spectrum reactivity against both the different lineage viruses of type O and the different viruses of type a. The m33 and m77 antibodies are specifically combined with the type-to-type conserved antigen epitope in the FMDV structural protein, compared with the existing antibodies, the sensitivity and specificity of a detection result are obviously improved, the method is suitable for detecting the structural protein antibodies after O type and A type FMDV infection or inactivated vaccine immunization, and the method is a novel method for monitoring the infection condition of FMD non-immune animal groups.

Description

General type foot-and-mouth disease virus structural protein antibody, preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a general foot-and-mouth disease virus structural protein antibody, a preparation method and application.

Background

Foot-and-mouth disease is an infectious disease of artiodactyl co-morbid animals caused by infection with Foot-and-mouth disease virus (FMDV). FMDV belongs to a member of the genus foot-and-mouth disease virus (aphtovirus) of the picornaviridae family (picornaviridae). FMDV currently has O, A, C, SAT1, SAT2, SAT3 (i.e. south african foot and mouth disease virus type 1, 2, 3) and a sia1 (asian type 1) 7 serotypes. There is little immune protection among the types, and animals infected with one type of foot-and-mouth disease can still be infected with another type of foot-and-mouth disease virus to cause diseases. The disease has serious influence on the breeding industry and the international animal product trade, so that the prevention and control of the disease are highly valued in various countries. The key to prevent and control foot-and-mouth disease is the application of high-efficiency vaccine and accurate diagnosis method.

Because the antigenicity of different subtypes in the same serotype are different in degree, and the serological cross reaction degree is different, the diagnosis and control of the foot-and-mouth disease are very difficult. The monoclonal antibody has the characteristics of capability of identifying a single antigen site, strong specificity of combining with an antigen, high homogeneity, single biological activity, easiness in standardization and capability of being produced in batches, and is widely applied to the field of biomedicine. Currently, type O and type a FMDs are prevalent in china. Therefore, the development of universal monoclonal antibodies to type O and type a FMDs has important implications for the diagnosis and prevention of type O and type a FMDs. However, because the FMDV antigen structure is complex, the antigenic sites and epitopes of different serotypes, genotypes and isolates are all different, there are wide antigenic variations, and there are also conserved antigen structures that determine the characteristics of the virus species, developing a broad-spectrum type foot-and-mouth disease virus antibody detection kit requires screening of broad-spectrum specific monoclonal antibodies that recognize conserved epitopes between types, but the present broad-spectrum specific monoclonal antibodies are rare, which brings certain obstacles to the establishment of a broad-spectrum FMDV antibody detection method.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a monoclonal antibody with broad-spectrum neutralization function on structural proteins of different pedigree strains of O-type and A-type foot-and-mouth disease viruses, a preparation method and application thereof, and the monoclonal antibody specifically comprises the following contents:

in a first aspect, the invention provides a monoclonal antibody of a general foot-and-mouth disease virus structural protein, wherein the monoclonal antibody is m33 or m77, the amino acid sequence of a heavy chain variable region of the monoclonal antibody m33 is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6.

In a second aspect, the present invention provides a gene fragment encoding the monoclonal antibody m33 of the first aspect, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody m33 is shown as SEQ ID NO.3, and the gene sequence encoding the light chain variable region of the monoclonal antibody m33 is shown as SEQ ID NO. 4.

In a third aspect, the present invention provides a gene fragment encoding the monoclonal antibody m77 of the first aspect, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody m77 is shown as SEQ ID NO.7, and the gene sequence encoding the light chain variable region of the monoclonal antibody m77 is shown as SEQ ID NO. 8.

In a fourth aspect, the present invention provides an expression vector comprising the gene fragment of the second aspect, or the expression vector comprising the gene fragment of the third aspect.

In a fifth aspect, the present invention provides a host cell comprising the expression vector of the fourth aspect, or a genome of the host cell having the gene segment of the second or third aspect integrated therein.

In a sixth aspect, the invention provides an immunoconjugate comprising:

(i) monoclonal antibody m33 and/or monoclonal antibody m77 as described above in relation to the first aspect;

(ii) and a coupling moiety selected from the group consisting of: a detectable label, a drug, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.

In a seventh aspect, the present invention provides an application of the monoclonal antibody m33 and/or the monoclonal antibody m77 described in the first aspect in preparing a reagent, or a test strip, or a kit for detecting foot-and-mouth disease virus.

In an eighth aspect, the present invention provides a monoclonal antibody composition of a general-purpose foot-and-mouth disease virus structural protein, comprising the monoclonal antibody m33 and the monoclonal antibody m77 described in the first aspect.

In a ninth aspect, the present invention provides an application of the monoclonal antibody composition of the above eighth aspect in the preparation of a reagent, or a test strip, or a kit for detecting foot-and-mouth disease virus.

In a tenth aspect, the present invention provides a universal foot-and-mouth disease virus structural protein solid phase competition ELISA detection kit, which comprises the monoclonal antibody m33 and/or the monoclonal antibody m77 described in the first aspect, or the monoclonal antibody composition described in the ninth aspect.

Preferably, the ELISA detection kit comprises an ELISA plate coated with the monoclonal antibody m33 and the inactivated foot-and-mouth disease virus antigen described in the first aspect, the monoclonal antibody m77 described in the first aspect, an antibody diluent, a concentrated washing solution, a color developing agent, and a stop solution;

or, the ELISA detection kit comprises an ELISA plate coated with the monoclonal antibody m77 and the foot-and-mouth disease virus inactivated antigen of the first aspect, the monoclonal antibody m33 of the first aspect, an antibody diluent, a concentrated washing solution, a color developing agent and a stop solution.

Preferably, the foot-and-mouth disease virus inactivated antigen is an O type foot-and-mouth disease virus inactivated antigen or an A type foot-and-mouth disease virus inactivated antigen.

Preferably, the ELISA test kit further comprises a positive control serum and a negative control serum.

Preferably, the working concentration of the monoclonal antibody m77 is 6 μ g/mL.

Preferably, the coating concentration of the monoclonal antibody m33 is 1 μ g/mL.

Preferably, the coating concentration of the O type foot-and-mouth disease virus inactivated antigen is 1: 16.

Preferably, the coating concentration of the type A foot-and-mouth disease virus inactivated antigen is 1: 8.

In an eleventh aspect, the invention provides a general solid-phase competitive ELISA detection method for foot-and-mouth disease virus structural proteins, which comprises the following steps:

coating the ELISA plate with the monoclonal antibody m33, adding the inactivated foot-and-mouth disease virus antigen into the ELISA plate, adding the serum to be detected and the monoclonal antibody m77, uniformly mixing and incubating;

or, the monoclonal antibody m77 of the first aspect is used for coating the ELISA plate, then the foot-and-mouth disease virus inactivated antigen is added into the ELISA plate, the serum to be detected and the monoclonal antibody m33 are added, mixed evenly and incubated;

after incubation, adding goat anti-mouse secondary antibody, adding TMB after incubation, developing in dark, and adding H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

And calculating the inhibition Percentage (PI), taking 40% of PI as a critical value, and when the PI of the serum to be detected is more than or equal to 40%, the detection result is positive, and when the PI of the serum to be detected is less than 40%, the detection result is negative.

Preferably, the method is:

(1) na with pH 9.6 was used₂CO₃/NaHCO₃Diluting the monoclonal antibody m33 by the coating solution, coating an enzyme label plate, and standing overnight at 4 ℃, wherein the coating concentration of the monoclonal antibody m33 is 1 microgram/mL;

(2) washing a plate by using PBST and drying the plate by patting, diluting the A type foot-and-mouth disease virus inactivated antigen by using PBST 1:8, or diluting the O type foot-and-mouth disease virus inactivated antigen by 1:16, adding the diluted antigen into an enzyme label plate, and incubating for 1h at 37 ℃;

(3) washing a plate by using PBST and drying the plate, diluting the serum to be detected by using the PBST, adding a monoclonal antibody m77 with the concentration of 6 mu g/mL, slightly shaking and uniformly mixing, and incubating for 1h at 37 ℃;

(4) washing the plate with PBST, patting dry, adding goat anti-mouse secondary antibody, and incubating for 1h at 37 ℃;

(5) washing the plate with PBST, drying, adding TMB, developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

(6) And (3) calculating the inhibition percentage PI, wherein the serum to be detected is positive when the PI of the serum to be detected is more than or equal to 40%, and the serum to be detected is negative when the PI of the serum to be detected is less than 40%.

Preferably, the method is:

(1) na with pH 9.6 was used₂CO₃/NaHCO₃Diluting the monoclonal antibody m77 by the coating solution, coating an enzyme label plate, and standing overnight at 4 ℃, wherein the coating concentration of the monoclonal antibody m77 is 6 mug/mL;

(2) washing a plate by using PBST and drying the plate by patting, diluting the A type foot-and-mouth disease virus inactivated antigen by using PBST 1:8, or diluting the O type foot-and-mouth disease virus inactivated antigen by 1:16, adding the diluted antigen into an enzyme label plate, and incubating for 1h at 37 ℃;

(3) washing a plate by using PBST and drying the plate, diluting the serum to be detected by using the PBST, adding a monoclonal antibody m33 with the concentration of 1 mu g/mL, slightly shaking and uniformly mixing, and incubating for 1h at 37 ℃;

(4) washing the plate with PBST, patting dry, adding goat anti-mouse secondary antibody, and incubating for 1h at 37 ℃;

(5) washing the plate with PBST, drying, adding TMB, developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

(6) And (3) calculating the inhibition percentage PI, wherein the serum to be detected is positive when the PI of the serum to be detected is more than or equal to 40%, and the serum to be detected is negative when the PI of the serum to be detected is less than 40%.

The invention has the beneficial effects that: the invention provides a general foot-and-mouth disease virus structural protein antibody m33 or m77, wherein the antibodies m33 and m77 are IgG2b monoclonal antibodies; the immunoblotting test shows that the representative strains of different lineages of the monoclonal antibodies m33 and m77 and O type foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93) and the representative strains of different lineages of A type foot-and-mouth disease (A/AF72, A/GDMM/2013, A/WH/CHA/09) have good reactivity and are general structural protein antibodies of the foot-and-mouth disease virus; the invention establishes a general foot-and-mouth disease virus structural protein solid phase competition ELISA detection kit and a detection method by using the monoclonal antibodies m33 and m77, compared with the existing general antibody detection method, the method obviously improves the sensitivity and specificity of the detection result, is suitable for the broad-spectrum detection of structural protein antibodies after foot-and-mouth disease infection or inactivated vaccine immunization, and is also a new method for monitoring the infection condition of non-immune areas of foot-and-mouth disease.

Drawings

FIG. 1WB test the reactivity of the universal monoclonal antibody m33 against different lineage representative strains of type O and type A FMDV;

FIG. 2WB test the reactivity of the universal monoclonal antibody m77 against different lineage representative strains of type O and type A FMDV;

FIG. 3 Indirect immunofluorescence assay to detect the reactivity of the universal monoclonal antibody m33 against different lineage representative strains of type O and type A FMDV;

FIG. 4 Indirect immunofluorescence assay to detect the reactivity of the universal monoclonal antibody m77 against different lineage representative strains of type O and type A FMDV.

Detailed Description

The following embodiments of the present invention are described in detail, and it should be noted that the following embodiments are exemplary only, and are not to be construed as limiting the present invention. In addition, all reagents used in the following examples are commercially available or can be synthesized according to texts or known methods, and are readily available to those skilled in the art for reaction conditions not listed, if not explicitly stated.

EXAMPLE 1 preparation of monoclonal antibodies m33 and m77

1. Preparation of hybridoma cells

1.1 immunization of mice

A5-6-week-old Balb/c mouse is immunized by taking O-type foot-and-mouth disease virus (O/Mya98)146s as an antigen in a mode of back subcutaneous multipoint injection, wherein the injection amount of each mouse is 100 mu g. The first immunization uses a Freund complete adjuvant, then the boosting immunization uses a Freund incomplete adjuvant, the immunization time interval is two weeks each time, the serum of the mouse is collected 1 week after the third immunization, the antibody titer is detected, when the antibody titer is not less than 1:1440, the impact immunization is carried out, namely the antigen is injected in the abdominal cavity, three days after the impact immunization, the spleen of the mouse is taken to be fused with myeloma cells.

1.2 cell fusion

Aseptically obtaining splenocytes of the immunized mice, mixing the splenocytes with myeloma cells according to the proportion of 10:1, adding 1mL of PEG 1500 within 1 minute at 37 ℃, after 1 minute of action, adding 1mL of DMEM culture solution preheated at 37 ℃ to stop the reaction, finishing the reaction within 1 minute, and after 1 minute of action, adding the solution for 5 times in total (the whole fusion process needs to shake a fusion tube), supplementing the preheated DMEM culture solution to 20mL, and after 15 minutes of action at 37 ℃, centrifuging at 800rpm for 7 minutes; discarding the supernatant, and resuspending with fetal bovine serum culture medium (adding HAT selection medium); adding the cells into a 96-well plate, wherein each well contains 50 mu L of the cells; the plates were incubated at 37 ℃ in 5% CO₂Culturing in an incubator. Half-changes were made on day 3 and day 6 with 20% fetal bovine serum culture medium (plus HAT selection medium) and supernatants were removed to detect specific antibodies when hybridoma cells were observed to grow to about 1/5% bottom of the wells.

1.3 selection of hybridoma cells

Using ELISA test to prepare cell suspension of the hybridoma cells which are detected to be positive by using 20% fetal bovine serum culture solution (added with HT selection medium, Sigma), counting, adjusting the cells to be 3-5 cells/mL, dripping 100 mu L of diluted cells into each hole, then adding 100 mu L of HT selection medium into each hole, placing at 37 ℃ and 5% CO₂An incubator; half-change of 20% fetal bovine serum culture medium (supplemented with HT selection medium, Sigma) was performed on days 3 and 6, and the formation of cell clones was observed to detect antibody activity in time. After subcloning to obtain positive monoclonal cells, they were subcultured on an expansion scale at 2X 10⁶Individual cells/branch were stored in liquid nitrogen.

2. Preparation of monoclonal antibodies m33 and m77

2.1 preparation of monoclonal antibodies m33 and m77 ascites

Sensitizing 7-8-week-old Balb/c mice with 0.5 mL/mouse by using sterilized liquid paraffin0.5mL (about 3.0X 10) is injected into the abdominal cavity within 10 to 18 days⁶Individual cells/mL). Gently Rou the abdomen of the mouse every day after injection, and extract ascites when the abdomen of the mouse has significantly risen laterally. Centrifuging ascites at 10000rpm for 10min, collecting supernatant, packaging, and storing at-20 deg.C.

2.2 purification of monoclonal antibodies m33 and m77

The purification of the ascites of the hybridoma mouse is carried out by adopting affinity chromatography, and the specific operation is as follows: thawing frozen ascites sample, centrifuging at 10000rpm and 4 deg.C for 10min, sucking clear liquid, and loading with 3 column volumes of loading buffer (Binding buffer formula: 20mM Na)₂HPO₄0.15M NaCl, pH8.0), purifying with Protein A affinity chromatography column, eluting with 0.1M glycine buffer solution (pH 2.5), adjusting pH of the eluted antibody to neutral with neutralizing buffer solution (1M Tris-HCl, pH8.5), and analyzing with SDS-PAGE gel and determining Protein content.

The results show that: the purified antibodies m33 and m77 have protein contents of 5.2mg/mL and 4.3mg/mL respectively, and can meet the requirements of clinical application.

2.3 subtype identification and sequencing of monoclonal antibodies m33 and m77

Subtype identification: the monoclonal antibody Ig subtype was identified according to the instructions of the murine monoclonal antibody subtype identification kit (purchased from Sigma Co.) and both subtypes m33 and m77 of the monoclonal antibody were determined to be IgG2 b. The sequences of the specific antibody IgG2b variable regions were determined as follows:

(1) total RNA extraction: get 10⁶Performing Trizol lysis on each cell, adding chloroform for layering to obtain RNA, washing with ethanol after isopropanol precipitation, drying, and dissolving the RNA with DEPC water;

(2) reverse transcription and PCR amplification: taking 500ng RNA, adding oligo (dT), Random, dNTP and 5 × RT buffer, reverse transcriptase (Takara company), terminating the reaction at 37 ℃ for 25min, 85 ℃ for 5sec and 4 ℃ to obtain cDNA, and then carrying out PCR amplification;

(3) sequencing and analysis: the amplified heavy and light chain variable regions were cloned into pMD18-T, sent to the company for sequencing, and analyzed for data alignment using the IMGT/V-QUEST database.

The DNA sequence of the variable region of the heavy chain of the monoclonal antibody m33 is sequenced as follows: caggtgcagctgaaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctcacctgcactgtcactggctactcaatcaccagtgattatgcctggaactggatccggcagtttccaggaaacaaactggagtggatgggctacatattctacagaggtagcactacccacaacccatctctcagaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattattgtgcaagggggctccacggtagtagcccgtttgcttactggggccaagggactctggtcactgtctcgagc (shown in SEQ ID NO. 3);

the DNA sequence encoding the light chain variable region of monoclonal antibody m33 is as follows: gacattgtgatgacccagtctcaaaaactcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcacaatgtgtatagtaatgtagtctggtatcaagagaaaccagggcaatctcctaatgcactgatttattcggcatcccaccggtacagtggagtccctgatcgcttcacaggcagtggacctgggacagatttcactctcaccatcagcaatgtgcagtctgaagacttggcaaagtctttctgtcagcaatgtaacagctatcctattcacgttcggctcgggcacaaagttggaaatcaa (shown in SEQ ID NO. 4);

according to the codon encoding rule, it is known that:

the amino acid sequence of the heavy chain variable region of the monoclonal antibody m33 is shown as follows: QVQLKESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIFYRGSTTHNPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGLHGSSPFAYWGQGTLVTVSS (shown in SEQ ID NO. 1);

the amino acid sequence of the variable region in the light chain of the monoclonal antibody m33 is shown as follows: DIVMTQSQKLMSTSVGDRVSVTCKASHNVYSNVVWYQEKPGQSPNALIYSASHRYSGVPDRFTGSGPGTDFTLTISNVQSEDLAKSFCQQCNSYPIHVRLGHKVGNQ (shown in SEQ ID NO. 2);

the DNA sequence of the variable region of the heavy chain of the monoclonal antibody m77 is sequenced as follows: gatgtgcagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgaagcctctggattcactttcagtagctataccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtactggaggtagtttcacctactatctagacagtgcgaagggccgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtattactgctcaaaagagggagaattctatggtaactacggggattactatgctatggactactggggtcaaggaacctcagtcaccgtctcgagc (shown in SEQ ID NO. 7);

the DNA sequence encoding the light chain variable region of monoclonal antibody m77 is as follows: gacatccagatgacccagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatacagggccagcaaaagtgtcagtacatctggctatagttatatgcactggaaccaacagaaaccaggacagccacccagactcctcatctatcttgtatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacattagggagcttacacgttcggaggggggaccagactggaaatcaaac (shown in SEQ ID NO. 8);

according to the codon encoding rule, it is known that:

the amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is represented by DVQLVESGGGLVKPGGSLKLSCEASGFTFSSYTMSWVRQTPEKRLEWVATISTGGSFTYYLDSAKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCSKEGEFYGNYGDYYAMDYWGQGTSVTVSS (represented by SEQ ID NO. 5);

the amino acid sequence of the variable region of the light chain of the monoclonal antibody m77 is shown as follows: DIQMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPDWKSN (shown in SEQ ID NO. 6).

Example 2 broad-spectrum reactivity assay of monoclonal antibodies m33 and m77

1. Immunoblot assay

BHK-21 cells were infected with different pedigree representative strains of type O foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93 strains) and different pedigree representative strains of type A foot-and-mouth disease (A/AF72, A/GDMM/2013 and A/WH/CHA/09) (the strains were all preserved in a national reference laboratory for foot-and-mouth disease), cells were harvested 6 hours after infection, samples were prepared for WB test, and immunoblotting was performed using the prepared monoclonal antibodies m33 and m77 as primary antibodies, respectively. The specific experimental procedures are as follows:

(1) electrophoresis: adding the denatured O-type and A-type FMDV antigens into 12% of preformed gel holes;

(2) loading the sample according to 10 mu L/hole, carrying out electrophoresis on the concentrated gel according to the constant voltage of 90V, and carrying out electrophoresis on the separation gel according to the constant voltage of 120V;

(3) film transfer: assembling a film transfer device, placing the film transfer device in an ice-water mixture, setting a constant current of 200mA for transferring a film for 2h, and transferring the sample subjected to electrophoretic separation from the gel onto a PVDF film;

(4) and (3) sealing: 5 percent of skimmed milk powder prepared from TBST solution is used as confining liquid, and the shaking table is sealed for 1 hour at room temperature;

(5) incubating the primary antibody: diluting monoclonal antibodies m33 and m77 to be detected to a final concentration of 2 mu g/mL by using 5% skimmed milk powder prepared by TBST solution, and incubating overnight at 4 ℃ on a shaking table;

(6) washing the membrane: washing the membrane three times by using TBST solution in a shaking way, wherein each time is ten minutes;

(7) incubation of secondary antibody: diluting goat anti-mouse antibody (1:5000) with 5% skimmed milk powder prepared from TBST solution, and incubating for 1 hr at room temperature on a shaking table;

(8) washing the membrane: washing the membrane three times by using TBST solution in a shaking way, wherein each time is ten minutes;

(9) and (3) developing: and (3) lightly and uniformly mixing the solution A and the solution B in the ECL luminescent solution according to the proportion of 1:1, uniformly dropwise adding the mixture on the PVDF membrane, reacting for 3min, and then sweeping the membrane.

As shown in the detection results of figures 1 and 2, the monoclonal antibodies m33 and m77 have good reactivity with representative strains of different lineages of type O foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93) and representative strains of different lineages of type A foot-and-mouth disease (A/AF72, A/GDMM/2013 and A/WH/CHA/09). The monoclonal antibodies m33 and m77 are general structural protein antibodies of the foot-and-mouth disease virus.

2. Indirect immunofluorescence assay:

respectively infecting BHK-21 cells with different pedigree representative strains of O-type foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93 strains) and A-type foot-and-mouth disease (A/AF72, A/GDMM/2013 and A/WH/CHA/09) (the strains are preserved by a national reference laboratory of foot-and-mouth disease), and judging the reaction condition of the prepared monoclonal antibodies m33 and m77 and FMDV antigen by fluorescence intensity under a fluorescence microscope, wherein the specific operation steps are as follows:

(1) culturing the cells: uniformly spreading BHK-21 cells into a 12-hole cell culture plate, and inoculating virus when the cells grow to about 70%;

(2) and (3) virus inoculation: respectively inoculating O-type strains of 3 lineages and A-type strains of 3 lineages into a 12-well plate according to the proportion of MOI (maximum oxygen index) 1, simultaneously establishing non-toxic normal cells as negative control, incubating the toxic cells in an incubator at 37 ℃, and inactivating cell supernatant when cytopathic effect reaches about 50%;

(3) fixing: adding paraformaldehyde into each well, fixing for 15min, and washing with PBS for 3 times;

(4) permeability: preparing a permeation solution containing 0.5% Triton X-100 with PBS solution, allowing cells to permeate for 10min, and washing with PBS for 3 times;

(5) incubating the primary antibody: washing with PBS for 3 times, diluting monoclonal antibodies m33 and m77 to be detected with PBS to a final concentration of 10 μ g/mL, adding 500 μ L/well, and incubating at 37 deg.C for 1h while using known positive murine antibody as positive control;

(6) incubation of secondary antibody: washing with PBS for 3 times, adding goat anti-mouse IgG-FITC fluorescent antibody (diluted 1: 1000), adding at 200 μ L/well, and incubating at 37 deg.C for 1 h;

(7) washed 5 times with PBS, 500. mu.L/well PBS was added to ensure cell surface wetting, and fluorescence was observed under a fluorescence microscope.

As shown in FIGS. 3 and 4, the monoclonal antibodies m33 and m77 of the invention have good reactivity with both the A-type strain of 3 lineages and the O-type strain of 3 lineages.

Example 3 establishment of Universal foot-and-mouth disease Virus structural protein solid phase competitive ELISA detection method

1. Determination of optimal reaction conditions of universal foot-and-mouth disease virus structural protein solid-phase competition ELISA detection method

1.1 determination of the optimal use concentration of the universal structural protein monoclonal antibody m33, the O type and the A type inactivated antigens:

the optimal use concentration of the universal structural protein monoclonal antibody m33, the O type and the A type inactivated antigens (stored in the laboratory) is determined by a matrix titration method. First, Na was used₂CO₃/NaHCO₃The coating solution (pH 9.6) was diluted in a dilution plate with m33 (lateral direction) of the universal monoclonal antibody at 8 μ g/mL in serial double ratios, and transferred to an elisa plate (50 μ L/well) at 4 ℃ overnight; discarding the liquid in the enzyme label plate, washing for 3 times by using PBST, and patting dry on absorbent paper; sequential double dilution of O-and A-type foot and mouth disease Virus inactivated antigen (longitudinal) (50. mu.L/well) starting at 1:2 Using PBST, 37 deg.CIncubating for 1 h; plate washing and drying (same as above); adding guinea pig antiserum working solution (stored in the laboratory) (50 μ L/well), and incubating at 37 deg.C for 1 h; plate washing and drying (same as above); adding goat anti-guinea pig secondary antibody (50 μ L/well), and incubating at 37 deg.C for 1 h; plate washing and drying (same as above); adding TMB (50 μ L/well), developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution (50. mu.L/well) and OD read with a microplate reader₄₅₀Value, taking OD₄₅₀The highest dilution factor corresponding to the universal antibody m33, the O type and the A type inactivated antigen is the optimal use concentration of the universal antibody m33 and the O type and the A type inactivated antigen when the value is in the range of 1.6-2.0.

The results show that the optimal coating concentration of the monoclonal antibody m33 is 1 mug/mL, the optimal using concentration of the A type inactivated antigen is 1:8, and the optimal using concentration of the O type inactivated antigen is 1: 16.

1.2 determination of the optimal concentration of monoclonal antibody m 77:

the optimal use concentrations of the monoclonal antibodies m33, type O and type A inactivated antigens determined above were used, and Na was used₂CO₃/NaHCO₃Coating solution (pH 9.6) coating m33 at the optimal concentration (50 μ L/well) at 4 ℃ overnight; plate washing and drying (same as above); adding O-type and A-type inactivated antigens with optimal use concentration into an enzyme label plate (50 mu L/hole), and incubating for 1h at 37 ℃; plate washing and drying (same as above); monoclonal m77 (transverse) (50. mu.L/well) was serially diluted in duplicate starting from 1:100 using PBST and incubated for 1h at 37 ℃; plate washing and drying (same as above); adding goat anti-mouse secondary antibody (50 μ L/well), and incubating at 37 deg.C for 1 h; plate washing and drying (same as above); adding TMB (50 μ L/well), developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution (50. mu.L/well) and OD read with a microplate reader₄₅₀Value, taking OD₄₅₀The highest dilution factor for monoclonal antibody m77 at a value of 1.6 was its optimal working concentration.

The results show that the optimal concentration of the monoclonal antibody m77 used is 6. mu.g/mL.

2. Operation steps of universal foot-and-mouth disease virus structural protein solid phase competition ELISA detection method

(1) Using Na₂CO₃/NaHCO₃Coating solution (pH 9.6) the coating solution diluted monoclonal antibody m33 to the optimum use concentration, and coatingELISA plate (50. mu.L/well), 4 ℃ overnight;

(2) washing the plate 5 times by using PBST, beating the plate dry, diluting O-type and A-type inactivated antigens by using PBST to working concentration, adding the diluted antigens into an enzyme label plate (50 mu L/hole), and incubating for 1h at 37 ℃;

(3) washing and drying the plate (same as above), using PBST to dilute the positive serum and the serum to be detected (1:4 times to 1:512) and the negative serum (1:4 times to 1:32) (50 mu L/hole), adding the monoclonal antibody m77 diluted to the working concentration into each hole, gently shaking and uniformly mixing, and incubating for 1h at 37 ℃;

(4) washing the plate and beating the plate dry (same as above), adding goat anti-mouse secondary antibody (50 mu L/hole), and incubating for 1h at 37 ℃;

(5) plate washing and drying (same as above); adding TMB (50 μ L/well), developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution (50. mu.L/well) and OD read with a microplate reader₄₅₀；

(6) The Percentage Inhibition (PI) is calculated by detecting the serum, the PI of 40% is taken as a critical value, if the PI of the detected serum is more than or equal to 40%, the highest dilution of the positive serum is the antibody titer of the serum, namely the titer, and the positive serum is determined. If the tested serum is less than 40%, the result is negative.

3. Specificity of universal foot-and-mouth disease virus structural protein solid phase competition ELISA method

The general foot-and-mouth disease virus structural protein solid-phase competition ELISA method established by the invention is used for measuring O type foot-and-mouth disease, A type foot-and-mouth disease, swine vesicular disease, seneca and swine fever positive reference serum, and the result shows that the general foot-and-mouth disease virus structural protein solid-phase competition ELISA method reacts specifically with the O type and A type foot-and-mouth disease reference positive serum but does not react with the swine vesicular disease, seneca and swine fever positive reference serum (Table 1).

TABLE 1 determination of the antibody titer of positive reference serum of foot-and-mouth disease, A-type foot-and-mouth disease, swine vesicular disease, seneca and swine fever by the general-purpose foot-and-mouth disease virus structural protein solid phase competition ELISA method

4. Sensitivity of universal foot-and-mouth disease virus structural protein solid phase competition ELISA method

The general foot-and-mouth disease virus structural protein solid-phase competition ELISA method established by the invention is used for measuring 75 parts of pig negative serum, 50 parts of pig O-type positive serum, 40 parts of pig A-type positive serum, 30 parts of cattle negative serum, 16 parts of cattle A-type positive serum and 18 parts of cattle O-type positive serum, and the result shows that the general foot-and-mouth disease virus structural protein solid-phase competition ELISA method has good sensitivity and specificity (see table 2)

TABLE 2 measurement of different-property serum by general foot-and-mouth disease virus structural protein solid-phase competition ELISA method

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

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Claims (10)

1. The monoclonal antibody of the general foot-and-mouth disease virus structural protein is characterized in that the monoclonal antibody is m33 or m77, the amino acid sequence of the heavy chain variable region of the monoclonal antibody m33 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6.

2. A gene fragment for encoding the monoclonal antibody m33 of claim 1, wherein the gene sequence for encoding the heavy chain variable region of the monoclonal antibody m33 is shown as SEQ ID NO.3, and the gene sequence for encoding the light chain variable region of the monoclonal antibody m33 is shown as SEQ ID NO. 4.

3. A gene fragment for encoding the monoclonal antibody m77 of claim 1, wherein the gene sequence for encoding the heavy chain variable region of the monoclonal antibody m77 is shown as SEQ ID NO.7, and the gene sequence for encoding the light chain variable region of the monoclonal antibody m77 is shown as SEQ ID NO. 8.

4. A monoclonal antibody composition of a structural protein of foot-and-mouth disease virus of the general type, comprising the monoclonal antibodies m33 and m77 according to claim 1.

5. An immunoconjugate, wherein the immunoconjugate comprises:

(i) the monoclonal antibody m33 and/or monoclonal antibody m77 of claim 1;

(ii) and a coupling moiety selected from the group consisting of: a detectable label, a drug, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.

6. The use of monoclonal antibody m33 and/or monoclonal antibody m77 of claim 1 in the preparation of a reagent, or a test strip, or a kit for the detection of foot-and-mouth disease virus.

7. A universal foot-and-mouth disease virus structural protein solid phase competition ELISA detection kit, characterized in that the kit comprises the monoclonal antibody m33 and/or the monoclonal antibody m77 of claim 1 or the monoclonal antibody composition of claim 4.

8. The ELISA detection kit of claim 7, wherein said kit comprises an ELISA plate coated with monoclonal antibody m33 and foot-and-mouth disease virus inactivated antigen, monoclonal antibody m77, an antibody diluent, a concentrated washing solution, a color-developing agent, and a stop solution; or the ELISA detection kit comprises an ELISA plate coated with a monoclonal antibody m77 and a foot-and-mouth disease virus inactivated antigen, a monoclonal antibody m33, an antibody diluent, a concentrated washing solution, a color developing agent and a stop solution.

9. The ELISA detection kit of claim 8 wherein said inactivated foot and mouth disease virus antigen is an inactivated foot and mouth disease virus type O antigen or an inactivated foot and mouth disease virus type A antigen.

10. A general solid-phase competition ELISA detection method for foot-and-mouth disease virus structural protein is characterized by comprising the following steps:

coating an ELISA plate with the monoclonal antibody m33 of claim 1, adding the inactivated foot-and-mouth disease virus antigen into the ELISA plate, adding the serum to be detected and the monoclonal antibody m77 of claim 1, mixing uniformly, and incubating; or, the monoclonal antibody m77 of claim 1 is used for coating an enzyme label plate, then the foot-and-mouth disease virus inactivated antigen is added into the enzyme label plate, the serum to be detected and the monoclonal antibody m33 of claim 1 are added, mixed evenly and incubated;

after incubation, adding goat anti-mouse secondary antibody, adding TMB after incubation, developing in dark, and adding H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

And calculating the inhibition Percentage (PI), taking 40% of PI as a critical value, and when the PI of the serum to be detected is more than or equal to 40%, the detection result is positive, and when the PI of the serum to be detected is less than 40%, the detection result is negative.

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