CN113189332A - Foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit and application thereof - Google Patents
Foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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- Biochemistry (AREA)
- Microbiology (AREA)
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Abstract
The invention relates to a foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit and application, belonging to the field of biotechnology, and the invention treats antigen by a concentration freeze-drying process of foot-and-mouth disease virus antigen; a novel enzyme-linked immunosorbent assay method is established by combining the dolphin anti-IgG-biotin and the HRP-SA, and the operation mode is improved. The problems of complex operation, multiple steps, long time, poor stability, easy error, easy inversion of pig serum samples, small production batch and the like of the foot-and-mouth disease virus liquid phase blocking ELISA antibody detection kit are solved. The kit is convenient to operate, time-reduced and mass-produced, the sensitivity and specificity of the classical method are maintained, the stability of transportation and storage is improved, the validity period of the kit is prolonged, and technical support is provided for the method to be used for foot-and-mouth disease immune antibody monitoring, vaccine immune effect evaluation, epidemiological investigation, immune guidance and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a liquid-phase blocking BSA-ELISA antibody detection kit for foot-and-mouth disease viruses and application thereof.
Background
Foot and Mouth Disease (FMD) is an acute, hot and highly contact infectious Disease which is easily caused by Foot and Mouth Disease Virus (FMDV) and is generated by artiodactyl animals such as pigs, cattle and sheep, infected animals are mainly clinically characterized by vesicular eruption on oral mucosa, hoof and breast skin, the Disease has multiple transmission ways and high speed, and each outbreak can cause huge economic loss to local livestock breeding. The world animal health Organization (OIE) lists the animal infectious diseases as the infectious diseases which need to be declared, and China also lists the animal infectious diseases as the first infectious diseases of the animals. Foot and mouth disease virus has seven serotypes O, A, Asia 1, C and south Africa 1, 2 and 3, and vaccine immunity can not ensure that all types are cross-protected. At present, China mainly carries out vaccination on O, A and Asia 1 type foot-and-mouth disease viruses.
The detection of the foot-and-mouth disease virus antibody by the liquid-phase blocking ELISA method is a reference method specified in the world animal health Organization (OIE) and international trade, and is also a recommended method for the immune effect evaluation and epidemiological investigation of the foot-and-mouth disease virus vaccine in China, and New veterinary certificates of two products, namely a foot-and-mouth disease virus O type liquid-phase blocking ELISA antibody detection kit and a foot-and-mouth disease virus Asia 1 type liquid-phase blocking ELISA antibody detection kit, are obtained by Lanzhou veterinary research institute.
The liquid phase blocking ELISA antibody detection kit for foot-and-mouth disease viruses, which is developed in the foot-and-mouth disease reference Laboratory of the world foot-and-mouth disease Laboratory (Pirbright Laboratory) and the foot-and-mouth disease reference Laboratory of the national Lanzhou veterinary research institute, has the problems of complicated test operation, long use time, difficulty in storage and the like in an operation instruction book recommended by a new veterinary medicine certificate, greatly influences the pleasure of operators, brings inconvenience to the popularization and the use of products, particularly has larger popularization resistance in enterprises lacking professional technicians, is easy to make mistakes in the operation process, and has poor storage stability of the kit due to unstable antigen storage.
How to optimize the operation of the foot-and-mouth disease virus liquid phase blocking ELISA antibody detection method and innovate the production process is the key to solve the problems. The improvement of the new operation flow and the production process of the kit should improve the convenience of the operation of the kit and the storage stability and the detection repeatability of the kit under the condition of ensuring the original sensitivity and specificity of the classical method.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit, and aims to provide application of the kit in vaccine antibody detection and preparation of products for detecting foot-and-mouth disease virus antibodies. The BSA-ELISA antibody detection kit realizes that the operation of a liquid phase blocking ELISA method is easily finished within a half working day, and solves the problems of complexity, overlong time, fatigue operation, instability, easy error and the like of the original operation.
In order to achieve the purpose, the invention adopts the specific scheme that:
a foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit comprises a 96-hole rabbit anti-coating plate, FMDV antigens, negative control serum, positive control serum, dolphin anti-biotin, HRP-SA, 25 xPBST, TMB substrate solution and stop solution;
the FMDV antigen is a serotype specific foot-and-mouth disease virus antigen, the foot-and-mouth disease virus antigen is concentrated by a PEG concentration and centrifugation technology and is tested and prepared to be uniform in antigen amount, 5% of trehalose and 0.01% of Proclin300 preservative are added, and the mixture is subpackaged and freeze-dried;
different color indicators were added to the negative control serum, positive control serum, pig anti-biotin and HRP-SA, respectively.
The foot-and-mouth disease virus antigen is obtained by inactivating foot-and-mouth disease virus or is obtained by demulsifying the foot-and-mouth disease inactivated vaccine.
The coating process of the 96-hole rabbit anti-coating plate specifically comprises the following steps: washing and sealing an integrated machine by using an ELISA plate, diluting serotype specific rabbit anti-FMDV serum by using a 0.05M carbonate buffer solution with the pH value of 9.6, coating the ELISA plate with the diluted serum, wherein each hole is 100 mu l, and standing overnight at room temperature; adding an enzyme label plate stabilizer into the mixture without washing, and incubating the mixture for 1 hour at room temperature, wherein the concentration of the enzyme label plate stabilizer is 150 mu l/hole; drying at 37 deg.C for 2 hr without washing, vacuum packaging, and storing at 4 deg.C.
The negative control serum was green, the positive control serum was red, the pig anti-biotin was orange, and the HRP-SA was blue.
The PEG concentration centrifugation technology comprises the following specific steps: adding 8% of PEG6000 and 4% of NaCL into the foot-and-mouth disease virus inactivated vaccine, stirring for 4 hours at 4 ℃, standing overnight at 4-8 ℃, centrifuging for 60 minutes in a 500ml centrifuge tube 6000rmp, removing supernatant, suspending antigen precipitate in the centrifuge tube by PBS, fixing the volume to 1/50 of the volume of the original antigen, adding cold trichloroethylene of the same body for emulsification and centrifugal degreasing, and absorbing antigen layer liquid; determining the optimal antigen concentration, diluting with PBS to 1:100 for antigen concentration according to the determined antigen concentration, adding 5% trehalose and 0.01% Proclin300 antiseptic, subpackaging 1.0 ml/tube into 1.5ml centrifuge tube, lyophilizing, collecting the antigen material in tube in loose shape, labeling, and storing at 4 deg.C.
The invention also claims the application of the BSA-ELISA antibody detection kit in the detection of the foot-and-mouth disease virus antibody, wherein the antibody refers to the antibody after the immunization of the foot-and-mouth disease virus vaccine; the detection method for detecting the antibody by adopting the kit comprises the following steps:
a. coating an ELISA plate to form a 96-hole rabbit anti-coating plate;
b. serum dilution and antigen reaction: recovering the FMDV antigen freeze-dried product by using sterile deionized water for later use, preserving the FMDV antigen freeze-dried product at 4 ℃ for 1 month, and diluting the FMDV antigen freeze-dried product to a working concentration when the FMDV antigen freeze-dried product is used temporarily; diluting 25 XPBST with deionized water to 1 time PBST for standby;
after PBST is added in advance on a U-shaped carrier plate, negative control serum, positive control serum and serum to be detected are added into corresponding holes, continuous gradient dilution of 2 times of corresponding initial times is carried out by a 60ul system according to requirements, then 60ul is added to the corresponding holes to dilute FMDV antigen to working concentration, at least 4-hole virus antigen contrast is arranged on each plate, the plates are all 120 mu l/hole, the plates are sealed, vibrated and incubated for 90 minutes at 37 ℃;
c. moving the plate: transferring the antigen-antibody mixture with sufficient effect into an ELISA plate coated with rabbit anti-foot-and-mouth disease virus antiserum in parallel at 100 ul/hole, and incubating for 30 minutes at 37 ℃;
d. dolphin anti-biotin conjugate, a guinea pig IgG anti-type specific FMDV-biotin marker: washing the ELISA plate reacted in the step c for 3-5 times by using PBST, spin-drying, adding the dolphin anti-biotin conjugate, 100 mu l/hole, sealing the plate, and incubating for 30 minutes at 37 ℃;
e. HRP-SA: washing the ELISA plate reacted in the step d with PBST for 3-5 times, spin-drying, adding an HRP-SA conjugate, 100 mu l/hole, sealing the plate, and incubating for 10 minutes at 37 ℃;
f. TMB substrate and termination reaction: washing the plate for 3-5 times, adding single-component TMB substrate solution, incubating at 37 deg.C for 10-15 min, and adding 50 μ l of 1.25M H2SO4Stopping the reaction, and reading a light absorption value under the wavelength of 450nm by using an enzyme-labeling instrument;
g. and (4) judging a result: and taking 50% of the average value of the OD values of the virus antigen control 4 wells as a critical value of 50% inhibition of the virus antigen, comparing the OD values of corresponding wells of the negative and positive control serum and the detected serum with the critical value, and obtaining the antibody titer of a certain serum, namely the average value of the antibody titers corresponding to two adjacent wells of which the OD value of the serum dilution well is slightly higher than the critical value and slightly lower than the critical value.
The invention also requests to protect the application of the kit in preparing products for detecting foot-and-mouth disease virus antibodies.
Has the advantages that:
the invention firstly breaks through the limit of the conventional enzymatic reaction principle of the liquid blocking ELISA kit at home and abroad, overcomes the defects of complicated original operation, time consumption, easy artificial test failure and the like of the liquid blocking ELISA method, maintains the inherent sensitivity and specificity of the classical method, improves the stability of the product, ensures that the foot-and-mouth disease virus liquid blocking BSA-ELISA kit has more ideal operation and production process and simpler and more convenient use, is beneficial to prolonging the service cycle of the classical method, and is beneficial to improving the product quality and the economic benefit.
According to the invention, the concentration and freeze-drying process treatment of the foot-and-mouth disease virus inactivated antigen is adopted, the antigen content is concentrated and improved, the working concentration of the antigen prepared, freeze-dried and redissolved in different batches is the same, and the stability of transportation and cold storage of the antigen is realized; a new enzyme-linked immunosorbent assay method is established by combining a technology of anti-foot-and-mouth disease guinea pig serum purified IgG labeled biotin (dolphin anti-IgG-biotin) with a technology of HRP labeled streptavidin (HRP-SA), the original technical route of preparing rabbit anti-guinea pig antiserum and labeling HRP is changed, the efficiency of enzymatic reaction is improved, the original sensitivity of the method is ensured, and the specificity of the method is improved; mixing 100ul of the detected serum and the antigen guided by the original operation of the kit in a carrier plate, acting overnight at 4 ℃, then taking 50ul of the antigen-antibody mixture, transferring the mixture to an ELISA rabbit anti-foot-and-mouth disease coated plate, incubating for 60 minutes at 37 ℃, optimizing to obtain a mixture of the detected serum and the antigen, incubating for 90 minutes at 37 ℃, then taking 100ul of the antigen-antibody mixture, transferring the mixture to the ELISA rabbit anti-foot-and-mouth disease coated plate, and incubating for 30 minutes at 37 ℃; after washing the plate, adding a dolphin anti-IgG-biotin working solution, and incubating for 30 minutes at 37 ℃; adding HRP-SA working solution after washing the plate, and incubating for 10 minutes at 37 ℃; after washing the plate, the single-component TMB substrate solution was added and incubated at 37 ℃ for 10-15 minutes, then stop solution was added to stop the reaction, and the absorbance was read at OD450 nm. The method can easily finish the operation of the liquid phase blocking ELISA method within half a working day, and solves the problems of complexity, overlong time consumption, operation fatigue, instability, easy error and the like of the original operation.
The method has the advantages that the biotin labeled antibody reacts with HRP-SA rapidly, the labeled raw materials are stable, the detection background value is high, and the like, so that the operation time of the liquid blocking ELISA kit is shortened to 3 hours, namely, the operation can be completed within a half working day, the inversion phenomenon which is easy to occur in the detection of pig serum is avoided, and the misjudgment of the antibody titer of the test result is avoided. The use of the ELISA plate washing and sealing integrated machine realizes the batch production of the rabbit anti-coating ELISA plate, and solves the problems that the manual coating can only be produced in small batch and the difference in batches is overlarge due to the manual coating. The green and red indicators are respectively added after the negative and positive control serum is prepared, the orange indicator and the blue indicator are added after the dolphin anti-IgG-biotin working solution is prepared and the enzyme-labeled antibody working solution is prepared, so that the visual appearance of the reagent components can be distinguished. The problem that the OPD is easy to lose efficacy when only being prepared at present is solved by using a TMB single-component substrate to replace an adjacent phenylenediamine (OPD) substrate, the storage stability of the substrate is realized, and the color development sensitivity is improved.
The invention creatively solves the problems of complex operation, multiple steps, long time consumption, poor stability, easy error, easy inversion of pig serum samples, small production batch and the like of the foot-and-mouth disease virus liquid phase blocking ELISA antibody detection kit. A novel enzyme-linked immunosorbent assay method (BSA-ELISA) established based on the combination of a biotin labeled antibody technology and HRP-SA ensures that the operation of the kit is convenient, the time is shortened, the batch production is realized, the sensitivity and the specificity of the classical method are maintained, the stability of transportation and storage is improved, the validity period (12 months) of the kit is prolonged, the service cycle of the classical method is prolonged, the product quality is improved, the economic benefit is improved, the workload of operators of the kit is reduced, the pleasure of users is increased, and the popularization and the application of the BSA-ELISA kit are facilitated to be expanded. The method is used for continuously providing technical support for foot-and-mouth disease immune antibody monitoring, vaccine immune effect evaluation, epidemiological investigation, immune guidance and the like.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1 foot-and-mouth disease Virus O-type liquid blocking BSA-ELISA kit development and application
1 materials and methods
1.1 preparation of foot-and-mouth disease Virus antigen, antiserum
1.1.1 concentration and lyophilization of FMDV antigen required by the kit: adding 8% PEG6000 and 4% NaCL into an inactivated vaccine (O/Mya98 strain) of foot-and-mouth disease virus with a proper volume, stirring for 4 hours at 4 ℃, standing overnight at 4-8 ℃, centrifuging for 60 minutes in a 500ml centrifuge tube 6000rmp, discarding supernatant, continuously adding non-centrifuged antigen for centrifugation, repeating the above operations when the antigen amount is large, finally suspending the antigen precipitate in the centrifuge tube by 0.01mol PBS with pH7.6, fixing the volume to 1/50 of the original antigen volume, adding trichloroethylene accumulated at the same side for emulsification and centrifugal degreasing, absorbing antigen layer liquid, determining the optimal use concentration (more than 100) of the antigen by a liquid phase blocking ELISA kit, diluting the antigen concentration to 1:100 for dilution by 0.01mol PBS according to the determined antigen concentration and adding 5% trehalose and 0.01% Proclin300 preservative, subpackaging the mixture into a 1.0ml centrifuge tube with the specification, freeze-drying the antigen material in the tube, wherein the antigen material is in a shape of a mass loose shape, labeling, and storing at 4 ℃.
1.1.2 immunization was prepared with FMDV 146S antigen: taking 100ml of antigen solution of inactivated foot-and-mouth disease (O/Mya98 strain), carrying out ultracentrifugation at 4 ℃ for 1h at 45000rmp, re-suspending the sediment at the bottom wall of a centrifugal tube to 2ml by using PBS with the pH value of 7.6, centrifuging for 2.5 hours at 35000rpm by using sucrose density gradient (45%, 35%, 25% and 15%), sampling according to 0.5ml, merging the same-level peaks, detecting by using a 260nm ultraviolet spectrophotometer, and collecting and merging the absorption peaks (second peaks) of 146S antigen of complete virus particles containing foot-and-mouth disease.
1.1.3O type rabbit anti-foot-and-mouth disease virus serum: selecting healthy rabbits of about 2.0kg, emulsifying the purified 146S antigen with equivalent Freund' S complete adjuvant, immunizing rabbits, bleeding in the carotid artery on day 30, separating serum, packaging, labeling, and freezing at-70 deg.C.
1.1.4O type guinea pig anti-foot-and-mouth disease virus serum: immunizing guinea pigs with FMDV purified 146S antigen homologous to rabbit antiserum, collecting blood from heart on day 30, separating serum, packaging, labeling, and freezing at-70 deg.C.
1.1.5O type guinea pig anti-foot-and-mouth disease virus serum IgG purification and biotinylation labeling: subjecting O-type guinea pig anti-foot-and-mouth disease virus serum prepared by immunization to affinity chromatography purification to obtain guinea pig serum IgG, diluting the guinea pig anti-IgG to 1mg/mL with 0.1mol/L sodium bicarbonate buffer solution (pH 8.0), labeling 1-2.5mL each time, interacting in a dialysis bag, adding the guinea pig anti-IgG to be biotinylated into the dialysis bag (MW CO 8000), and placing in 0.1mol/L sodium bicarbonate buffer solution (pH 8.0) for full dialysis; 120ul of a 1mg/mL solution of NHSB (N-hydroxysuccinimide biotin) per mg of protein (i.e., 1mg NHSB dissolved in 1mL LDMSO) was added and stirred at room temperature for 2-4 hours, 9.6. mu.L of 1mol/L NH4Cl (1. mu.L per 25. mu.g NHSB) was added and stirred at room temperature for 10 minutes. Removing free biotin with 0.01mol/L buffer system with pH of 7.2, sucking up the labeled substance in the bag, adding 50% glycerol and 0.01% Proclin300, subpackaging, and freezing at-70 deg.C.
1.1.6HRP-SA horseradish peroxidase labeled streptavidin, a commercial reagent, purchased.
1.1.7 type O foot-and-mouth disease antibody positive serum: hyperimmune serum prepared by immunizing cattle with inactivated vaccine of foot and mouth disease homologous to rabbit antiserum is first immunized and then boosted once at 30 days, and after second immunization, blood is collected 2 weeks and serum is separated, subpackaged, labeled and frozen at-70 ℃.
1.1.8 foot-and-mouth disease antibody negative sera: to purchase newborn bovine serum that is negative for foot-and-mouth disease antibodies.
1.2 methods
2.1 type O foot-and-mouth disease antibody positive serum titer determination
And (3) measuring by using a liquid phase blocking ELISA detection kit (Lanzhou veterinary research institute) for the foot-and-mouth disease O-type antibody.
2.2 determination of working concentration of antiserum
The rabbit anti-foot-and-mouth disease virus serum and the HRP-guinea pig anti-foot-and-mouth disease virus serum IgG are used for sequentially determining the working concentration of each reagent by taking the titer of the positive serum antibody as a reference.
2.3 working concentration after reconstitution of lyophilized antigen (1:100)
After the freeze-dried antigen is recovered, the working concentration is 1:100, the test is carried out according to the liquid phase blocking ELISA operation, and O type positive serum control, A type and Asia 1 type hyperimmune serum control are set.
2.4 operating step optimization
2.4.1 serum dilution and antigen reaction
Adding 1-fold PBST with the pH value of 7.4 into a carrier reaction plate at the bottom of a U-shaped hole according to the amount of 60 mu l per hole, carrying out 2-fold continuous dilution on negative and positive control serum and detected serum according to requirements, then adding 60 mu l of O-type foot-and-mouth disease virus antigen diluted to the working concentration (1:100) into all the holes, arranging at least 4-hole virus antigen control (adding 60 mu l of PBST in advance) into each reaction plate, finally, ensuring that the liquid amount in an ELISA reaction plate is 120 mu l per hole, sealing the plate and sealing the membrane, vibrating, and incubating for 90 minutes at 37 ℃.
2.4.2 moving plate
100ul of the antigen-antibody mixture after sufficient action is taken and transferred to an ELISA rabbit anti-foot-and-mouth disease coating plate, and the mixture is incubated for 30 minutes at 37 ℃.
2.4.3 addition of Dolphin anti-IgG-Biotin
Washing the ELISA plate obtained by the reaction for 3-5 times by using PBST, drying, adding a working solution of a dolphin anti-IgG-biotin antibody, 100 mu l/hole, sealing the plate, and incubating for 30 minutes at 37 ℃.
2.4.4 enzyme-labeled streptavidin (HRP-SA)
Washing the ELISA plate obtained by the reaction for 3-5 times by using PBST, drying, adding enzyme-labeled streptavidin (HRP-SA) working solution, incubating for 10 minutes at 37 ℃ with 100 mu l/hole and a sealing plate.
2.4.5TMB substrate and enzyme-labeled antibody for reaction termination (HRP-guinea pig anti-foot-and-mouth disease virus serum IgG)
Washing the plate for 3-5 times, adding single-component TMB substrate solution, incubating at 37 deg.C for 10-15 min, adding 1.25% H50 μ l per well2SO4And (5) stopping the reaction, and reading a light absorption value under the wavelength of 450nm by using an enzyme-labeling instrument.
2.4.6 determination of results
And taking 50% of the average value of the OD values of the virus antigen control 4 wells as a critical value of 50% of the blocked virus antigen, comparing the OD values of corresponding wells of the negative control serum and the positive control serum and the detected serum with the critical value, and obtaining the antibody titer of a certain serum, namely the average value of the antibody titers corresponding to the OD value of the serum dilution well slightly higher than the critical value and slightly lower than the critical value (two adjacent wells).
2.5 compliance (sensitivity and specificity) with original operating methods of liquid blocking ELISA kits
The vaccine for the foot-and-mouth disease of the type O is used for immunizing 60 parts of animal serum (20 parts of cattle, 20 parts of sheep and 20 parts of pigs), 1 part of Asia 1 type foot-and-mouth disease inactivated vaccine immune bovine serum, 1 part of A type foot-and-mouth disease inactivated vaccine immune bovine serum and 30 parts of foot-and-mouth disease antibody negative animals (15 parts of cattle and 15 parts of pigs). And carrying out parallel detection with the original operation method of the liquid phase blocking ELISA kit.
2.6 repeatability
And repeating the measurement of 30 parts of the foot-and-mouth disease O type inactivated vaccine immune animal serum by using an inhibition ELISA method for 3 times.
3 results
3.1 type O foot-and-mouth disease Positive serum antibody titer determination results
The O-type foot-and-mouth disease positive serum is determined to be 720 (512-1024) by a liquid phase blocking ELISA method, and the antibody titer of the O-type foot-and-mouth disease positive serum used as a positive control serum for inhibiting ELISA is controlled to be 720 (512-1024) as well.
3.2 measurement of antiserum working concentration
The working concentration of the O-type rabbit antiserum, determined by a liquid blocking ELISA method, was 1:1000, working concentration of biotinylated O-type guinea pig antiserum IgG 1:1000, the working concentration of HRP-SA is 1: 1000; coating an ELISA plate with O-type rabbit antiserum at a working concentration, and storing at 4 ℃; diluting O-type guinea pig antiserum IgG labeled by biotin with working concentration of 1:1000 to prepare working solution, and storing at 4 ℃; HRP-SA is diluted to prepare working solution with working concentration of 1:10000 and stored at 4 ℃.
3.3 measurement of working concentration (1:100) after reconstitution of lyophilized antigen
The working concentration of the recovered freeze-dried antigen is 1:100, and the freeze-dried antigen and the original operation of liquid phase blocking ELISA are subjected to parallel comparison test, and O type positive serum control, A type and Asia 1 type hyperimmune serum control are set. The positive control antibody titer is consistent with the known value, the A type and Asia 1 type hyperimmune serum antibody titer is not more than 64, and the OD value of the 4-hole virus antigen is in the range of 1.0-2.0.
3.4 optimization results of the operating Steps
The operation and production process of the kit are optimized on the principle of keeping the sensitivity and specificity of a liquid phase blocking classical method consistent, the optimized operation of the kit is that 120ul of mixed system of detected serum and antigen is incubated for 90 minutes at 37 ℃ in a carrier plate, then 100ul of antigen-antibody mixture is taken and is transferred to an ELISA rabbit anti-foot-and-mouth disease coating plate for further incubation for 30 minutes at 37 ℃, and after the plate is washed, a dolphin anti-IgG-biotin working solution is added and is incubated for 30 minutes at 37 ℃; adding HRP-SA working solution after washing the plate, and incubating for 10 minutes at 37 ℃; after washing the plate, the single-component TMB substrate solution was added and incubated at 37 ℃ for 10-15 minutes, then stop solution was added to stop the reaction, and the absorbance was read at OD450 nm. The test serum titer was calculated by using 50% of the mean value of the OD values of the virus antigen control 4 wells as the cut-off value at which the virus antigen was blocked by 50%.
3.5 results in accordance with the original protocol of the liquid blocking ELISA kit (sensitivity and specificity)
The O-type liquid phase blocking ELISA method antibody detection kit for the foot-and-mouth disease produced by Lanzhou veterinary research institute has the advantages that the bovine and sheep serum antibody titer is more than or equal to 128 and is judged as the O-type foot-and-mouth disease antibody positive, and the pig serum antibody titer is more than or equal to 64 and is judged as the O-type foot-and-mouth disease antibody positive; the optimized liquid blocking ELISA method still keeps the bovine and ovine serum antibody titer more than or equal to 128 and judges as O-type foot-and-mouth disease antibody positive, and the porcine serum antibody titer more than or equal to 64 and judges as O-type foot-and-mouth disease antibody positive. And (3) determining the serum antibody titer of 30 parts of foot-and-mouth disease antibody negative animals and 60 parts of O-type foot-and-mouth disease inactivated vaccine immunized animals: the difference of the antibody titer measured by the liquid blocking BSA-ELISA method and the original operation method of the liquid blocking ELISA kit is within 1 titer, and the sensitivity coincidence rate is 100%; 1 part of A type inactivated vaccine immune animal (the A type antibody titer is more than 1024) and 1 part of Asia 1 type inactivated vaccine immune animal (the Asia 1 type antibody titer is more than 1024), the optimized O type antibody titer detected by the liquid phase blocking ELISA method is less than 64, and the detection result is consistent with the original operation titer detection result of the liquid phase blocking ELISA kit.
3.6 repeatability
30 parts of O-type foot-and-mouth disease inactivated vaccine is repeatedly detected for 3 times by an inhibition ELISA method, the antibody titer is completely consistent with 24 parts, the difference is 4 parts within one titer, and the difference is more than 1 titer and is 2 parts, which indicates that the method has good repeatability.
4 conclusion
4.1 the new detection method of the liquid blocking BSA-ELISA kit established by the invention keeps consistent with the original operation sensitivity and specificity of the liquid blocking ELISA.
4.2 the new method of the liquid phase blocking BSA-ELISA kit is to establish a new enzyme-linked immunosorbent assay method by combining the technology of anti-foot-and-mouth disease guinea pig serum purification IgG biotinylation labeling (pig anti-IgG-biotin) and the technology of HRP labeling streptavidin (HRP-SA), change the original technical route of re-labeling the HRP required for preparing rabbit anti-guinea pig antiserum, improve the efficiency of enzymatic reaction, omit the reaction step of HRP-rabbit anti-guinea pig serum in the re-reaction of the HRP-rabbit anti-guinea pig antiserum after the anti-foot-and-mouth disease virus guinea pig antiserum reaction in the original operation, solve the inversion phenomenon easily occurring in the detection of pig serum titer, avoid the misjudgment phenomenon of titer, complete the new operation of the kit within 3 hours, react with the antigen and antibody of the original operation method of the liquid phase blocking ELISA kit overnight, complete the whole operation within 2 days, time is greatly saved.
4.3 compared with the liquid blocking ELISA kit, the liquid blocking BSA-ELISA method has the advantages that the antigen preparation is provided in a freeze-dried product form, the working concentration is uniform (1:100), the transportation and the stability of the antigen refrigeration storage are facilitated, the product quality is improved, and the operation errors are reduced.
4.4 compared with the original liquid blocking ELISA kit, the new method of liquid blocking BSA-ELISA kit has the advantages that the addition of the negative and positive control serum, the dolphin anti-biotin and the HRP-SA through the color indicator enables the components of the kit to be distinguished conveniently in the eye view, and the occurrence of misoperation is reduced.
4.5 the research provides reliable technological innovation technical support for the optimization and the updating of the foot-and-mouth disease virus O-type liquid blocking ELISA antibody detection kit.
5. Foot-and-mouth disease virus O-type liquid phase blocking BSA-ELISA antibody detection kit specification
5.1 kit name: foot-and-mouth disease virus O-type liquid phase blocking BSA-ELISA antibody detection kit
5.2 uses: is used for foot-and-mouth disease O-type antibodies in foot-and-mouth disease virus susceptible animals such as pigs, cattle, sheep, and the like.
5.2 specification: 5 x 96 wells/box, 50-100 samples can be detected; the kit can detect 100-200 samples by 10 multiplied by 96 holes per box.
5.3, preservation: 2-8 ℃, validity period: and 12 months.
5.2 kit Components
5.3 operating procedure
5.3.1 preparation before experiment
Diluting 25 × washing solution (PBST) to 1 × washing solution with deionized water or ultrapure water, and washing the serum and the reaction plate; redissolving the FMDV O-type antigen freeze-dried product to 1mL by using sterile deionized water for later use, and using the FMDV O-type antigen freeze-dried product within 1 month at the temperature of 4 ℃ until the FMDV O-type antigen freeze-dried product is used up, wherein the use concentration is 1: 100; the kit was allowed to come to room temperature for more than 30 minutes before the assay was started to bring the components back to room temperature.
5.3.2 according to the sample layout (taking 10 samples as an example), on a 96-well U-shaped plate, adding the serum to be detected in A1 and A2.. A10 wells, 30 μ l of serum and 30 μ l of PBST in each well, adding 60 μ l of PBST in other sample dilution wells, carrying out 2-fold serial dilution, wherein the initial ratio is 1: 4, 60 μ l of virus antigen is added in each well, and the initial dilution of the serum after the antigen is added is 1: 8; adding 60 mu l of positive control serum into A11 hole, diluting 2 times to H11 in series, starting at 1:8, adding 60 mu l of virus antigen into each hole, and the initial dilution of the serum after adding the antigen is 1: 16; adding 60 mu l of negative control serum into A12 hole, diluting 2 times to B12 with the initial ratio of 1: 2, adding 60 mu l of virus antigen into each hole, and the initial dilution of the serum after adding the antigen is 1: 4; e12, F12, G12 and H12 were added 60. mu.l of viral antigen per well (60. mu.l of PBST was added in advance) as 4 viral antigen control wells. The reaction plate was gently shaken, sealed with a sealing plate membrane, and incubated at 3 ℃ for 90 minutes.
5.3.3 continuously washing the coated ELISA plate for 3-5 times by using a washing solution, and patting the coated ELISA plate on absorbent paper to be dry. Transferring the serum/virus antigen mixed solution and the virus antigen contrast solution on the serum/virus antigen reaction plate into corresponding holes on an enzyme label plate, sealing the plate by a sealing plate membrane at 100 mu l/hole, and incubating for 30 minutes at 37 ℃.
And 5.3.4 continuously washing the enzyme label plate for 3-5 times by using a washing solution, and patting the enzyme label plate on absorbent paper to be dry. Add 100. mu.l/well of Dolphin anti-biotin working solution, seal the plate with sealing plate membrane, incubate for 30 minutes at 37 ℃.
5.3.5 the ELISA plate is continuously washed for 3-5 times by using a washing solution, and the plate is patted dry on absorbent paper. 100 μ l/well of HRP-SA working solution was added, the plate was sealed with a sealing plate, and incubated at 37 ℃ for 10 minutes.
5.3.6 washing the plate for 3-5 times, drying on absorbent paper, adding TMB substrate solution 50 μ l per well, and incubating at 37 deg.C for 10-15 min.
5.3.7 stop the reaction by adding 50. mu.l of stop solution to each well.
5.3.8 OD450nm values were read on a microplate reader.
5.4 determination of results
5.4.1 test establishment conditions
The OD492nm value of at least 2 holes of the foot-and-mouth disease O-type virus antigen control is in the range of 1.0-2.0; the titer of the foot-and-mouth disease O type negative control serum antibody is less than 1: 8; the titer of the foot-and-mouth disease O type positive control serum antibody is 1: 512-1: 2048.
5.4.2 serum antibody titers
Antibody titers were expressed as 50% endpoint dilutions, with 50% of the mean OD450nm value of the antigen control wells being the cut-off value. The wells with the OD450nm value greater than the critical value are negative wells, and the wells with the OD450nm value less than or equal to the critical value are positive wells. The highest dilution of the positive hole of the serum to be detected is the antibody titer of the serum, and the final dilution of the serum is calculated by using a Karber method (shown in the following table).
Karber method formula: ㏒ X-N-D (S-0.5) X represents the serum antibody titer; n-the log of the highest dilution for all positive wells (calculated from the last dilution at which a negative well appeared); d is a logarithmic value of the dilution factor; s is the sum of the ratio of positive wells at each dilution (calculated from the last dilution at which a negative well appeared).
5.4.3 determination
The titer of the bovine and sheep serum antibodies is more than or equal to 1: 128, and the bovine and sheep serum antibodies are judged to be positive for the foot-and-mouth disease O-type antibody; the pig serum antibody titer is more than or equal to 1: 64, and the pig serum antibody is judged to be positive for the foot-and-mouth disease O-type antibody.
Example 2 foot-and-mouth disease Virus A-type liquid blocking BSA-ELISA kit development and application
1 materials and methods
1.1 preparation of foot-and-mouth disease Virus antigen, antiserum
1.1.1 concentration and lyophilization of FMDV antigen required by the kit: adding a proper volume of foot-and-mouth disease virus inactivated vaccine (AF72 strain) into 8% PEG6000 and 4% NaCL, stirring at 4 ℃ for 4 hours, standing at 4-8 ℃ overnight, centrifuging for 60 minutes in a 500ml centrifuge tube 6000rmp, discarding supernatant, continuously adding non-centrifuged antigen for centrifugation, repeating the above operations when the antigen amount is large, finally suspending the antigen precipitate in the centrifuge tube by using 0.01mol PBS with pH7.6, fixing the volume to 1/50 of the original antigen volume, adding cold trichloroethylene for emulsification and centrifugal degreasing, absorbing antigen layer liquid, determining the optimal use concentration (more than 100) of the antigen by using a liquid phase blocking ELISA kit, diluting the antigen concentration to 1:100 diluted use by using 0.01mol PBS with pH7.6 according to the determined antigen concentration, adding 5% trehalose and 0.01% Proclin300 preservative, subpackaging 1.5ml centrifuge tubes with 1.0 ml/tube, freeze-drying, and the antigen substance in the tubes is in a loose shape, labeling, and storing at 4 ℃.
1.1.2 immunization was prepared with FMDV 146S antigen: taking 100ml of antigen solution of inactivated foot-and-mouth disease (AF72 strain), carrying out ultracentrifugation at 45000rmp for 1h at 4 ℃, re-suspending the sediment at the bottom wall of a centrifugal tube to 2ml by using PBS with the pH value of 7.6, centrifuging for 2.5 h by using a sucrose density gradient (45%, 35%, 25% and 15%) 35000rpm, sampling according to 0.5ml, merging the same-level peaks, detecting by using a 260nm ultraviolet spectrophotometer, and collecting and merging the absorption peaks (second peaks) of the 146S antigen of the complete virus particles containing the foot-and-mouth disease.
1.1.3 type a rabbit anti-foot-and-mouth disease virus serum: selecting healthy rabbits of about 2.0kg, emulsifying the purified 146S antigen with equivalent Freund' S complete adjuvant, immunizing rabbits, bleeding in the carotid artery on day 30, separating serum, packaging, labeling, and freezing at-70 deg.C.
1.1.4 type a guinea pig anti-foot-and-mouth disease virus serum: immunizing guinea pigs with FMDV purified 146S antigen homologous to rabbit antiserum, collecting blood from heart on day 30, separating serum, packaging, labeling, and freezing at-70 deg.C.
1.1.5 type A guinea pig anti-foot-and-mouth disease virus serum IgG purification and biotinylation labeling: a type A guinea pig anti-foot-and-mouth disease virus serum prepared by immunization is purified by affinity chromatography to obtain guinea pig serum IgG, the guinea pig anti-IgG is diluted to 1mg/mL by 0.1mol/L sodium bicarbonate buffer solution (pH 8.0), the labeling amount is 1-2.5mL each time, the interaction is carried out in a dialysis bag, the guinea pig anti-IgG to be biotinylated is added into the dialysis bag (MW CO 8000) and placed in 0.1mol/L sodium bicarbonate buffer solution (pH 8.0) for full dialysis; 120ul of a 1mg/mL solution of NHSB (N-hydroxysuccinimide biotin) per mg of protein (i.e., 1mg NHSB dissolved in 1mL LDMSO) was added and stirred at room temperature for 2-4 hours, 9.6. mu.L of 1mol/L NH4Cl (1. mu.L per 25. mu.g NHSB) was added and stirred at room temperature for 10 minutes. Removing free biotin with 0.01mol/L buffer system with pH of 7.2, sucking up the labeled substance in the bag, adding 50% glycerol and 0.01% Proclin300, subpackaging, and freezing at-70 deg.C.
1.1.6HRP-SA horseradish peroxidase labeled streptavidin, a commercial reagent, purchased.
1.1.7 type a foot-and-mouth disease antibody positive sera: hyperimmune serum prepared by immunizing cattle with inactivated vaccine of foot and mouth disease homologous to rabbit antiserum is first immunized and then boosted once at 30 days, and after second immunization, blood is collected 2 weeks and serum is separated, subpackaged, labeled and frozen at-70 ℃.
1.1.8 foot-and-mouth disease antibody negative sera: to purchase newborn bovine serum that is negative for foot-and-mouth disease antibodies.
1.2 methods
2.1 type A foot-and-mouth disease antibody positive serum titer determination
And (3) measuring by using a liquid phase blocking ELISA detection kit (Lanzhou veterinary research institute) for the foot-and-mouth disease type A antibody.
2.2 determination of working concentration of antiserum
The rabbit anti-foot-and-mouth disease virus serum and the HRP-guinea pig anti-foot-and-mouth disease virus serum IgG are used for sequentially determining the working concentration of each reagent by taking the titer of the positive serum antibody as a reference.
2.3 working concentration after reconstitution of lyophilized antigen (1:100)
After the freeze-dried antigen is recovered, the working concentration is 1:100, the test is carried out according to the liquid phase blocking ELISA operation, and A type positive serum control, O type and Asia 1 type hyperimmune serum control are set.
2.4 operating step optimization
2.4.1 serum dilution and antigen reaction
Adding 1-fold PBST with the pH value of 7.4 into a carrier reaction plate at the bottom of a U-shaped hole according to the amount of 60 mu l per hole, carrying out 2-fold continuous dilution on negative and positive control serum and detected serum according to requirements, then adding 60 mu l of A-type foot-and-mouth disease virus antigen diluted to the working concentration (1:100) into all the holes, arranging at least 4-hole virus antigen control (adding 60 mu l of PBST in advance) into each reaction plate, finally, ensuring that the liquid amount in an ELISA reaction plate is 120 mu l per hole, sealing the plate and sealing the membrane, vibrating, and incubating for 90 minutes at 37 ℃.
2.4.2 moving plate
100ul of the antigen-antibody mixture after sufficient action is taken and transferred to an ELISA rabbit anti-foot-and-mouth disease coating plate, and the mixture is incubated for 30 minutes at 37 ℃.
2.4.3 addition of Dolphin anti-IgG-Biotin
Washing the ELISA plate obtained by the reaction for 3-5 times by using PBST, drying, adding a working solution of a dolphin anti-IgG-biotin antibody, 100 mu l/hole, sealing the plate, and incubating for 30 minutes at 37 ℃.
2.4.4 enzyme-labeled streptavidin (HRP-SA)
Washing the ELISA plate obtained by the reaction for 3-5 times by using PBST, drying, adding enzyme-labeled streptavidin (HRP-SA) working solution, incubating for 10 minutes at 37 ℃ with 100 mu l/hole and a sealing plate.
2.4.5TMB substrate and enzyme-labeled antibody for reaction termination (HRP-guinea pig anti-foot-and-mouth disease virus serum IgG)
Washing the plate for 3-5 times, adding single-component TMB substrate solution, incubating at 37 deg.C for 10-15 min, adding 1.25% H50 μ l per well2SO4And (5) stopping the reaction, and reading a light absorption value under the wavelength of 450nm by using an enzyme-labeling instrument.
2.4.6 determination of results
And taking 50% of the average value of the OD values of the virus antigen control 4 wells as a critical value of 50% of the blocked virus antigen, comparing the OD values of corresponding wells of the negative control serum and the positive control serum and the detected serum with the critical value, and obtaining the antibody titer of a certain serum, namely the average value of the antibody titers corresponding to the OD value of the serum dilution well slightly higher than the critical value and slightly lower than the critical value (two adjacent wells).
2.5 compliance (sensitivity and specificity) with original operating methods of liquid blocking ELISA kits
The vaccine for foot-and-mouth disease type A is prepared from (by weight parts) A type foot-and-mouth disease inactivated vaccine 60 parts of animal serum (20 parts of cattle, 20 parts of sheep and 20 parts of pig), Asia 1 type foot-and-mouth disease inactivated vaccine 1 part of immune bovine serum, O type foot-and-mouth disease inactivated vaccine 1 part of immune bovine serum, and foot-and-mouth disease antibody negative animal 30 parts (15 parts of cattle and 15 parts of pig). And carrying out parallel detection with the original operation method of the liquid phase blocking ELISA kit.
2.6 repeatability
30 parts of the aftosa A type inactivated vaccine immune animal serum is repeatedly measured for 3 times by using an inhibition ELISA method.
3 results
3.1 type A foot-and-mouth disease Positive serum antibody titer determination results
The type A foot-and-mouth disease positive serum is determined to be 720 (512-1024) by a liquid phase blocking ELISA method, and the antibody titer of the type A foot-and-mouth disease positive serum used as a positive control serum for inhibiting ELISA is controlled to be 720 (512-1024) as well.
3.2 measurement of antiserum working concentration
The working concentration of the A-type rabbit antiserum is 1:1000, working concentrations of biotinylated-labeled type a guinea pig antiserum IgG were 1:1000, the working concentration of HRP-SA is 1: 1000; coating an ELISA plate with the A-type rabbit antiserum at a working concentration, and storing at 4 ℃; diluting A type guinea pig antiserum IgG labeled by biotin with working concentration of 1:1000 to prepare working solution, and storing at 4 ℃; HRP-SA is diluted to prepare working solution with working concentration of 1:10000 and stored at 4 ℃.
3.3 measurement of working concentration (1:100) after reconstitution of lyophilized antigen
The working concentration of the recovered freeze-dried antigen is 1:100, and a parallel comparison test is carried out with the original operation of liquid phase blocking ELISA, and A type positive serum control, A type and Asia type 1 hyperimmune serum control are set. The positive control antibody titer is consistent with the known value, the A type and Asia 1 type hyperimmune serum antibody titer is not more than 64, and the OD value of the 4-hole virus antigen is in the range of 1.0-2.0.
3.4 optimization results of the operating Steps
The operation and production process of the kit are optimized on the principle of keeping the sensitivity and specificity of a liquid phase blocking classical method consistent, the optimized operation of the kit is that 120ul of mixed system of detected serum and antigen is incubated for 90 minutes at 37 ℃ in a carrier plate, then 100ul of antigen-antibody mixture is taken and is transferred to an ELISA rabbit anti-foot-and-mouth disease coating plate for further incubation for 30 minutes at 37 ℃, and after the plate is washed, a dolphin anti-IgG-biotin working solution is added and is incubated for 30 minutes at 37 ℃; adding HRP-SA working solution after washing the plate, and incubating for 10 minutes at 37 ℃; after washing the plate, the single-component TMB substrate solution was added and incubated at 37 ℃ for 10-15 minutes, then stop solution was added to stop the reaction, and the absorbance was read at OD450 nm. The test serum titer was calculated by using 50% of the mean value of the OD values of the virus antigen control 4 wells as the cut-off value at which the virus antigen was blocked by 50%.
3.5 results in accordance with the original protocol of the liquid blocking ELISA kit (sensitivity and specificity)
A type foot-and-mouth disease A liquid blocking ELISA method antibody detection kit produced by Lanzhou veterinary research institute, wherein the A type foot-and-mouth disease antibody is judged to be positive by the bovine and sheep serum antibody titer being more than or equal to 128, and the A type foot-and-mouth disease antibody is judged to be positive by the pig serum antibody titer being more than or equal to 64; the optimized liquid blocking ELISA method still keeps the bovine and ovine serum antibody titer more than or equal to 128 and the A type foot-and-mouth disease antibody positive, and the porcine serum antibody titer more than or equal to 64 and the A type foot-and-mouth disease antibody positive. And (3) determining the serum antibody titer of 30 parts of foot-and-mouth disease antibody negative animals and 60 parts of A-type foot-and-mouth disease inactivated vaccine immunized animals: the difference of the antibody titer measured by the liquid blocking BSA-ELISA method and the original operation method of the liquid blocking ELISA kit is within 1 titer, and the sensitivity coincidence rate is 100%; 1 part of O type inactivated vaccine immune animal (the O type antibody titer is more than 1024) and 1 part of Asia 1 type inactivated vaccine immune animal (the Asia 1 type antibody titer is more than 1024), the optimized liquid phase blocking ELISA method for detecting the A type antibody titer is less than 64, and the detection result is consistent with the original operation titer detection result of the liquid phase blocking ELISA kit.
3.6 repeatability
30 parts of A-type foot-and-mouth disease inactivated vaccine are repeatedly detected for 3 times by an inhibition ELISA method, the antibody titer is completely consistent with 24 parts, the difference is 4 parts within one titer, and the difference is more than 1 titer and is 2 parts, which indicates that the method has good repeatability.
4 conclusion
4.1 the new detection method of the liquid blocking BSA-ELISA kit established by the invention keeps consistent with the original operation sensitivity and specificity of the liquid blocking ELISA.
4.2 the new method of the liquid phase blocking BSA-ELISA kit is to establish a new enzyme-linked immunosorbent assay method by combining the technology of anti-foot-and-mouth disease guinea pig serum purification IgG biotinylation labeling (pig anti-IgG-biotin) and the technology of HRP labeling streptavidin (HRP-SA), change the original technical route of re-labeling the HRP required for preparing rabbit anti-guinea pig antiserum, improve the efficiency of enzymatic reaction, omit the reaction step of HRP-rabbit anti-guinea pig serum in the re-reaction of the HRP-rabbit anti-guinea pig antiserum after the anti-foot-and-mouth disease virus guinea pig antiserum reaction in the original operation, solve the inversion phenomenon easily occurring in the detection of pig serum titer, avoid the misjudgment phenomenon of titer, complete the new operation of the kit within 3 hours, react with the antigen and antibody of the original operation method of the liquid phase blocking ELISA kit overnight, complete the whole operation within 2 days, time is greatly saved.
4.3 compared with the liquid blocking ELISA kit, the liquid blocking BSA-ELISA method has the advantages that the antigen preparation is provided in a freeze-dried product form, the working concentration is uniform (1:100), the transportation and the stability of the antigen refrigeration storage are facilitated, the product quality is improved, and the operation errors are reduced.
4.4 compared with the original liquid blocking ELISA kit, the new method of liquid blocking BSA-ELISA kit has the advantages that the addition of the negative and positive control serum, the dolphin anti-biotin and the HRP-SA through the color indicator enables the components of the kit to be distinguished conveniently in the eye view, and the occurrence of misoperation is reduced.
4.5 the research provides reliable technological innovation technical support for the optimization and the updating of the foot-and-mouth disease virus A-type liquid blocking ELISA antibody detection kit.
5. Foot-and-mouth disease virus A type liquid phase blocking BSA-ELISA antibody detection kit specification
5.1 kit name: foot-and-mouth disease virus A-type liquid phase blocking BSA-ELISA antibody detection kit
5.2 uses: is used for foot-and-mouth disease O-type antibodies in foot-and-mouth disease virus susceptible animals such as pigs, cattle, sheep, and the like.
5.2 specification: 5 x 96 wells/box, 50-100 samples can be detected; the kit can detect 100-200 samples by 10 multiplied by 96 holes per box.
5.3, preservation: 2-8 ℃, validity period: and 12 months.
5.2 kit Components
5.3 operating procedure
5.3.1 preparation before experiment
Diluting 25 × washing solution (PBST) to 1 × washing solution with deionized water or ultrapure water, and washing the serum and the reaction plate; re-dissolving FMDV A type antigen freeze-dried product to 1mL by using sterile deionized water for later use, and preserving at 4 ℃ for 1 month until the FMDV A type antigen freeze-dried product is used up, wherein the use concentration is 1: 100; the kit was allowed to come to room temperature for more than 30 minutes before the assay was started to bring the components back to room temperature.
5.3.2 according to the sample layout (taking 10 samples as an example), on a 96-well U-shaped plate, adding the serum to be detected in A1 and A2.. A10 wells, 30 μ l of serum and 30 μ l of PBST in each well, adding 60 μ l of PBST in other sample dilution wells, carrying out 2-fold serial dilution, wherein the initial ratio is 1: 4, 60 μ l of virus antigen is added in each well, and the initial dilution of the serum after the antigen is added is 1: 8; adding 60 mu l of positive control serum into A11 hole, diluting 2 times to H11 in series, starting at 1:8, adding 60 mu l of virus antigen into each hole, and the initial dilution of the serum after adding the antigen is 1: 16; adding 60 mu l of negative control serum into A12 hole, diluting 2 times to B12 with the initial ratio of 1: 2, adding 60 mu l of virus antigen into each hole, and the initial dilution of the serum after adding the antigen is 1: 4; e12, F12, G12 and H12 were added 60. mu.l of viral antigen per well (60. mu.l of PBST was added in advance) as 4 viral antigen control wells. The reaction plate was gently shaken, sealed with a sealing plate membrane, and incubated at 3 ℃ for 90 minutes.
5.3.3 continuously washing the coated ELISA plate for 3-5 times by using a washing solution, and patting the coated ELISA plate on absorbent paper to be dry. Transferring the serum/virus antigen mixed solution and the virus antigen contrast solution on the serum/virus antigen reaction plate into corresponding holes on an enzyme label plate, sealing the plate by a sealing plate membrane at 100 mu l/hole, and incubating for 30 minutes at 37 ℃.
And 5.3.4 continuously washing the enzyme label plate for 3-5 times by using a washing solution, and patting the enzyme label plate on absorbent paper to be dry. Add 100. mu.l/well of Dolphin anti-biotin working solution, seal the plate with sealing plate membrane, incubate for 30 minutes at 37 ℃.
5.3.5 the ELISA plate is continuously washed for 3-5 times by using a washing solution, and the plate is patted dry on absorbent paper. 100 μ l/well of HRP-SA working solution was added, the plate was sealed with a sealing plate, and incubated at 37 ℃ for 10 minutes.
5.3.6 washing the plate for 3-5 times, drying on absorbent paper, adding TMB substrate solution 50 μ l per well, and incubating at 37 deg.C for 10-15 min.
5.3.7 stop the reaction by adding 50. mu.l of stop solution to each well.
5.3.8 OD450nm values were read on a microplate reader.
5.4 determination of results
5.4.1 test establishment conditions
The OD492nm value of at least 2 holes of the foot-and-mouth disease A type virus antigen control is in the range of 1.0-2.0; the titer of the foot-and-mouth disease A type negative control serum antibody is less than 1: 8; the titer of the foot-and-mouth disease A type positive control serum antibody is 1: 512-1: 2048.
5.4.2 serum antibody titers
Antibody titers were expressed as 50% endpoint dilutions, with 50% of the mean OD450nm value of the antigen control wells being the cut-off value. The wells with the OD450nm value greater than the critical value are negative wells, and the wells with the OD450nm value less than or equal to the critical value are positive wells. The highest dilution of the positive hole of the serum to be detected is the antibody titer of the serum, and the final dilution of the serum is calculated by using a Karber method (shown in the following table).
Karber method formula: ㏒ X-N-D (S-0.5) X represents the serum antibody titer; n-the log of the highest dilution for all positive wells (calculated from the last dilution at which a negative well appeared); d is a logarithmic value of the dilution factor; s is the sum of the ratio of positive wells at each dilution (calculated from the last dilution at which a negative well appeared).
5.4.3 determination
The titer of the antibody of the cattle and sheep serum is more than or equal to 1: 128, and the cattle and sheep serum is judged to be positive by the foot-and-mouth disease A type antibody; the titer of the pig serum antibody is more than or equal to 1: 64, and the pig serum antibody is judged to be positive for the foot-and-mouth disease A type antibody.
6. The specification of an ELISA detection kit for detecting Asia 1 type antibody liquid phase blocking of foot and mouth disease virus is enclosed in 2015 edition 'animal pharmacopoeia'.
Foot-and-mouth disease virus Asia 1 type antibody liquid blocking ELISA detection kit description animal non-prescription medicine.
[ veterinary drug nomenclature ]
General name: foot-and-mouth disease virus Asia 1 type antibody liquid phase blocking ELISA detection kit
The commodity name is as follows: is free of
English name: liquid-phase Blocking ELISA Kit for Detecting Food and mouse Disease Virus Type Asia 1Antibodies Chinese Pinyin: koutiyibigdu Yazhouyixing Kangti Yexiangzuan ELISA Jianeseshijihe
[ main Components and content ] ELISA plates (1 piece, coated Asia 1 type rabbit antiserum for foot and mouth disease); asia 1 type virus antigen of foot and mouth disease (2 bottles, 6 ml/bottle, working concentration 1: 6); foot-and-mouth disease Asia type 1 guinea pig antiserum (1 tube, 70. mu.l/tube, working concentration 1: 1000); rabbit anti-guinea pig IgG-horseradish peroxidase conjugate (1 tube, 70. mu.l/tube, working concentration 1: 1000); 3% hydrogen peroxide (1 tube, 1 ml/tube); asia 1 type positive control serum for foot and mouth disease (1 tube, diluted concentration 1:8, 1 ml/tube); asia 1 type negative control serum for foot and mouth disease (1 tube, 1 ml/tube); 96-well serum/virus antigen reaction plates (2 blocks); substrate buffer (1 vial, 50 ml/vial); 25 times concentrated washing (25 x PBST, 3 bottles, 60 ml/bottle); diluted guinea pig antiserum (1 vial, 50 ml/vial); stop solution (1 bottle, 50 ml/bottle); o-phenylenediamine tablets (OPD, 1 tablet, 20 mg/tablet); a sealing film (10 sheets); transfer tanks (5); instructions for use (1 part).
[ PROPERTIES ] has perfect sealing, no damage and no leakage. The kit comprises a coating ELISA plate, a foot-and-mouth disease Asia 1 type virus antigen, a foot-and-mouth disease Asia 1 type guinea pig antiserum, a rabbit anti-guinea pig IgG-horseradish peroxidase conjugate, 3% hydrogen peroxide, a foot-and-mouth disease Asia 1 type positive control serum, a foot-and-mouth disease Asia 1 type negative control serum, a 25-fold concentrated washing solution, a guinea pig antiserum diluent, a stop solution, a substrate buffer solution, an o-phenylenediamine tablet and the like.
[ ACTION AND USE ] is used for detecting Asia 1-type virus antibodies of foot-and-mouth disease in the serum of artiodactyl animals such as cattle, sheep, pigs and the like, and is suitable for detecting immune antibodies of animal vaccines.
[ DOSAGE AND DECISION ]
1 preparation of reagent (preparation before clinical use)
1.1 washing solution 25 times concentrated washing solution (PBST)1 part, adding deionized water or distilled water 24 parts and mixing, namely the washing solution with working concentration.
1.2 coating buffer (0.05mol/L Na2CO3/NaHCO3, pH9.6) 1 capsule of carbonate buffer was dissolved in 100ml of deionized water and dissolved sufficiently. Labeling, storing at 2-8 ℃, and using up within 30 days.
1.3 substrate solution A substrate buffer solution sheet of the kit was first dissolved in 100ml of deionized water, and then 50ml of the solution was taken and 1 sheet of o-phenylenediamine (OPD) was added. Dissolving completely, packaging (5.0 ml/bottle), and storing at-20 deg.C in dark. When in use, 10 mul of 3 percent hydrogen peroxide (H2O2) is added into each 1.0ml of solution in the reagent box.
1.4 other reagents, namely foot-and-mouth disease Asia 1 type virus antigen, guinea pig antiserum and rabbit anti-guinea pig IgG-horseradish peroxidase conjugate, are respectively diluted by washing solution, guinea pig antiserum diluent and washing solution according to the working concentration indicated by the label.
1.5 dilution of the serum sample to be tested the serum to be tested is mixed with a washing solution in a volume ratio (1: 1). 1.6 dilution of Asia 1 type positive control serum of foot and mouth disease, mixing the Asia 1 type positive serum of foot and mouth disease with washing solution according to the volume ratio (1: 3).
2 operating procedure
2.1 foot-and-mouth disease Asia 1 rabbit antiserum is diluted to working concentration by using coating buffer solution, and then an enzyme label plate (96 holes/plate) is added, 50 mu l/hole is formed, and the plate is sealed by using a sealing plate film and stays overnight at room temperature.
2.2 according to the sample layout (taking 10 samples as an example), adding the serum to be detected into wells A1 and A2.. A10 on a 96-well U-shaped plate, wherein each well is 50 mu l, each serum is subjected to 2 times of repeated serial dilution, the initial dilution is 1: 4, each well is added with 50 mu l of virus antigen, and the initial dilution of the serum after the antigen is added is 1: 8; adding 50 mu l of positive control serum into A11 hole, diluting 2 times to H11 in series, starting at 1:8, adding 50 mu l of virus antigen into each hole, and the initial dilution of the serum after adding the antigen is 1: 16; adding 50 mu l of negative control serum into A12 hole, diluting 2 times to B12 with the initial ratio of 1: 2, adding 50 mu l of virus antigen into each hole, and the initial dilution of the serum after adding the antigen is 1: 4; e12, F12, G12 and H12 were added with 100. mu.l of virus antigen per well as 4 virus antigen control wells. The reaction plate was gently shaken, sealed with a sealing plate membrane and left overnight at 2-8 ℃.
2.3 continuously washing the coated enzyme label plate for 5 times by using a washing solution, and patting the coated enzyme label plate dry on absorbent paper. Transferring the serum/virus antigen mixed solution and the virus antigen contrast solution on the serum/virus antigen reaction plate into corresponding holes on an enzyme label plate, sealing the plate by a sealing plate membrane at 50 mu l/hole, and incubating for 60 minutes at 37 ℃.
2.4 washing the ELISA plate with washing solution for 5 times, and drying on absorbent paper. Adding diluted foot-and-mouth disease Asia 1 type guinea pig antiserum with working concentration, sealing with sealing plate membrane, and incubating at 37 deg.C for 60 min, wherein each well contains 50 μ l of the antiserum.
2.5 Wash plate 5 times, pat dry on absorbent paper, add diluted working concentration of rabbit anti-guinea pig IgG-horseradish peroxidase conjugate 50. mu.l per well, seal plate with sealing plate membrane, incubate 60 minutes at 37 ℃.
2.6 Wash the plate 5 times, pat dry on absorbent paper, add 50. mu.l of substrate solution per well, incubate for 15 minutes at 37 ℃.
2.7 stop the reaction by adding 50. mu.l of stop solution to each well.
2.8 the OD492nm values of each well were read on a microplate reader.
3 determination of results
3.1 test conditions are satisfied, and the OD492nm value of at least 2 holes of foot-and-mouth disease Asia 1 type virus antigen control is in the range of 1.0-2.0; the Asia 1 type negative control serum antibody titer of the foot and mouth disease is less than 1: 8; the Asia 1 type positive control serum antibody titer of the foot and mouth disease is 1: 512-1: 2048.
3.2 serum antibody titers were expressed as 50% end point dilution, with 50% of the mean OD492nm value of the antigen control wells being the cut-off value. The wells with the OD492nm value larger than the critical value are negative wells, and the wells with the OD492nm value smaller than or equal to the critical value are positive wells. The highest dilution of the positive hole of the serum to be detected is the antibody titer of the serum, and the final dilution of the serum is calculated by using a Karber method (shown in the following table).
Karber method formula: ㏒ X-N-D (S-0.5) X represents the serum antibody titer; n-the log of the highest dilution for all positive wells (calculated from the last dilution at which a negative well appeared); d is a logarithmic value of the dilution factor; s is the sum of the ratio of positive wells at each dilution (calculated from the last dilution at which a negative well appeared).
3.3 determination
3.3.1 if the serum antibody titer is not less than 1: 128, the Asia 1 type antibody of the foot and mouth disease is judged to be positive.
3.3.2 if the serum antibody titer is close to 1: 128, it is judged to be suspicious. The test can be repeated for 1 time.
[ notes ] to provide a novel therapeutic agent
(1) The kit is refrigerated and transported at the temperature of 2-8 ℃.
(2) The thawed OPD solution is added with hydrogen peroxide and then is used up for 1 time, and the rest solution cannot be reused.
(3) The number of times of freezing and thawing of the virus antigen cannot exceed 3 times.
[ Specification ] 10 96-well plates/boxes
The Asia 1-type virus antigen of foot-and-mouth disease is preserved at the temperature below 20 ℃ below zero, the rest components of the kit are preserved at the temperature of 2-8 ℃, and the period of validity is 6 months.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
Claims (7)
1. A foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit is characterized in that: comprises a 96-hole rabbit anti-coating plate, FMDV antigen, negative control serum, positive control serum, dolphin anti-biotin, HRP-SA, 25 Xwashing solution, TMB substrate solution and stop solution; the FMDV antigen is a serotype specific foot-and-mouth disease virus antigen, the foot-and-mouth disease virus antigen is concentrated by a PEG concentration and centrifugation technology and is tested and prepared to be uniform in antigen amount, 5% of trehalose and 0.01% of Proclin300 preservative are added, and the mixture is subpackaged and freeze-dried;
different color indicators were added to the negative control serum, positive control serum, pig anti-biotin and HRP-SA, respectively.
2. The kit of claim 1, wherein: the foot-and-mouth disease virus antigen is obtained by inactivating foot-and-mouth disease virus or is obtained by demulsifying the foot-and-mouth disease inactivated vaccine.
3. The kit of claim 1, wherein: the coating process of the 96-hole rabbit anti-coating plate specifically comprises the following steps: washing and sealing an integrated machine by using an ELISA plate, diluting serotype specific rabbit anti-FMDV serum by using a 0.05M carbonate buffer solution with the pH value of 9.6, coating the serum on the ELISA plate at 100 mu l/hole, and standing overnight at room temperature; adding an enzyme label plate stabilizer without washing and patting, and incubating for 1 hour at room temperature in 150 mul/hole; drying at 37 deg.C for 2 hr without washing, vacuum packaging, and storing at 4 deg.C.
4. The kit of claim 1, wherein: the negative control serum was green, the positive control serum was red, the pig anti-biotin was orange, and the HRP-SA was blue.
5. The kit of claim 1, wherein: the PEG concentration centrifugation technology comprises the following specific steps: adding 8% of PEG6000 and 4% of NaCL into the foot-and-mouth disease virus inactivated vaccine, stirring for 4 hours at 4 ℃, standing overnight at 4-8 ℃, centrifuging for 60 minutes in a 500ml centrifuge tube 6000rmp, removing supernatant, suspending antigen precipitate in the centrifuge tube by PBS, fixing the volume to 1/50 of the volume of the original antigen, adding cold trichloroethylene of the same body for emulsification and centrifugal degreasing, and absorbing antigen layer liquid; determining the optimal antigen concentration, diluting with PBS to 1:100 for antigen concentration according to the determined antigen concentration, adding 5% trehalose and 0.01% Proclin300 antiseptic, subpackaging 1.0 ml/tube into 1.5ml centrifuge tube, lyophilizing, collecting the antigen material in tube in loose shape, labeling, and storing at 4 deg.C.
6. The use of the kit according to any one of claims 1 to 5 in the detection of antibodies to foot-and-mouth disease virus, said antibodies being antibodies after immunization with a foot-and-mouth disease virus vaccine; the detection method for detecting the antibody by adopting the kit comprises the following steps:
a. coating an ELISA plate to form a 96-hole rabbit anti-coating plate;
b. serum dilution and antigen reaction: recovering the FMDV antigen freeze-dried product by using sterile deionized water for later use, preserving the FMDV antigen freeze-dried product at 4 ℃ for 1 month, and diluting the FMDV antigen freeze-dried product to a working concentration when the FMDV antigen freeze-dried product is used temporarily; diluting 25 XPBST with deionized water to 1 time PBST for standby;
after PBST is added to a U-shaped carrier plate in advance, adding negative control serum, positive control serum and detected serum into corresponding holes, performing 2-time continuous gradient dilution of corresponding initial times by a 60ul system according to requirements, then adding 60ul of FMDV antigen diluted to working concentration, setting at least 4-hole virus antigen control on each plate, sealing the plates, oscillating, and incubating for 90 minutes at 37 ℃;
c. moving the plate: transferring the antigen-antibody mixture with sufficient effect into an ELISA plate coated with rabbit anti-foot-and-mouth disease virus antiserum in parallel at 100 ul/hole, and incubating for 30 minutes at 37 ℃;
d. dolphin anti-biotin conjugate, a guinea pig IgG anti-type specific FMDV-biotin marker: washing the ELISA plate reacted in the step c for 3-5 times by using PBST, spin-drying, adding the dolphin antibody-biotin conjugate, 100 mu l/hole, sealing the plate, and incubating for 30 minutes at 37 ℃;
e. HRP-SA: washing the ELISA plate reacted in the step d for 3-5 times by using PBST, spin-drying, adding an HRP-SA conjugate, sealing the plate, and incubating for 10 minutes at 37 ℃;
f. TMB substrate and termination reaction: washing the plate for 3-5 times, adding a single-component TMB substrate solution, incubating at the temperature of 37 ℃ for 10-15 minutes, and then adding 1.25M H of 50 mul into each hole2SO4Stopping the reaction, and reading a light absorption value under the wavelength of 450nm by using an enzyme-labeling instrument;
g. and (4) judging a result: and taking 50% of the average value of the OD values of the virus antigen control 4 wells as a critical value of 50% inhibition of the virus antigen, comparing the OD values of corresponding wells of the negative and positive control serum and the detected serum with the critical value, and obtaining the antibody titer of a certain serum, namely the average value of the antibody titers corresponding to two adjacent wells of which the OD value of the serum dilution well is slightly higher than the critical value and slightly lower than the critical value.
7. Use of a kit according to any one of claims 1 to 5 for the preparation of a product for the detection of foot and mouth disease virus antibodies.
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