CN109232720B

CN109232720B - Foot-and-mouth disease O type virus sIgA antibody ELISA detection kit and application thereof

Info

Publication number: CN109232720B
Application number: CN201811068030.0A
Authority: CN
Inventors: 潘丽; 吕建亮; 张中旺; 罗虹; 王永录; 张永光
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2021-11-23
Anticipated expiration: 2038-09-13
Also published as: CN109232720A

Abstract

本发明公开了一种口蹄疫O型病毒sIgA抗体的ELISA检测试剂盒及其应用。所述的试剂盒包括口蹄疫O型病毒广谱多表位重组抗原包被的酶标反应板，100×浓缩酶标抗体、酶标抗体稀释液、样品稀释液、浓缩洗涤液、显色液、终止液、阳性对照样品和阴性对照样品。所述的口蹄疫O型病毒广谱多表位重组抗原由中国型(Cathay)、泛亚型(PanAsia)、缅甸98三个谱系口蹄疫O型病毒代表毒株的主要中和性抗原表位组成，因此提高了试剂盒检测的灵敏性和特异性，并且适用于不同的口蹄疫O型病毒感染的检测。本发明提供的试剂盒适于检测猪、牛、羊三种易感动物黏膜分泌物中的sIgA抗体，对于防治口蹄疫O型病毒的传播和感染具有重要的意义。The invention discloses an ELISA detection kit for foot-and-mouth disease O-type virus sIgA antibody and its application. The kit includes an enzyme-labeled reaction plate coated with a broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease type O virus, 100× concentrated enzyme-labeled antibody, enzyme-labeled antibody diluent, sample diluent, concentrated washing solution, chromogenic solution, Stop solution, positive control samples and negative control samples. The described foot-and-mouth disease O-type virus broad-spectrum multi-epitope recombinant antigen is composed of the main neutralizing antigenic epitopes of representative strains of three lineages of foot-and-mouth disease O-type virus of China type (Cathay), pan-subtype (PanAsia) and Myanmar 98, Therefore, the sensitivity and specificity of the kit detection are improved, and it is suitable for the detection of different foot-and-mouth disease O-type virus infections. The kit provided by the invention is suitable for detecting sIgA antibodies in the mucosal secretions of three susceptible animals, such as pigs, cattle and sheep, and is of great significance for preventing and treating the spread and infection of the foot-and-mouth disease type O virus.

Description

Foot-and-mouth disease O type virus sIgA antibody ELISA detection kit and application thereof

Technical Field

The invention relates to an indirect ELISA detection kit for foot-and-mouth disease O-type virus sIgA antibody and a detection method thereof. The invention belongs to the technical field of biological detection.

Background

Foot-and-Mouth Disease (FMD) is an infectious Disease caused by Foot-and-Mouth Disease virus characterized by the development of vesicular Disease in the Mouth and hooves of affected animals. Foot-and-Mouth Disease Virus (FMDV) belongs to the family picornaviridae, with a single-stranded RNA at the viral center. The serotype can be divided into 7 serotypes, namely A serotype, O serotype, C serotype, SAT1 serotype, SAT2 serotype, SAT3 serotype (namely south Africa 1, 2 and 3 serotypes) and Asial (Asia 1 serotype), and animals infected with FMDV of one serotype can still be infected with FMDV of another serotype to cause diseases. The MDV nucleic acid molecule is a single-strand positive-strand RNA molecule, the genome has about 8500 bases in total length, only one large open reading frame is contained, the structural proteins (VP1, VP2, VP3 and VP4) and the non-structural proteins (Lab, 2A, 2B, 2C, 3A, 3B, 3C and 3D) of the virus are generated by stepwise cleavage of the expressed polyprotein, the structural proteins are assembled to generate the capsid structure of the virus, and the non-structural proteins are involved in the interaction between the virus and a host, inhibit the transcription and translation mechanism of the host cell and participate in the replication process of the virus. The viral capsid protein is the main immunogen for inducing the production of neutralizing antibodies, and a plurality of conformational and linear neutralizing epitopes exist on the structural protein. The sequence analysis research of the monoclonal antibody and the immune escape mutant shows that at least 5 functionally independent neutralizing epitopes exist in the O-type FMDV, and the sections forming the antigenic sites mainly comprise the G-H loop of VP1 and C-terminal amino acid thereof, amino acids at positions 31, 70-73, 75 and 77 of VP2, and amino acids at positions 43 and 44 of the VP1B-C loop; amino acid 58 of VP3, amino acid 1149 and the like are involved in the formation of five neutralizing epitopes. The 5 neutralizing epitopes are conformational epitopes except that site 1, which is composed of a VP1G-H loop, is a linear epitope. The amino acids in the G-H loop of VP1 in the four structural proteins VP1, VP2, VP3, and VP4 of FMDV are easily mutated, but arginine-glycine-aspartic acid (Arg-Gly-Asp (RGD)) is highly conserved. The G-H loop spans approximately 20 amino acid residues at position 140-160 of VP 1. The VP1 protein contains not only major antigenic sites (141-160aa,200-213aa and 21-40aa), but also host recognition sites for FMD virus. Therefore, the protein expressed and purified by the expression vector constructed by selecting the region gene can be used as a good alternative coating antigen for preparing an antibody detection kit.

The production process of the multi-epitope antigen prepared by a prokaryotic expression system does not relate to live viruses, and the risk of virus dispersion does not exist; not only can the epitope sequence be adjusted at any time according to the change of the epidemic strains, but also different antigen sequences can be used, and the broad spectrum of the antigen is increased. Aiming at representative foot-and-mouth disease O-type strains O/Akesu/58(NCBI accession number: AF511039.1), O/TAW/81/97(NCBI accession number: AJ296321.1), O/Tibet/99(NCBI accession number: AJ318830.1) and O/Mya98(NCBI accession number: AJ303521.1) of three lineages of Chinese (Cathay), Panica and Myanmar 98, the invention precisely analyzes VP1 structural proteins of the 4 strains by adopting a bioinformatics method, screens out dominant antigen epitopes, expresses and purifies by using a prokaryotic expression system to obtain a main neutral antigen epitope region of the representative foot-and-mouth disease O-type strains, prepares a more updated coating antigen, and establishes an indirect foot-and-mouth disease O virus sIgA antibody ELISA detection method and a detection kit thereof.

The sIgA antibody is a main effector of mucosal immunity, is widely present in secretion such as colostrum, tears, saliva and the like, can neutralize viruses at the first time, and can further activate systemic immune response by means of a mucous membrane so as to prevent the invasion of the viruses. Its main functions include inhibiting adhesion, immunological elimination, dissolving bacteria, neutralizing virus, mediating antibody-dependent cell-mediated cytotoxicity (ADCC), resisting inflammation, promoting natural factor action and regulating mucosal immunoreaction. The anti-foot-and-mouth disease virus sIgA antibody is a specific mark generated in respiratory tracts and digestive tracts after foot-and-mouth disease virus infection or vaccine mucosal immunization, and simultaneously reflects the effect of the mucosal immunization vaccine. At present, no kit capable of effectively detecting the foot-and-mouth disease virus sIgA antibody exists in the market, and in view of the deep research on a mucous membrane immune mechanism and a mucous membrane immune vaccine in recent years, an ELISA kit for detecting the level of the foot-and-mouth disease virus sIgA antibody in mucous membrane secretions is developed, so that the foot-and-mouth disease virus infection and the mucous membrane immune state can be more accurately reflected, meanwhile, the dynamic change rule of the foot-and-mouth disease virus sIgA antibody is monitored by using the method, and theoretical and experimental bases are provided for the foot-and-mouth disease mucous membrane immune vaccine and the establishment of a matched detection method.

Disclosure of Invention

The invention aims to provide a detection kit and a method for foot-and-mouth disease O-type virus sIgA antibody against the defects in the prior art.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the invention uses DNASAR software to accurately analyze the 4 strain VP1 structural proteins aiming at foot-and-mouth disease O type representative strains O/Akesu/58(NCBI accession number: AF511039.1), O/TAW/81/97(NCBI accession number: AJ296321.1), O/Tibet/99(NCBI accession number: AJ318830.1) and O/Mya98(NCBI accession number: AJ303521.1) of three lineages of China (Cathay), Panya and Myanmar 98, and adopts a bioinformatics method to screen out main neutralizing epitope, and the series sequence of the designed epitope genes is as follows: (O/Akesu/58) VP1132-160-GGGS-VP 1193-213-GGGS- (O/TAW/81/97) VP1132-160-GGGS-VP 1193-213-GGGS- (O/Tibet/99) VP1132-160-GGGS-VP 1193-213-GGGS- (O/Mya98) VP1132-160-GGGS-VP 1193-213-6 XHis connected among the epitopes by GGGS, and finally a 6 histidine tag sequence is added to facilitate the purification of the expressed protein. The designed sequence is handed over to Nanjing GenScript company for optimization and synthesis of the coding sequence.

The broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease O-type virus obtained by the invention comprises main neutralizing antigen epitope sequences at the 132 th and 193 rd positions in VP1 proteins of foot-and-mouth disease O-type virus representative strains O/Akesu/58, O/TAW/81/97, O/Tibet/99 and O/Mya98, and the amino acid sequence of the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease O-type virus is shown as SEQ ID NO. 2.

The nucleotide sequence for coding the foot-and-mouth disease O type virus broad-spectrum multi-epitope recombinant antigen is also in the protection scope of the invention, preferably, the nucleotide sequence is shown as SEQ ID NO. 1.

Furthermore, the invention also provides application of the foot-and-mouth disease O type virus broad-spectrum multi-epitope recombinant antigen in preparation of a foot-and-mouth disease O type virus sIgA antibody detection reagent. Preferably, the reagent is a kit.

Furthermore, the invention provides an ELISA detection kit for foot-and-mouth disease O-type virus sIgA antibodies, which comprises an ELISA plate coated by the foot-and-mouth disease O-type virus broad-spectrum multi-epitope recombinant antigen, 100 multiplied concentrated enzyme-labeled antibodies, enzyme-labeled antibody diluent, sample diluent, concentrated washing solution, developing solution, stop solution, positive control samples and negative control samples.

Preferably, the foot-and-mouth disease O type virus broad-spectrum multi-epitope recombinant antigen is obtained by expression of a prokaryotic expression system.

Preferably, the enzyme label plate coated by the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease type O virus is prepared by the following method:

(1) preparation and coating of coating antigens

The recombinant protein inclusion body obtained by the expression of the prokaryotic expression system is renatured and purified by Ni-NTA to obtain the foot-and-mouth disease O-type virus broad-spectrum multi-epitope recombinant antigen, the amino acid sequence of which is shown as SEQ ID NO.2, carbonate buffer solution with pH9.6 is used for diluting to 3 mu g/ml during coating, an ELISA plate is coated according to 100 mu l/hole, and the mixture is kept still overnight in a refrigerator at 4 ℃;

(2) sealing and preservation of enzyme label plate

Washing the overnight-coated ELISA plate for 4 times by using PBST, adding 100 mu l of 10mM PBS blocking solution containing 0.5 w/v% BSA into each hole, standing at 37 ℃ for 2h, discarding the blocking solution, washing by using PBST for 3 times to obtain an ELISA plate pre-coated with antigen, adding 100 mu l of 6 v/v% horse serum-containing ELISA plate stabilizer into each hole, standing at room temperature for 30min, naturally drying after discarding the solution, vacuum-sealing by using an aluminum foil bag, and storing at 4 ℃;

wherein, the enzyme label plate stabilizer is prepared by adding 5g BSA, 10g sucrose and 20g trehalose into 1000ml0.01mol/LPBS (pH7.4) and gently shaking until dissolution, adding 0.05 w/v% Procline300, and storing at 4 ℃ for later use.

Preferably, the positive control sample is a nasal swab sample of 7-21d pigs, cattle and sheep which are subjected to foot-and-mouth disease O type virus challenge, and the positive control sample is a sample with an OD450 value of 1.5 +/-0.05 after mixing and serial dilution; the negative sample is

FMDV NS kit for detecting whether foot-and-mouth disease virus non-structural protein antibody is negative, and foot-and-mouth disease A type, O type and AsiaI liquid phase blocking ELISA kit for detecting serum antibody titer<1/4, detecting negative nasal swabs of pig, cattle and sheep by FMDV specific PCR, mixing, diluting, detecting samples with OD450 value of 0.1 + -0.05, and aseptically packaging.

Preferably, the 100 × concentrated enzyme-labeled antibody is a monoclonal antibody of mouse-anti-pig, cow or sheep IgA marked by Horse Radish Peroxidase (HRP), and is diluted by 100 times with an enzyme-labeled antibody diluent used, wherein the enzyme-labeled antibody diluent is a 0.01mol/LPBS buffer solution containing 1 v/v% of glycerol, 0.5 w/v% of bovine serum albumin, 1 w/v% of casein and 0.05 w/v% of Procline300 and has a pH value of 7.4.

Wherein, preferably, the sample diluent is a diluent containing 0.5M NaCl, 2.68mM KCl and 2.79mM KH₂PO₄、8.1mM Na₂HPO₄A mixed solution of 5g/L casein, 0.05% Tween20 and 10mM PBS; the color developing solution is TMB color developing solution, and the stop solution is 1moL/L H₂SO₄A solution; the concentrated washing solution is 10 x PBST solution, i.e. containing 0.5 v/V% Tween20 0.1mol/L PBS solution, pH7.6, use the dilution of 10 times.

Preferably, when the kit of the invention is used for detecting the foot-and-mouth disease O type virus sIgA antibody, the detection is carried out according to the following steps:

(1) sample dilution

Diluting the sample to be detected, the positive control and the negative control by the volume ratio of 1: 2, diluting;

(2) washing plate

Taking an ELISA plate coated with a main neutralizing epitope (TB/O) protein of foot-and-mouth disease O virus representative strain from a refrigerator at 4 ℃, opening an aluminum foil bag, taking out the ELISA plate, washing the plate for 3 times by using diluted washing liquid, and drying by using absorbent paper for 3min each time;

(3) sample application

Respectively adding the diluted sample to be detected, the positive control and the negative control into an enzyme label plate, incubating for 45min at 37 ℃ with 100 mu L of each hole;

(4) washing machine

Taking out the enzyme-linked immunosorbent assay plate, throwing off a sample, rinsing for 4 times by using a washing solution, and patting dry by using absorbent paper;

(5) adding enzyme-labeled antibody

Adding diluted horse radish peroxidase-labeled mouse anti-pig, cow or sheep IgA monoclonal antibody at 100 μ L/hole, and working at 37 deg.C for 30 min;

(6) adding a substrate:

throwing off the enzyme-labeled antibody, rinsing with a washing solution for 4 times, drying with absorbent paper, adding a developing solution for developing at 37 ℃ for 15min, taking out, and immediately adding a stop solution;

(7) determination of results

And (3) reading the OD450nm value on the microplate reader, judging that the OD450nm value of the sample to be detected is more than or equal to 2.5 times of the average value of the negative control samples as positive, and judging that the sample to be detected is less than 2.5 times of the average value of the negative control samples as negative.

The technical points of the invention are as follows:

1. the foot-and-mouth disease virus O type broad-spectrum multi-epitope recombinant antigen expressed by a prokaryotic expression system is used as a coating antigen, an enzyme-labeled mouse anti-pig, cow or sheep IgA monoclonal antibody is used as a secondary antibody, after a sample to be detected reacts with the coating antigen, the level of foot-and-mouth disease virus sIgA antibody is evaluated by further reaction of the enzyme-labeled antibody and comparison with a reference positive nasal swab and a reference negative nasal swab.

2. The envelope antigen is a recombinant protein which is purified after being expressed by escherichia coli pET-30a (+) pronucleus and contains main neutralizing antigen epitopes of a foot-and-mouth disease O type representative strain, the antigen consists of the main neutralizing antigen epitopes of three pedigree foot-and-mouth disease O type virus representative strains of China (Cathay), Panasia (Panasia) and Myanmar 98, and the envelope antigen contains a host recognition site (RGD tripeptide motif) of the foot-and-mouth disease virus and has broad spectrum.

3. The secondary antibody is a mouse anti-pig, bovine or sheep IgA monoclonal antibody marked by a horseradish peroxidase (HRP) marking kit.

4. The reference positive sample is a mixture of nasal swabs of pigs, cattle and sheep infected with foot-and-mouth disease 7-21 days after laboratory virus challenge; the negative sample is the channel

FMDV NS kit for detecting foot-and-mouth disease virus non-structural protein antibodyNegative in body, liquid phase blocking ELISA kit for A type, O type and AsiaI foot-and-mouth disease to detect antibody titer<1/4, FMDV specific PCR detects a mixture of swine, bovine and ovine nasal swabs that are negative for antigen.

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

1) the invention discloses a method for detecting foot-and-mouth disease O-type virus sIgA antibody and a detection kit thereof, which provides an effective method for evaluating the immune effect of foot-and-mouth disease mucous membrane by coating antigen and HRP-marked monoclonal antibody of mouse anti-pig, cattle and sheep IgA, provides a new method for early diagnosis of foot-and-mouth disease infection, and can quickly and accurately detect the foot-and-mouth disease A-type virus sIgA antibody in pig, cattle and sheep nasal swabs;

2) because the mutation frequency of the foot-and-mouth disease virus epidemic virus in recent years is higher and higher, the invention expresses the dominant epitope of 4 foot-and-mouth disease O type representative strains as the coating antigen through a prokaryotic expression system, and the antigen consists of main neutralizing epitopes of three pedigree foot-and-mouth disease O type virus representative strains of China (Cathay), Panasia (Panasia) and Burma 98, thereby preparing a new coating antigen with broad spectrum and improving the detection capability of the kit on the existing strains;

3) the sample collection operation is simple, the labor consumption is low, and the stress on animals is low;

4) the method is simple and convenient to operate, needs short time, and can be used for detecting a large number of samples and evaluating the mucosal immune effect.

Drawings

FIG. 1 is an SDS-PAGE image of EB/O protein provided in accordance with an embodiment of the present invention;

in the figure: lane M₁: protein molecular weight standards; lane 1: a BSA protein; lane 2: EB/O protein;

FIG. 2 shows the results of protein purification provided in the examples of the present invention.

In the figure: lane M: protein molecular weight standards; lanes 1, 2: the protein was eluted.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.

Example 1 preparation and Assembly of foot-and-mouth disease O type Virus sIgA antibody ELISA detection kit

Preparation of a kit:

1. designing the main neutralizing antigen epitope (EB/O) of foot-and-mouth disease O type virus representative strain:

the foot-and-mouth disease virus structural protein VP1 is the dominant antigen of the virus, and both the separated and purified natural VP1 protein and the recombinant expression product can induce the organism to produce protective neutralizing antibody with type specificity. The full length of the foot-and-mouth disease virus VP1 gene consists of 639 nucleotides, encodes a protein with 213 amino acids, and the main antigen epitope thereof is concentrated in the amino acid segment at the 140-position and 160-position and the amino acid segment at the 200-position and 213-position. The invention uses DNAStar biological software to analyze VP1 gene sequences of 4 foot-and-mouth disease O type virus representative strains O/Akesu/58(NCBI accession number: AF511039.1), O/TAW/81/97(NCBI accession number: AJ296321.1), O/Tibet/99(NCBI accession number: AJ318830.1) and O/Mya98(NCBI accession number: AJ303521.1) isolated in China, and determines amino acid segments at positions 132-. The tandem sequence of the designed epitopes was:

(O/Akesu/58) VP1132-160-GGGS-VP 1193-213-GGGS- (O/TAW/81/97) VP1132-160-GGGS-VP 1193-213-GGGS- (O/Tibet/99) VP1132-160-GGGS-VP 1193-213-GGGS- (O/Mya98) VP1132-160-GGGS-VP 1193-213-6 XHis connected among the epitopes by GGGS, and finally a 6 histidine tag sequence is added to facilitate the purification of the expressed protein.

The finally obtained nucleotide sequence for coding the main neutralizing antigen epitope (EB/O) antigen is shown as SEQ ID No.1, the amino acid sequence is shown as SEQ ID No.2, and the multi-epitope gene sequence is synthesized by Nanjing Kingsry Biotech company.

2. Expression of foot-and-mouth disease type O virus main neutralizing antigen epitope (EB/O) protein:

cloning 705bp (shown in SEQ ID NO. 1) of a main neutralizing antigen epitope (EB/O) coding gene of the foot-and-mouth disease O-type virus to a pET-30a (+) prokaryotic expression vector, correctly performing enzyme digestion and sequencing, transforming a positive plasmid to a BL21DE3plysS strain, screening a monoclonal by a kanamycin-resistant TB plate, and shaking the strain in a TB culture medium overnight at 37 ℃; transferring the overnight bacterial liquid to a fresh TB culture medium according to the volume ratio (v/v) of 1:100, shaking at 37 ℃ and 200rpm for 3h until the OD600 value reaches about 4, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mmol/L, continuing to shake for 3h, and centrifuging at 6000rpm to collect the bacterial liquid. Reducing the culture temperature to 30 ℃; adding IPTG inducer to the final concentration of 0.5mM, and continuing shaking culture at 30 ℃ for 3-4 h; centrifuging at 8000rpm for 3min, collecting thallus, suspending in 50mL precooling NTA-0 buffer solution, and ice-cooling for 30 min; ultrasonically crushing the thallus, centrifuging at 16000rpm and 4 ℃ for 50min, and collecting the supernatant and the precipitate; a small amount of the supernatant and the precipitate were subjected to SDS-PAGE, and the protein expression results are shown in FIG. 1. The remaining supernatant and precipitate were left at 4 ℃ for further use.

3. Protein purification and renaturation:

resuspending the pellet in 50mL NTA-0 buffer solution, adding DTT to a final concentration of 1mM, ultrasonically promoting the dissolution of the hybrid protein, centrifugally collecting the pellet, repeating the steps for three times until the supernatant is transparent, resuspending the pellet in PBS, ultrasonically treating the pellet, centrifugally removing the supernatant, resuspending the inclusion body in 6M guanidine hydrochloride, and adding DTT to a final concentration of 5 mM; shaking at 37 deg.C for 3 hr until inclusion body is completely dissolved, and centrifuging to remove supernatant. Then the protein is dialyzed and renatured at low temperature by using protein renaturation liquid, 3M guanidine hydrochloride with 2 times of volume is used for diluting the protein solution, the protein solution is dropwise added into 200mL renaturation liquid (pH8.0) at the temperature of 4 ℃, the rotating speed is adjusted to be maximum, the stirring is carried out for 24 hours, the protein solution is taken out in a dialysis bag, the concentration is carried out by using PEG60000, and then the dialysis is carried out by using PBS buffer solution overnight. And (4) purifying the renatured protein again by using an anion exchange column Ni-NTA, and collecting the protein. The protein purification results are shown in FIG. 2.

4. Preparation of coating antigen and selection of optimal coating concentration:

the purified protein was serially diluted with carbonate buffer (pH9.6) to 6. mu.g/mL, 3. mu.g/mL, 1.5. mu.g/mL, 0.75. mu.g/mL, 0.38. mu.g/mL, 0.19. mu.g/mL, two columns were coated at each concentration, applied to a 96-well microplate at 100. mu.L/well, and coated overnight at 4 ℃. As a result, the concentration of the coating antigen was determined to be the optimum coating concentration (Table 1) because the positive value OD450 was more than 1.0 at a concentration of 3. mu.g/mL and the value of P/N was the largest.

TABLE 1 chessboard titration antigen working concentration (OD450nm)

Coating concentration (μ g/mL)	6.00	3.00	1.50	0.75	0.38	0.19
							Positive sample (P)	1.122	1.084	0.916	0.746	0.653	0.554
Negative sample (N)	0.138	0.098	0.092	0.087	0.079	0.073
							P/N	8.130	11.06	9.956	8.575	8.266	7.589

5. Sealing of the ELISA plate:

the plate was washed 4 times with PBST (0.01 mol/L PBS containing 0.5 v/v% Tween20, pH 7.6), 100. mu.l of 10mM PBS containing 0.5 w/v% BSA was added to each well, and the plate was allowed to stand at 37 ℃ for 2 hours. Removing the sealing liquid, washing for 3 times by PBST (Poly-p-phenylene benzobisoxazole) to obtain an ELISA plate pre-coated with the antigen, adding 100 mu l of ELISA plate stabilizer containing 6 v/v% horse serum into each hole, standing at room temperature for 30min, naturally drying after removing the liquid, sealing in vacuum by using an aluminum foil bag, and storing at 4 ℃;

wherein, the enzyme label plate stabilizer is prepared by adding 5g BSA, 10g sucrose and 20g trehalose into 1000ml0.01mol/L PBS (pH7.4) and gently shaking for dissolving, adding 0.05 w/v% Procline300, and storing at 4 ℃ for later use.

6. Preparing standard negative and positive nasal test strips:

the positive control sample is a sample obtained by collecting nasal swabs of pigs, cattle and sheep 7-21d after the O-type foot-and-mouth disease virus is attacked, and detecting that the OD450 value is 1.5 +/-0.05 after mixing and serial dilution; the negative control sample is

7. Enzyme-labeled antibody (10X)

And the horse radish peroxidase-labeled mouse anti-pig, cow or sheep IgA secondary antibody is diluted by 10 times for use.

8. Sample diluent

The sample dilution contains 0.5M NaCl, 2.68mM KCl, 2.79mM KH₂PO₄、8.1mM Na₂HPO₄A mixed solution of 5g/L casein, 0.05% v/vTween20 and 10mM PBS.

9、PBST(10×)

0.1mol/LPBS solution containing 0.5 v/v% Tween20, pH 7.6. The composition is diluted 10 times for use.

10. Color developing solution and stop solution

The color development liquid is TMB color development liquid, and the stop solution is 1moL/LH₂SO₄And (3) solution.

(II) assembling and storing of kit

The kit was assembled according to the kit contents listed in Table 2, assembled, and stored at 4 ℃.

TABLE 2 ELISA test kit content

(III) method of Using the kit (detection method)

(1) Diluting: the PBST (10X) wash was diluted to 250ml with sterile water, and the enzyme-labeled antibody (10X) was diluted to 5ml with a sample diluent.

(2) Washing the plate: and opening the packaging bag, taking out the enzyme label plate pre-coated with the antigen, washing the plate for 4 times by using the diluted PBST, and drying by using absorbent paper for 3min each time.

(3) Sample adding: samples were mixed at a ratio of 1: diluting by 2 times, adding 100ul of each well into a reaction plate, adding 100 mu L of positive control and negative control per well, and repeating the positive control and the negative control for two times respectively. Incubate at 37 ℃ for 45 min.

(4) Adding an enzyme-labeled antibody: the sample was spun off, rinsed 4 times with PBST, blotted dry with absorbent paper, and diluted horseradish peroxidase-labeled mouse-anti-pig, bovine or ovine IgA secondary antibodies were added at 100. mu.L/well and incubated at 37 ℃ for 30 min.

(5) Adding a substrate: the enzyme-labeled antibody was spun off, rinsed 4 times with PBST, and patted dry on absorbent paper. Adding TMB developing solution, incubating at 37 deg.C for 15min, and adding 100 μ l/well reaction stop solution.

(6) Reading OD on microplate reader₄₅₀Numerical values.

Reading OD on microplate reader₄₅₀Numerical value, OD of sample to be measured_450nmThe value of the average value of the negative control serum is more than or equal to 2.5 times of the average value of the negative control serum, and the average value of the negative control serum is less than 2.5 times of the average value of the negative control serum, so that the test result is positive.

Example 2 sensitivity and specificity test of foot-and-mouth disease O-type Virus sIgA antibody ELISA detection kit

1. Sensitivity test:

the kit prepared in example 1 was used to test 100 nasal test sample, which were collected from the affected pig after challenge using O/Akesu/58, O/TAW/81/97, O/Tibet/99 and O/Mya98 experiments, each virus sample was 25 cases, and no immunization was performed before challenge, and the test method was the same as example 1. The sensitivity of the method was analyzed by calculating the positive detection rate. The ELISA detection result shows that the samples are 93 positive parts and 7 negative parts, and the positive detection rate is 93%. The kit has better sensitivity and can detect the infection of different foot-and-mouth disease O-type viruses. The results are shown in Table 3.

TABLE 3 ELISA kit sensitivity test

Detection method	Number of samples (parts)	Number of positive (number of copies)	Negative number (number)	Positive rate (%)
					Indirect ELISA	100	93	7	93％

2. Specific experiments:

the kit prepared in the embodiment 1 is used for detecting a Pig Epidemic Diarrhea Virus (PEDV) infected piglet intestinal mucosa flushing fluid sample, a pig breeding and respiratory syndrome virus (PRRSV) antibody positive sample, a Porcine Circovirus (PCV) specific IgA antibody positive sample and a swine fever virus (CSFV) specific IgA antibody positive sample, 90 parts of A-type FMDV infected pig nose swab samples are added for detection aiming at the cross infection conditions of A-type FMDV and O-type FMDV, and the specificity of the method is analyzed by taking the A-type FMDV positive sample as a positive control. The ELISA detection result shows that 90 parts of A-type FMDV infected pig nose swab samples are positive for 2 parts, suspicious for 10 parts and negative for 78 parts, and the coincidence rate is 86.7%. And the kit has no cross reaction with IgA antibodies of Porcine Epidemic Diarrhea Virus (PEDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Circovirus (PCV) and classical swine fever virus, thereby ensuring that the diagnostic kit has good sensitivity and specificity at the same time and fundamentally ensuring the accuracy and reliability of results.

3. Repeatability test

6 parts of positive nasal mucosa test sample and 1 part of negative nasal mucosa test sample with different antibody levels are selected, the same batch of antigen coated ELISA plates are adopted, 4 batches of repeated tests are carried out at different time, the detection result is counted, and the intra-batch variation coefficient is less than 9 percent (1.41 to 8.64 percent). The experiment proves that the variation coefficient of the same nasal mucosa test sample in the antigen-coated ELISA plate prepared in the same batch is very small, and the repeatability is good. The antigen-coated ELISA plates prepared in 4 batches are used for batch repeated tests at the same time, the detection results are counted, and the batch variation coefficient is less than 8% (2.28% -7.13%), so that the variation coefficient of the same nasal mucosa sample in the antigen-coated ELISA plates prepared in different batches is very small, and the indirect ELISA method established by the invention has very good repeatability (Table 4).

TABLE 4 repeatability tests

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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

<120> foot-and-mouth disease O type virus sIgA antibody ELISA detection kit and application thereof

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<170>Patent-In 3.5

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Met Gly Ser Cys Arg Tyr Ser Asn Ser Asn Val Ser Asn Val Ser Gly Asp Leu Gln Val 20

Leu Ala Gln Lys Ala Ala Arg Ala Leu Pro Gly Gly Gly Ser Ala Ile His Pro Ser Glu 40

Ala Arg His Lys Gln Lys Ile Val Ala Pro Ala Lys Gln Leu Leu Gly Gly Gly Ser Gly 60

Ser Ser Lys Tyr Gly Asp Thr Ser Thr Asn Asn Val Arg Gly Asp Leu Gln Val Leu Ala 80

Gln Lys Ala Glu Arg Thr Leu Pro Gly Gly Gly Ser Ala Ile Gln Pro Ser Asp Ala Arg 100

His Lys Gln Arg Ile Val Ala Pro Ala Lys Gln Leu Leu Gly Gly Gly Ser Gly Asn Cys 120

Lys Tyr Gly Glu Ser Pro Val Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 140

Ala Ala Arg Thr Leu Pro Gly Gly Gly Ser Ala Ile His Pro Ser Glu Ala Arg His Lys 160

Gln Lys Ile Val Ala Pro Val Lys Gln Leu Leu Gly Gly Gly Ser Gly Asn Cys Lys Tyr 180

Ala Glu Gly Ser Leu Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala 200

Arg Pro Leu Pro Gly Gly Gly Ser Ala Val His Pro Asp Glu Ala Arg His Lys Gln Lys 220

Ile Val Ala Pro Val Lys Gln Ser Leu His His His His His His 235

Claims

1. The broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease O-type virus is characterized by comprising main neutralizing antigen epitope sequences of a 132 th-position 160 th-position and a 193 th-position 213 th-position in VP1 protein of foot-and-mouth disease O-type virus representative strains O/Akesu/58, O/TAW/81/97, O/Tibet/99 and O/Mya98, wherein the amino acid sequence of the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease O-type virus is shown as SEQ ID NO. 2.

2. A polynucleotide encoding the foot-and-mouth disease type O virus broad-spectrum multi-epitope recombinant antigen of claim 1.

3. The polynucleotide of claim 2, wherein the sequence of said polynucleotide is set forth in SEQ ID No. 1.

4. The use of the broad-spectrum multi-epitope recombinant antigen of foot-and-mouth disease type O virus of claim 1 in the preparation of a reagent for detecting foot-and-mouth disease type O virus sIgA antibody.

5. An ELISA detection kit for foot-and-mouth disease O-type virus sIgA antibody, which is characterized by comprising the foot-and-mouth disease O-type virus broad-spectrum multi-epitope recombinant antigen coated ELISA plate, 100 Xconcentrated ELISA antibody, ELISA antibody diluent, sample diluent, concentrated washing solution, developing solution, stopping solution, positive control sample and negative control sample of claim 1.

6. The foot-and-mouth disease O type virus sIgA antibody ELISA detection kit of claim 5, wherein the foot-and-mouth disease O type virus broad-spectrum multi-epitope recombinant antigen is obtained by expression of a prokaryotic expression system.

7. The kit for ELISA detection of sIgA antibody for foot-and-mouth disease virus O according to claim 5, wherein the ELISA plate coated with the broad-spectrum multi-epitope recombinant antigen for foot-and-mouth disease virus O is prepared by the following method:

(1) preparation and coating of coating antigens

(2) sealing and preservation of enzyme label plate

Washing the overnight-coated ELISA plate for 4 times by using PBST, adding 100 mu l of 10mM PBS blocking solution containing 0.5% w/vBSA into each hole, standing at 37 ℃ for 2h, discarding the blocking solution, washing by using PBST for 3 times to obtain an ELISA plate pre-coated with antigen, adding 100 mu l of 6% v/v horse serum-containing ELISA plate stabilizer into each hole, standing at room temperature for 30min, discarding the solution, naturally drying, vacuum-sealing by using an aluminum foil bag, and storing at 4 ℃;

wherein, the enzyme label plate stabilizer is prepared by adding 5g BSA, 10g sucrose and 20g trehalose into 1000ml PBS with 0.01mol/L pH7.4, gently shaking for dissolving, adding 0.05% w/v Procline300, and storing at 4 ℃ for later use.

8. The ELISA detection kit for sIgA antibody against foot-and-mouth disease virus O according to claim 5, wherein the positive control sample is a sample obtained by collecting nasal swabs of pigs, cattle and sheep 7-21d after the foot-and-mouth disease virus O is attacked, and detecting the sample with OD450 value of 1.5 +/-0.05 after mixing and serial dilution; the negative sample is

9. The kit for ELISA detection of sIgA antibody for foot-and-mouth disease virus O according to claim 5, wherein the 100X concentrated enzyme-labeled antibody is a monoclonal antibody against porcine, bovine or ovine IgA labeled with horseradish peroxidase (HRP), and when used, the kit is diluted 100-fold with an enzyme-labeled antibody diluent, and the enzyme-labeled antibody diluent is 0.01mol/L PBS buffer containing 1% v/v glycerol, 0.5% w/v bovine serum albumin, 1% w/v casein and 0.05% w/v Procline300, and has pH of 7.4.

10. The ELISA test kit for detecting sIgA antibody against foot-and-mouth disease virus O according to claim 5, wherein said sample diluent comprises 0.5M NaCl, 2.68mM KCl, 2.79mM KH₂PO₄、8.1mM Na₂HPO₄A mixed solution of 5g/L casein, 0.05% Tween20 and 10mM PBS; the color developing solution is TMB color developing solution, and the stop solution is 1moL/L H₂SO₄A solution; the concentrated washing solution is 10 x PBST solution, i.e. containing 0.5% v/vTween20 0.1mol/L PBS solution, pH7.6, use the dilution of 10 times.