CN109187993B - Foot-and-mouth disease type A virus sIgA antibody ELISA detection kit and application thereof - Google Patents
Foot-and-mouth disease type A virus sIgA antibody ELISA detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses an ELISA detection kit for foot-and-mouth disease type A virus sIgA antibody and application thereof. The kit comprises an enzyme-labeled reaction plate coated by broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease A-type virus, 100 multiplied concentrated enzyme-labeled antibody, enzyme-labeled antibody diluent, sample diluent, concentrated washing solution, developing solution, stop solution, a positive control sample and a negative control sample. The broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease A type virus consists of dominant epitopes (TB/A) of 3 foot-and-mouth disease A representative strains separated in China, so that the sensitivity and specificity of the detection of the kit are improved, and the kit is suitable for detecting different foot-and-mouth disease A type virus infections. The kit provided by the invention is suitable for detecting sIgA antibodies in mucosal secretions of three susceptible animals including pigs, cattle and sheep, and has important significance for preventing and treating the spread and infection of the foot-and-mouth disease type A virus.
Description
Technical Field
The invention relates to an indirect ELISA detection kit for foot-and-mouth disease type A 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 FMDV infection target is more than 70, the latency period is 1-7 days, the spread speed is high, the disease is acute, viruses mainly enter an animal body through a respiratory tract and a digestive tract, firstly enter the throat and tonsil of the animal, and then enter the blood circulation system of the animal, the clinical symptoms are that the infected animal suffers from lassitude, sialic acid, and blisters appear in the mouth, hoof, nose and nipple areas, and almost all tissues, organs, secretions and the like contain FMDV in the FMD latency period. The disease is the first of the 15 animal epidemics list specified by the international animal health Organization (OIE), causing significant economic losses to the livestock industry and international animal trade.
The MDV genome has about 8500 basic groups in total length and only contains one large open reading frame, the expressed polyprotein is gradually cracked to generate structural proteins (VP1, VP2, VP3 and VP4) and non-structural proteins (Lab, 2A, 2B, 2C, 3A, 3B, 3C and 3D) of the virus, the structural proteins are assembled to generate a capsid structure of the virus, and the non-structural proteins participate 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 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. In addition, the production process of the multi-epitope antigen prepared by a prokaryotic expression system does not relate to live viruses, so that 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.
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. The main functions of the cell-mediated anti-inflammatory agent are adhesion inhibition, immune elimination, bacterial lysis, virus neutralization, antibody-dependent cell-mediated cytotoxicity (ADCC), inflammation resistance, natural factor action promotion and mucosal immune response regulation. 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 type A virus sIgA antibody aiming at 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 carry out sequence analysis on VP1 gene of 3 foot-and-mouth disease virus A type representative strains (A/GDMM/2013, A/HuBWH/2009, A/GSLX/62) isolated in China, determines dominant antigen epitope, and introduces a spacer sequence KKK, (G4S)2 and GPGPG which can ensure that each epitope structure is correctly displayed between two adjacent epitopes, thereby constructing a series sequence of multi-epitope gene (TB/A), wherein the series sequence is A/GDMM/2013[ (20-85) -KKK- (133-173) - (G4S)2- (194-213) ]. The nucleotide sequence of the polyepitope gene was synthesized by the national Biotech company of Kinsrui.
The broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease A virus obtained by the invention comprises main neutralizing antigen epitope sequences of 20 th-85 th, 133 th-172 th and 193 th-212 th positions in VP1 protein of a representative strain A/GDMM/2013, A/HuBWH/2009, A/GSLX/62 of the foot-and-mouth disease A virus, and the amino acid sequence of the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease A virus is shown as SEQ ID No. 2.
The nucleotide sequence for coding the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease type A virus 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 A type virus broad-spectrum multi-epitope recombinant antigen in preparation of a foot-and-mouth disease A type virus sIgA antibody detection reagent.
A kit for detecting foot-and-mouth disease A type virus sIgA antibody ELISA comprises an enzyme label plate coated by foot-and-mouth disease A type virus broad-spectrum multi-epitope recombinant antigen, 100 multiplied concentrated enzyme-labeled antibody, enzyme-labeled antibody diluent, sample diluent, concentrated washing solution, developing solution, stopping solution, a positive control sample and a negative control sample.
Preferably, the foot-and-mouth disease A 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 A type 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 A 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/L PBS (pH7.4) and gently shaking for dissolving, 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 type A 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 foot-and-mouth disease virusLiquid phase blocking ELISA kit for detecting serum antibody titer of foot-and-mouth disease A type, O type and AsiaI type with non-structural protein antibody as negative<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 used after being diluted by 100 times by an enzyme-labeled antibody diluent, wherein the enzyme-labeled antibody diluent is 0.01mol/L PBS 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 pH 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, pH 7.6, use the dilution of 10 times.
Preferably, when the kit is used for detecting the foot-and-mouth disease type A virus sIgA antibody, the detection method comprises 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 the ELISA plate coated with the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease virus A from a refrigerator at 4 ℃, opening the 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) foot-and-mouth disease virus A dominant antigen epitope protein (TB/A) expressed by a prokaryotic expression system is used as a coating antigen, an enzyme-labeled mouse anti-pig, cattle and sheep IgA monoclonal antibody is used as a second antibody, after a sample to be detected reacts with the coating antigen, the level of the 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 coating antigen is a multi-epitope protein purified after prokaryotic expression of Escherichia coli pET-30a (+), and the protein contains main antigen sites (141-160aa, 200-213aa and 21-40aa) of the foot-and-mouth disease virus and a host recognition site (RGD tripeptide motif) of the foot-and-mouth disease virus.
3) The secondary antibody is a mouse anti-pig, cattle and sheep IgA monoclonal antibody marked by a horseradish peroxidase (HRP) marking kit.
4) The reference positive sample is a nasal swab of a pig, a cow or a sheep infected with foot-and-mouth disease 7-21 days after the laboratory toxin attack; the negative sample is the channel
FMDV NS kit for detecting whether foot-and-mouth disease virus non-structural protein antibody is negative, and A-type, O-type and AsiaI foot-and-mouth disease liquid blocking ELISA kit for detecting antibodyPotency of the drug<1/4, FMDV specific PCR detects nasal swabs of pigs, cattle and sheep 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 A 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 mouse anti-pig, cattle and sheep IgA monoclonal antibodies, 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 uses the dominant antigen epitope of 3 foot-and-mouth disease A representative viruses expressed by a prokaryotic expression system as the coating antigen to prepare a more updated coating antigen and improve 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 pattern of TB/A protein provided by an example of the present invention;
in the figure: lane M1: protein molecular weight standards; lane 1: a BSA protein; lane 2: TB/A protein
FIG. 2 shows the results of protein purification provided in the examples of the present invention.
In the figure: lane M2: protein molecular weight standards; lane 3: 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 type A Virus sIgA antibody ELISA detection kit
Preparation of a kit:
1. screening and designing dominant epitope (TB/A) of foot-and-mouth disease type A virus:
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 DNASAR biological software to analyze VP1 gene sequences of 3 foot-and-mouth disease virus A type representative strains A/GDMM/2013(GenBank accession number: KF450794.1), A/HuBWH/2009(GenBank accession number: JF792355.1) and A/GSLX/62(GenBank accession number: AJ131666.1) isolated in China, and determines amino acid sections with dominant antigen epitopes of 20-85, 133-172 and 193-212 positions. In order to prevent new epitopes from appearing in the process of constructing genes, spacer sequences KKK, (G4S)2 and GPGPG which can ensure the correct structure of each epitope are introduced between two adjacent epitopes, and the tandem sequence of the multi-epitope genes is as follows:
A/GDMM/2013/[(20-85)-KKK-(133-172)-(G4S)2-(193-212)]-GPGPG-A/HuBWH/2009/[(20-85)-KKK-(133-172)-(G4S)2-(193-212)]-GPGPG-A/GSLX/62/[(20-85)-KKK-(133-173)-(G4S)2-(194-213)]
the nucleotide sequence of the multi-epitope gene is shown as SEQ ID No.1, the coded amino acid sequence is shown as SEQ ID No.2, and the nucleotide sequence of the multi-epitope gene is synthesized by Kinsley Biotech company.
2. Expression of foot-and-mouth disease type A virus dominant antigen epitope protein:
cloning a coding gene 1287bp (shown in SEQ ID NO. 1) of dominant antigen epitope protein of foot-and-mouth disease A type virus to a pET-30a (+) prokaryotic expression vector, after enzyme digestion and sequencing are correct, converting a positive plasmid into a BL21DE3plys 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)
5. Preparing standard negative and positive nasal test strips:
the positive control sample is a nasal swab sample of 7-21d pigs, cattle and sheep which are subjected to foot-and-mouth disease type A virus challenge, and the positive control sample is a sample with an OD450 value of 1.5 +/-0.05 after being mixed and serially diluted; the negative control 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.
6. Determination of the optimal confining liquid:
the ELISA plate was blocked with different blocking solutions for 1h at 37 ℃ and the best blocking solution was selected, the results are shown in Table 2.
TABLE 2 Elisa plate was blocked with different blocking solutions (OD450nm)
The results showed that the positive OD450 value was greater than 1.0 and the P/N value was the greatest when 10mM PBS containing 0.5 w/v% BSA was selected as the blocking solution, thus determining the blocking solution as the optimal blocking solution.
7. 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 to obtain the ELISA plate pre-coated with the antigen, adding 100 μ l of ELISA plate stabilizer containing 6 v/v% horse serum into each hole, standing at room temperature for 30min, removing the liquid, naturally drying, vacuum sealing with aluminum foil bag, and storing at 4 deg.C.
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.
8. Enzyme-labeled antibody (10X)
And the horseradish peroxidase-labeled mouse anti-pig, cow and sheep IgA secondary antibodies are diluted by 10 times for use.
9. 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.
10、PBST(10×)
0.1mol/LPBS solution containing 0.5 v/v% Tween20, pH 7.6. The composition is diluted 10 times for use.
11. Color developing solution and stop solution
The color developing solution is TMB color developing solution, and the stop solution is 1moL/L H2SO4And (3) solution.
(II) assembling and storing of kit
The kit was assembled according to the kit contents listed in Table 3, assembled, and stored at 4 ℃.
TABLE 3 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 measured450nmThe 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, specificity, repeatability experiment of foot-and-mouth disease type A Virus sIgA antibody ELISA detection kit
1. Sensitivity test:
the kit prepared in example 1 was used to detect 90 nasal test sample collected after experimental challenge, which was not immunized before challenge, and the sensitivity of the method was analyzed by calculating the positive detection rate as in example 1. ELISA detection results show that the positive 88 parts and the negative 2 parts of the samples have a positive detection rate of 97.78%. The kit of the invention has better sensitivity, and the result is shown in table 4.
TABLE 4ELISA kit sensitivity test
| Detection method | Number of samples (parts) | Number of positive (number of copies) | Negative number (number) | Positive rate (%) |
| Indirect ELISA | 90 | 88 | 2 | 97.78％ |
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, and for the cross infection conditions of A-type FMDV and O-type FMDV, 84 parts of O-type FMDV infected pig nose swab samples are added for detection, and the specificity of the method is analyzed by taking the A-type foot-and-mouth disease sIgA antibody positive sample as a positive control. The ELISA detection result shows that 84 parts of O-type FMDV infected pig nose swab samples are positive 1 part, suspicious 7 parts and negative 76 parts, and the coincidence rate is 90.5%. 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 (CSFV), 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. And (3) repeatability experiment:
6 parts of positive nasal mucosa test sample with different antibody levels and 1 part of negative nasal mucosa test sample are selected, the same batch of antigen coated ELISA plates are adopted, 4 times of batch repeated tests are carried out at different time, the detection result is counted, the batch variation coefficient is less than 7 percent (1.53 percent to 6.41 percent), and the result shows that the variation coefficient of the same nasal mucosa test sample in the antigen coated ELISA plates 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% (1.27% -7.92%), 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 5).
TABLE 5 results of repeated experiments
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 A type virus sIgA antibody ELISA detection kit and application thereof
<160>2
<170>Patent-In 3.5
<210>1
<211>1287
<212>DNA
<213>artificial sequence
<400>1
atgggcgaga cccaggcgca gcgtcgttac cacaccgatg ttggtttcct gatggaccgt 60
ttcgtgcaga tcaagccggt tggcccgacc cacgtgatcg acctgatgca gacccaccaa 120
catggtctgg ttggcgcgat gctgcgtgcg gcgacctact atttcagcga tctggagatt 180
gtggttaacc acaccggtaa caagaaaaag accagcaaat atagcgcgcc gcagaaccgt 240
cgtggtgacc tgggtccgct ggcggcgcgt ctggcggcgc aactgccggc gagcttcaac 300
tttggcgcga tccgtgcgac cgaaatccgt ggtggcggtg gcagcggtgg cggtggcagc 360
gaagtgagca gccaggatcg tcacaaacaa aagatcattg cgccggcgaa gcaactgctg 420
ggtccgggtc cgggtggtga aacccaggtt caacgtcgtc accacaccga cgtgagcttc 480
atcatggatc gttttgtgca aattaagccg gttagcccga cccatgttat tgatctgatg 540
caaacccatc agcatggtct ggttggcgct atgctgcgcg cggcgaccta ttacttcagc 600
gatctggaaa tcgttgttaa ccacaccggt cgtaaaaaga aaaccagcaa atatagcgct 660
cctgcgactc gtcgtggtga cctgggcagc ctggcggcgc gcctggcggc gcagctgccg 720
gcgagcttta actatggtgc gatccgtgcg accgagattc aaggtggcgg tggcagcggt 780
ggcggtggca gcgaagtgac cagccaagac cgccataagc agaaaattat cgctcctgcg 840
aagcaactgc tgggcccggg cccgggtggt gaaacccaag ttcaacgtcg tcaacacacc 900
aacgtgggtt tcatcatgga ccgttttgtt aagattccga gccagagccc gacccacgtg 960
atcgatctga tgcaaaccca ccagcatggt ctggttggcg cattattacg cgcggcgacc 1020
tattatttta gcgacctgga aatcgtggtt cgtcacgacg ataacaagaa aaagaccacc 1080
aaatatagca ccggtaacgc gggtcgtcgt ggtgatctgg gcagcctggc tgctcgtgtt 1140
gctgctcagc tgccggcgag cttcaatttc ggcgcgattc gcgcgaccgt tatccatggt 1200
ggcggtggca gcggtggcgg tggcagcaag gttaccagcc aggaccgtca taaacaacgc 1260
atcatcgcgc cggcgaaaca actgctg1287
<210>2
<211>429
<212>PRT
<213>artificial sequence
<400>2
Met Gly Glu Thr Gln Ala Gln Arg Arg Tyr His Thr Asp Val Gly Phe Leu Met Asp Arg 20
Phe Val Gln Ile Lys Pro Val Gly Pro Thr His Val Ile Asp Leu Met Gln Thr His Gln 40
His Gly Leu Val Gly Ala Met Leu Arg Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile 60
Val Val Asn His Thr Gly Asn Lys Lys Lys Thr Ser Lys Tyr Ser Ala Pro Gln Asn Arg 80
Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn 100
Phe Gly Ala Ile Arg Ala Thr Glu Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 120
Glu Val Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln Leu Leu 140
Gly Pro Gly Pro Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser Phe 160
Ile Met Asp Arg Phe Val Gln Ile Lys Pro Val Ser Pro Thr His Val Ile Asp Leu Met 180
Gln Thr His Gln His Gly Leu Val Gly Ala Met Leu Arg Ala Ala Thr Tyr Tyr Phe Ser 200
Asp Leu Glu Ile Val Val Asn His Thr Gly Arg Lys Lys Lys Thr Ser Lys Tyr Ser Ala 220
Pro Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro 240
Ala Ser Phe Asn Tyr Gly Ala Ile Arg Ala Thr Glu Ile Gln Gly Gly Gly Gly Ser Gly 260
Gly Gly Gly Ser Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala 280
Lys Gln Leu Leu Gly Pro Gly Pro Gly Gly Glu Thr Gln Val Gln Arg Arg Gln His Thr 300
Asn Val Gly Phe Ile Met Asp Arg Phe Val Lys Ile Pro Ser Gln Ser Pro Thr His Val 320
Ile Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Leu Leu Arg Ala Ala Thr 340
Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg His Asp Asp Asn Lys Lys Lys Thr Thr 360
Lys Tyr Ser Thr Gly Asn Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val 380
Ala Ala Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg Ala Thr Val Ile His Gly 400
Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Val Thr Ser Gln Asp Arg His Lys Gln Arg 420
Ile Ile Ala Pro Ala Lys Gln Leu Leu 429
Claims (9)
1. The broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease A virus is characterized by comprising a main neutralizing antigen epitope sequence of 20 th-85 th, 133 th-172 th and 193 th-212 th positions in VP1 protein of a representative strain A/GDMM/2013, A/HuBWH/2009, A/GSLX/62 of the foot-and-mouth disease A virus, wherein the amino acid sequence of the broad-spectrum multi-epitope recombinant antigen of the foot-and-mouth disease A virus is shown as SEQ ID No. 2.
2. The polynucleotide for coding the foot-and-mouth disease type A virus broad-spectrum multi-epitope recombinant antigen of claim 1, wherein the sequence of the polynucleotide is shown as SEQ ID No. 1.
3. The use of the broad-spectrum multi-epitope recombinant antigen of foot-and-mouth disease type A virus of claim 1 in the preparation of a reagent for detecting foot-and-mouth disease type A virus sIgA antibody.
4. An ELISA detection kit for foot-and-mouth disease type A virus sIgA antibody, which is characterized by comprising the foot-and-mouth disease type A 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 according to claim 1.
5. The kit for ELISA detection of sIgA antibody for foot-and-mouth disease type A virus of claim 4, wherein said foot-and-mouth disease type A virus broad-spectrum multi-epitope recombinant antigen is obtained by expression in a prokaryotic expression system.
6. The kit for ELISA detection of sIgA antibody for foot-and-mouth disease type A virus of claim 4, wherein the ELISA plate coated with the broad-spectrum multi-epitope recombinant antigen for foot-and-mouth disease type A 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 A 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/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 1000ml0.01mol/LPBS pH7.4, gently shaking to dissolve, adding 0.05% w/v Procline300, and storing at 4 ℃ for later use.
7. The ELISA detection kit for sIgA antibody against foot-and-mouth disease virus A according to claim 4, 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 A is attacked, and detecting the sample with 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 FMDV specific PCR detection of negative antigen nasal swabs of pig, cattle and sheep, mixing, diluting, and detectingAnd measuring the sample with the OD450 value of 0.1 +/-0.05, and performing sterile subpackaging for later use.
8. The kit for ELISA detection of sIgA antibody for foot-and-mouth disease type A virus according to claim 4, 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.
9. The ELISA test kit for detecting sIgA antibody against foot-and-mouth disease virus A according to claim 4, 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/V Tween20 0.1mol/LPBS solution, pH 7.6, use the dilution of 10 times.
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| CN110526959A (en) * | 2019-08-29 | 2019-12-03 | 中国农业科学院兰州兽医研究所 | A kind of foot-and-mouth disease virus antigen, the gene for expressing foot-and-mouth disease virus antigen and its recombinant vector and Recombinant Lactococcus lactis |
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| CN113267621B (en) * | 2021-05-14 | 2021-12-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN114705857B (en) * | 2022-05-16 | 2023-03-21 | 北京亿森宝生物科技有限公司 | Microporous plate type chemiluminescence detection kit for pig foot-and-mouth disease virus O-type and A-type antibodies and application thereof |
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