阅读说明：本技术 一种猪伪狂犬病病毒gD、gE蛋白的抗体联检试剂盒及其制备方法与应用 (Antibody joint detection kit for gD and gE proteins of porcine pseudorabies virus, and preparation method and application thereof ) 是由 田克恭 昌静峰 李玉芳 张许科 于 2020-05-13 设计创作，主要内容包括：本发明涉及一种含猪伪狂犬病病毒gD、gE蛋白抗体的抗体联检试剂盒,该联检试剂盒包括一个或多个猪伪狂犬病病毒gD、gE蛋白抗体检测芯片、酶标试剂,该检测芯片包被有猪伪狂犬病病毒gD蛋白、gE蛋白、质控品、空白对照点,该酶标试剂为含有酶标记猪伪狂犬病病毒gD、及gE蛋白单克隆抗体的溶液,该质控品为羊抗鼠多克隆抗体,其包被量为1～4ng/点。本发明制备的试剂盒在反应载体上设有质控品,不需要在再试剂盒中设有阴性对照、阴性对照,快速检测,操作简便,且高度集成,一键式智能化数据处理,可用于检测猪群中猪伪狂犬病病毒感染状态、评估猪群中猪伪狂犬病病毒疫苗免疫效果及鉴别新引种猪猪伪狂犬病病毒感染情况,便于制定免疫程序及早期净化。(The invention relates to an antibody joint inspection kit containing porcine pseudorabies virus gD and gE protein antibodies, which comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chips and an enzyme labeling reagent, wherein the detection chips are coated with porcine pseudorabies virus gD protein, gE protein, a quality control product and a blank control point, the enzyme labeling reagent is a solution containing enzyme labeled porcine pseudorabies virus gD and gE protein monoclonal antibodies, the quality control product is goat anti-mouse polyclonal antibodies, and the coating amount is 1-4 ng/point. The kit prepared by the invention is provided with the quality control product on the reaction carrier, does not need to be provided with negative control and negative control in a secondary kit, is rapid in detection, simple and convenient to operate, highly integrated, and capable of being used for detecting the infection state of the porcine pseudorabies virus in a swinery, evaluating the immune effect of the porcine pseudorabies virus vaccine in the swinery and identifying the infection condition of the newly introduced porcine pseudorabies virus, and is convenient for establishing an immune program and early purifying.)

1. An antibody joint inspection kit for porcine pseudorabies virus gD and gE proteins, wherein the antibody joint inspection kit comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chips and an enzyme labeling reagent, the porcine pseudorabies virus gD and gE protein antibody detection chips are coated with porcine pseudorabies virus gD proteins, porcine pseudorabies virus gE proteins, quality control products and blank control points, the quality control products are goat anti-mouse polyclonal antibodies, the blank control points are detection background values, and the enzyme labeling reagent is a solution containing an enzyme labeling porcine pseudorabies virus gD protein monoclonal antibody and an enzyme labeling porcine pseudorabies virus gE protein monoclonal antibody; the coating amount of the goat anti-mouse polyclonal antibody is 1-4 ng/point; the quality control point has three points, and the coating amount of the quality control point 1 is more than or equal to 1 ng/point and less than 2 ng/point; the coating amount of the quality control product point 2 is 2 ng/point; the coating amount of the quality control product point 3 is more than 2 ng/point and less than or equal to 4 ng/point.

2. The antibody joint inspection kit according to claim 1, wherein the porcine pseudorabies virus gD protein coated by the porcine pseudorabies virus gD and gE protein antibody detection chip is the gD protein of porcine pseudorabies virus HN1201 strain, and the coated porcine pseudorabies virus gE protein is the gE protein of porcine pseudorabies virus HN1201 strain;

the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody in the enzyme-labeled reagent is enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 or/and 3B6, the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody is enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1, and when the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody is enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibodies 5G7 and 3B6, the content ratio of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 to the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 3B6 is 1: 1;

the coating amount of the porcine pseudorabies virus gD protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 0.05-10 ng/point, preferably, the coating amount of the porcine pseudorabies virus gD protein is 0.25-8 ng/point, and more preferably, the coating amount of the porcine pseudorabies virus gD protein is 4 ng/point;

the coating amount of the porcine pseudorabies virus gE protein is 0.02-8 ng/point, preferably the coating amount of the porcine pseudorabies virus gE protein is 0.1-4 ng/point, and more preferably the coating amount of the porcine pseudorabies virus gE protein is 1 ng/point;

the coating amount of the goat anti-mouse polyclonal antibody is 1 ng/point, 2 ng/point and 4 ng/point;

further preferably, the coating amount of the porcine pseudorabies virus gD protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 4 ng/point or 2 ng/point, and the coating amount of the porcine pseudorabies virus gE protein is 2 ng/point or 1 ng/point;

the IFA titer of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and 3B6 in the enzyme-labeled reagent is 1: 3200-1: 6400, the IFA titer of the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 is 1: 3200-1: 6400, the enzyme-labeled reagent is a PBS solution containing 20% V/V fetal calf serum in final volume, and the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6 in final volume of 0.05% V/V and the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 in final volume of 0.05% V/V are also contained.

3. The antibody joint inspection kit of claim 1, wherein the antibody joint inspection kit further comprises a sample diluent, a substrate solution, and a washing solution; the sample diluent is a PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/V Proclin300, the substrate solution is a TMB solution, and the washing solution is a PBS solution containing 1% V/V Tween 20; preferably, the sample diluent is PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.1% W/V Casein and 1% W/V Proclin300, and the substrate solution is 1-step Ultra TMB.

4. The antibody joint inspection kit of claim 2, wherein the porcine pseudorabies virus gD, gE protein antibody detection chip is further coated with CSFV E2 protein;

the coating amount of the CSFV E2 protein is 0.05-12.8 ng/point; preferably, the coating amount of the CSFV E2 protein is 0.2-6.4 ng/dot; preferably, the coating amount of the CSFV E2 protein is 6.4 ng/dot;

further preferably, the porcine pseudorabies virus gD and gE protein antibody detection chip is coated with 4.0 ng/dot porcine pseudorabies virus gD protein, 1.0 ng/dot porcine pseudorabies virus gE protein and 6.4 ng/dot CSFV E2 protein;

the enzyme-labeled reagent also comprises an enzyme-labeled CSFV E2 monoclonal antibody 15A9 with the final volume of 0.05% V/V, and the IFA titer of the enzyme-labeled antibody 15A9 is 1: 6400-1: 25600.

5. The antibody joint inspection kit according to claim 1 to 4, wherein the coating amounts of the goat anti-mouse polyclonal antibody of the quality control point (1), the quality control point (2) and the quality control point (3) in the detection chip are 1 ng/point, 2 ng/point, 4 ng/point and a blank control point (4) as background detection points, the porcine pseudorabies virus gD protein is spotted on the point (5), the porcine pseudorabies virus gE protein is spotted on the point (6), and the CSFV 2 protein is spotted on the point (7); the sampling points of the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) are respectively fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points (5), (6) and (7) can be respectively sampled at other positions of the detection chip, the distance between the points and the edge of any one of the other points is more than or equal to 700 mu m, and the shortest distance between the middle point and the point edge of the sampling points (1) to (7) is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank reference point sample point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point to the left edge and the right edge of the detection chip subunit are not less than 5 mm;

the antibody joint inspection kit comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chip subunits, an independent detection hole is formed by a bottom end, an upper grid and a detection chip which is arranged between the bottom end and the upper grid and clamped by the bottom end and the upper grid, the detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip.

6. A method for preparing the antibody joint inspection kit of claims 1-5, wherein the method comprises the following steps:

respectively preparing porcine pseudorabies virus gD protein and gE protein or porcine pseudorabies virus gD protein, gE protein and CSFV E2 protein, respectively diluting the proteins together with an goat anti-mouse polyclonal antibody by using a sample solution, and spotting the diluted solutions on a membrane to prepare an antibody detection chip of the porcine pseudorabies virus gD and gE protein or an antibody detection chip of the porcine pseudorabies virus gD, gE protein and CSFV E2 protein;

step (2) forming independent detection holes by a bottom end, an upper grid and the antibody detection chip in the step (1) arranged between the bottom end and the upper grid, wherein the antibody detection chip is clamped by the bottom end and the upper grid, one detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip;

respectively marking the porcine pseudorabies virus gD protein monoclonal antibody and the gE protein monoclonal antibody or the porcine pseudorabies virus gD protein monoclonal antibody, the gE protein monoclonal antibody and the CSFV E2 protein monoclonal antibody by using enzyme, diluting the mixture by using an enzyme-labeled diluent according to the final volume of 0.05 percent V/V, and uniformly mixing the diluted mixture to be used as an enzyme-labeled reagent, wherein the enzyme-labeled diluent contains a PBS (phosphate buffer solution) solution of fetal bovine serum with the final volume of 20 percent V/V;

preparing a sample diluent, a washing solution and a substrate solution; and

and (5) assembling the porcine pseudorabies virus gD and gE protein antibody detection chip subunit prepared in the step (2) or the porcine pseudorabies virus gD, gE protein and CSFV E2 protein antibody detection chip subunit or subunit microarray thereof, the enzyme labeling reagent prepared in the step (3), and the sample diluent, the washing solution and the substrate solution prepared in the step (4) into the antibody joint inspection kit.

7. The method according to claim 6, wherein the spotting solution in the step (1) is prepared by uniformly mixing a 5% glycerol solution, a 5% sorbitol solution, a 0.05% triton solution, a DMSO solution and a PBS (pH6.8) solution according to a volume ratio of 10: 15: 0.1: 50: 100; diluting the goat anti-mouse polyclonal antibody to 50 mug/ml, 100 mug/ml and 200 mug/ml respectively by using the sample solution, and using the diluted goat anti-mouse polyclonal antibody as quality control point sample solution to be respectively spotted on the points (1), (2) and (3); using the sample solution as a blank control sample solution to sample a spot (4), using PRVgD sample solution to sample a spot (5), using PRVgE sample solution to sample a spot (6), using CSFV E2 sample solution to sample a spot (7);

the sampling points of the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) are respectively fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points (5), (6) and (7) are respectively sampled on the detection chip and have a distance of more than or equal to 700 mu m from the edge of any one point, the shortest distance between the middle point and the point edge of the sampling points (1) to (7) is also more than or equal to 700 mu m, the distances from the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) to the upper edge and the lower edge of a detection chip subunit are more than or equal to 8mm, and the distances from the left edge and the right edge of the detection chip subunit are more than or equal to 5 mm;

in the step (3), the porcine pseudorabies virus gD protein monoclonal antibody is porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6, the porcine pseudorabies virus gE protein monoclonal antibody is porcine pseudorabies virus monoclonal antibody 11H1, the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6 and the enzyme-labeled porcine pseudorabies virus monoclonal antibody 11H1 are diluted by the enzyme-labeled diluent according to the final volume of 0.05 percent V/V and then mixed uniformly, and the mixture is filtered by 0.22 mu m and packaged in an aseptic manner; or in the step (3), the porcine pseudorabies virus gD protein monoclonal antibody is porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6, the porcine pseudorabies virus gE protein monoclonal antibody is porcine pseudorabies virus monoclonal antibody 11H1, the classical swine fever virus E2 protein monoclonal antibody is porcine pestivirus E2 protein monoclonal antibody 15A9, the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6, the enzyme-labeled porcine pseudorabies virus monoclonal antibody 11H1 and the enzyme-labeled porcine pestivirus E2 protein monoclonal antibody 15A9 are diluted by the enzyme-labeled diluent and mixed uniformly according to the final antibody volume of 0.05% V/V, and the mixture is filtered by 0.22 mu m and packaged in an aseptic mode; when the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody is enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and 3B6, the content ratio of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 to the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 3B6 is 1: 1; and

the sample diluent in the step (4) is PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/V Proclin300, and is filtered by 0.22 mu m and packaged in an aseptic manner;

the washing solution is PBS solution containing 1% V/V Tween 20, and is filtered by 0.22 μm, aseptically subpackaged, and diluted by 20 times with purified water during detection;

the substrate solution is TMB (3,3',5,5' -tetramethylbenzidine) solution and is packaged in an aseptic mode.

8. Use of the antibody joint inspection kit according to claims 1-5 for the preparation of a non-immunological diagnosis; preferably, the applications in the non-immune diagnosis comprise epidemiological analysis, qualitative and quantitative porcine pseudorabies virus antibody detection on isolated tissues, international live pig trade, import and export inspection and quarantine, and porcine pseudorabies vaccine quality control.

Technical Field

The invention relates to an antibody bigeminy detection kit and a antibody bigeminy detection kit of porcine pseudorabies virus gD and gE proteins, as well as a preparation method and application thereof, and belongs to the field of biological medicines.

Background

Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease with fever, extreme itching (except for pigs), encephalomyelitis as the main symptoms in various domestic animals and wild animals such as pigs, cows, sheep, etc., caused by the porcine herpesvirus type I (Suid herpesvirus I) in the alphasubfamily of Herpesviridae. Pigs are the primary reservoir and source of infection for the disease. Porcine pseudorabies virus can infect pigs of different ages, but pregnant sows and suckling piglets are the most serious: resulting in miscarriage, stillbirth and mummy in pregnant sows; the fattening pigs show respiratory tract symptoms when infected with the porcine pseudorabies virus, and have low mortality rate when free of complications; the piglets suffer from nervous symptoms, paralysis and exhaustion death, the death rate is almost 100 percent, the morbidity of the weaned piglets is about 40 percent, and the death rate can reach 20 percent, thereby causing huge economic loss to the healthy development of the domestic and even global pig industry and becoming one of the major infectious diseases seriously harming the healthy development of the pig industry.

In the prevention and treatment work of the pseudorabies, monitoring and detecting the level of the pseudorabies virus antibody in the serum of the pig is a very important link. In the prior art, the detection of the immune effect of the porcine pseudorabies virus vaccine needs a neutralization test determination process which takes a long time, and the simple, convenient and rapid detection of the immune effect of the porcine pseudorabies virus live vaccine and the quality detection of the live vaccine are urgently needed in clinic.

The porcine pseudorabies is prevented mainly by a porcine pseudorabies virus gE gene deletion attenuated vaccine, and an antibody aiming at gE protein cannot be detected in the serum of an immunized pig by an enzyme-linked immunosorbent assay (ELISA), so that the difference provides conditions for differential diagnosis by utilizing the serum to carry out pseudorabies wild strain infection and gE gene deletion vaccine immunization. Negative reference substances, positive reference substances or standard substances are set in a traditional ELISA kit to serve as quality control substances in the kit, and the internal control substances need to be subjected to complex procedures such as animal screening, special animal rooms, professional feeding, immunization, sampling, inactivation, calibration, subpackaging and the like. Particularly, clinically, the disease condition is complex, the vaccine immune pigs, the wild strain infected pigs and the nonimmune and non-infection negative pigs are always in a living state, and a method for scientifically evaluating the immune condition of a swinery, the protective profile of antibodies, the wild virus infection and the progress condition of the wild virus infection is urgently needed clinically so as to better guide the breeding.

The Chinese patents CN103792373A and CN106405082A utilize colloidal gold detection technology to prepare colloidal gold test strips for simultaneously detecting PRVgD and gE antibodies, but the detection result cannot be accurately quantified, and the health state or early, medium or late infection of pigs or a swinery can be comprehensively judged according to the PRVgD antibody level and the PRVgE antibody level in the detection result, so that the clinical establishment of an immunization strategy and a purification measure can be guided.

Classical swine fever (classic swine fever) is a highly contagious disease caused by Classical Swine Fever Virus (CSFV), is characterized by strong infectivity and high lethality rate, and pigs of different ages, sexes and breeds can be infected, so that the Classical swine fever is one of the main infectious diseases closely concerned by grain and agricultural organizations and governments in the world. Clinically, the disease and the porcine pseudorabies are very easy to be infected mixedly. In consideration of actual production, multiple immunizations stimulate animal bodies to influence growth of the animals, increase labor intensity in feeding and increase labor cost, and therefore, a bigeminal vaccine is developed, but research on the immune interference phenomenon of the bigeminal vaccine of swine fever and porcine pseudorabies (master academic paper of northeast university of Hebei agriculture, 2016) finds that the generation of antibodies at the early stage of swine fever can be interfered by the porcine pseudorabies after the bigeminal vaccine of swine fever and porcine pseudorabies is used for immunizing piglets at the same time. The experimental results are monitored by corresponding kits to avoid the morbidity or mortality of the pigs caused by unrealized immune effect, the existing market kits are independent kits, and the operation and calculation are time-consuming and labor-consuming in monitoring and long in time-consuming.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides an antibody joint inspection kit for porcine pseudorabies virus gD and gE proteins, wherein the antibody joint inspection kit comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chips and an enzyme labeling reagent, the porcine pseudorabies virus gD and gE protein antibody detection chips are coated with porcine pseudorabies virus gD protein, porcine pseudorabies virus gE protein, a quality control product and blank control points, the quality control product is an goat anti-mouse polyclonal antibody, the blank control points are detection background values, and the enzyme labeling reagent is a solution containing an enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody and an enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody; the coating amount of the goat anti-mouse polyclonal antibody is 1-4 ng/point; the quality control point has three points, and the coating amount of the quality control point 1 is more than or equal to 1 ng/point and less than 2 ng/point; the coating amount of the quality control product point 2 is 2 ng/point; the coating amount of the quality control product point 3 is more than 2 ng/point and less than or equal to 4 ng/point.

Compared with the traditional ELISA kit which is provided with negative control, positive control or standard substance as the quality control substance in the kit, the joint inspection kit provided by the invention is technically improved, the quality control substance is arranged on the reaction carrier, the quality control substance is a commercialized goat anti-mouse polyclonal antibody, and the negative control are not required to be arranged in the kit, namely, the complicated preparation steps of immunization or toxicity attack of animals, serum collection and the like, and the cost of manpower and animal test sites are not required to be screened in advance, and the convenience and the accuracy of the operation of the kit are greatly improved. According to the invention, researches show that the goat anti-mouse polyclonal antibody in a certain content range can be used as a quality control product without setting negative control and negative control, and the goat anti-mouse polyclonal antibody is used as a reaction control for no antibody reaction in a single detection hole, is used for evaluating the effectiveness of a test and reflects the reaction degree of an enzyme labeling reagent in the absence of blocking. The goat anti-mouse polyclonal antibody can be purchased commercially or prepared by a conventional method, and the operation is simple.

The coating amount of the goat anti-mouse polyclonal antibody can be 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 and 4.0 ng/point.

In one embodiment of the present invention, in the joint inspection kit of the present invention, the porcine pseudorabies virus gD protein coated by the porcine pseudorabies virus gD and gE protein antibody detection chip is the gD protein of porcine pseudorabies virus HN1201 strain, and the coated porcine pseudorabies virus gE protein is the gE protein of porcine pseudorabies virus HN1201 strain; the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody in the enzyme-labeled reagent is enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 or/and 3B6, the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody is enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1, and when the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody is enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibodies 5G7 and 3B6, the content ratio of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 to the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 3B6 is 1: 1; the coating amount of the porcine pseudorabies virus gD protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 0.05-10 ng/point, the coating amount of the porcine pseudorabies virus gE protein is 0.02-8 ng/point, and the coating amount of the goat anti-mouse polyclonal antibody is 1 ng/point, 2 ng/point and 4 ng/point; the IFA titer of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and 3B6 in the enzyme-labeled reagent is 1: 3200-1: 6400, the IFA titer of the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 is 1: 3200-1: 6400, the enzyme-labeled reagent is a PBS solution containing 20% V/V fetal calf serum in final volume, and the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6 in final volume of 0.05% V/V and the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 in final volume of 0.05% V/V are also contained.

Through experimental research, the invention defines the specific porcine pseudorabies virus gD protein coating amount, the specific porcine pseudorabies virus gE protein coating amount, the specific coating amount of the goat anti-mouse polyclonal antibody, the specific titer and content of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody and the specific titer and content of the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody, and realizes the high-sensitivity detection of the serum antibody in the sample.

The porcine pseudorabies virus gD protein and the gE protein can be prepared by molecular biology means, including but not limited to eukaryotic expression system expression (such as baculovirus expression and CHO expression), prokaryotic expression system expression (such as escherichia coli expression) and yeast expression system expression.

The sample application volume of the porcine pseudorabies virus gD protein and the gE protein is 20 nl/point.

The porcine pseudorabies virus gD protein coating amount can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 7.6, 7.7, 6, 7.8, 7.9, 4.0, 4.1, 4.2, 4.3, 4.4.5, 4.6, 7.5, 6, 7.6, 7, 8, 7.5, 6, 7.5, 8, 7.6.6, 7, 8, 6, 8, 7.0, 8.5, 7.5, 8.6, 6, 7.0, 8.5, 8, 8.7.6.6, 7.8.8, 7.6, 8, 7.0, 8, 7.7.7.7.6, 8, 8.5, 8.6.6, 8.7.7.8.8.8.7.7.8, 8, 7.6, 8, 7.8.8.6, 8, 8.6, 7.8.6, 7.6, 8.7, 7.8.8, 8, 8.8.6, 8, 8.6, 8.7.7, 8, 8.6, 7, 8, 7.0, 8, 8.7.7.6, 8.6, 7.6, 8.6, 7, 7.6, 8, 7, 8, 7, 7.6, 8.6, 8.8.8.6, 8.7.6, 8, 7, 8, 8.7, 7, 8, 8.6, 7.6, 7.7.6, 8.7, 7, 7.8.8, 8.8.8.8.8.7, 8.0, 8.8.8.8.8.7.7.8.8, 8.7.6, 7.7.8.6, 8.8.8.6, 8.6, 7.6, 8.6, 7, 7.6, 7.8.8.8.8.8.8.8.7.8.8.7, 8, 8.8.8.6, 8.8.8.8.8.8.8.8.8.8.8, 8.8, 8, 8.8.8.8.8.8, 8, 8.8.8.8.8.8.8.8.7, 7.8.6, 7, 8, 8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8, 7.8.8.8, 7, 7.8.8.8.8.8.8.8.8.8.8, 7.8.8, 8, 7, 7.8, 7, 7.8.8.8.8.

The porcine pseudorabies virus gE protein coating amount can be 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.2, 4.5, 4.6, 4.7, 4.5, 6, 6.7, 6, 7.8, 3.9, 4.0, 4.5, 6.6, 7.5, 6, 6.5, 6, 7.5, 6, 6.5, 6, 7.6, 6, 6.7, 6, 6.6.6, 6, 7, 6, 6.7.6, 7.6, 6, 7.6, 6, 6.6.6.7.6, 7.6, 6.6.6, 7.7.6, 7, 7.6, 6.6.6, 7.6, 7.6.6.6.6, 7.7.6, 7.6, 7.6.6, 6, 7, 7.6, 7.6.6, 6, 6.6, 7.6, 7.6.6.6, 6, 7.6, 6, 7.6, 6, 7, 6, 7.6, 6, 6.6, 7.6, 6, 7.6, 7.6.6.6, 6, 7.6.6.6.6.6, 7.6, 6, 6.6.7.7.7.6, 6, 6.6.6.6.6, 6, 6.7.7.6.6, 7.6.6.6, 7.6.6.6.6.6.6, 6, 7.6.6, 6, 7.6, 7.6.6, 7.6, 7.7.7.7.7.7.7.6, 6, 6.6, 6, 6.6.6, 6.7.7.6, 6, 7.6, 6, 6.6, 6, 6.7.7.7.7.7.7.7.6.7.7.6, 6, 6.6.6, 6, 6.6, 6.

The porcine pseudorabies virus gD protein monoclonal antibodies 5G7 and 3B6 are disclosed in Chinese patent CN 109206509A; the porcine pseudorabies virus gE protein monoclonal antibody 11H1 is disclosed in Chinese patent CN 109959788A. The IFA titer of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and 3B6 is 1: 3200-1: 6400, and the IFA titer of the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 is 1: 3200-1: 6400.

As a preferred embodiment of the invention, the coating amount of the porcine pseudorabies virus gD protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 0.25-8 ng/point.

As a more preferable embodiment of the invention, the coating amount of the porcine pseudorabies virus gD protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 4 ng/point.

As a preferred embodiment of the invention, the coating amount of the porcine pseudorabies virus gE protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 0.1-4 ng/point.

As a more preferable embodiment of the invention, the coating amount of the porcine pseudorabies virus gE protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 1 ng/point.

As a preferred embodiment of the invention, the coating amount of the porcine pseudorabies virus gD protein in the porcine pseudorabies virus gD and gE protein antibody detection chip is 4 ng/point or 2 ng/point, and the coating amount of the porcine pseudorabies virus gE protein is 2 ng/point or 1 ng/point.

The high sensitivity of detection is ensured by further optimizing and selecting the protein coating amount.

As an embodiment of the invention, the labeled enzyme in the joint inspection kit is horseradish peroxidase, alkaline phosphatase or beta-D-galactosidase.

In a preferred embodiment of the present invention, the joint inspection kit further comprises a sample diluent, a substrate solution, and a washing solution; the sample diluent is a PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/V Proclin300, the substrate solution is a TMB solution, and the washing solution is a PBS solution containing 1% V/V Tween 20.

As a more preferred embodiment of the present invention, the sample diluent is PBS solution containing 10% V/V fetal bovine serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.1% W/V Casein, 1% W/V Proclin 300.

In a more preferred embodiment of the present invention, the substrate solution is 1-step Ultra TMB.

The substrate solution is 1-step Ultra TMB, is a ready-made single-component horseradish peroxidase (HRP) substrate, and the main component of the substrate solution is soluble TMB (3,3',5,5' -tetramethylbenzidine) which reacts with HRP to generate insoluble deep blue precipitate.

The invention provides further optimized selection of components and contents in the sample diluent, the substrate solution and the washing solution, and ensures high sensitivity of detection.

As a preferred embodiment of the invention, the porcine pseudorabies virus gD and gE protein antibody detection chip is further coated with CSFV E2 protein; the coating amount of the CSFV E2 protein is 0.05-12.8 ng/point; the enzyme-labeled reagent also comprises an enzyme-labeled CSFV E2 monoclonal antibody 15A9 with the final volume of 0.05% V/V, and the IFA titer of the enzyme-labeled antibody 15A9 is 1: 6400-1: 25600.

The swine fever E2 protein can be prepared by molecular biology means, including but not limited to eukaryotic expression system expression (such as baculovirus expression and CHO expression), prokaryotic expression system expression (such as Escherichia coli expression) and yeast expression system expression.

The spotting volume of the hog cholera E2 protein was 20 nl/spot.

The swine fever E2 protein coating amount can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4, 4.5, 4.6, 4.5, 4, 4.5, 7, 6, 6.6, 5, 6, 7.6, 6, 7.6, 6, 7, 6, 5, 7.6, 6, 7.6, 6, 7, 6, 7.7.7, 6, 7.6, 7.7, 6, 8, 7.7.7.7, 6, 8, 6, 7, 7.9.9, 8, 7, 6, 7.9.9.9.9.9, 6, 8, 7.9.9.9, 7.9, 6, 7, 6, 7.9.9, 6, 8, 7, 7.9.9, 7.9, 8, 6, 8, 7.9, 8, 7, 7.9, 6, 8, 7, 6, 8, 6, 7, 6, 8, 7, 7.9, 8, 9.9, 8, 9, 8, 7, 6, 8, 6, 9, 7, 8, 7, 8, 6, 7.8.8, 9.8, 9, 8, 9.8, 7, 6, 7, 8, 9.0, 9, 9.8.8, 7.8.8.8, 9.8.8, 9, 8, 9, 7, 8, 6, 8.8, 9, 8.8.8, 8, 8.8.8, 9.8.8, 8, 9.8, 8.8, 8, 9.8.8, 9.8, 9, 9.8.8, 9, 8, 8.8, 8, 8.8, 9, 9.8.8, 9, 8, 6, 8, 9, 8, 8.8, 8, 8.8.8, 8, 8.8.8.8, 8, 8.8, 8.8.8.8, 8, 8.8.8.8.8, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8 ng/point.

The swine fever virus E2 protein monoclonal antibody 15A9 is disclosed in Chinese patent CN 105837686A. The IFA titer of the enzyme-labeled classical swine fever virus E2 protein monoclonal antibody 15A9 is 1: 6400-1: 25600.

In a more preferred embodiment of the present invention, the coating amount of the CSFV E2 protein is 0.2 to 6.4 ng/dot.

As a further preferable embodiment of the invention, the coating amount of the CSFV E2 protein is 6.4 ng/dot.

As a more preferable embodiment of the invention, the porcine pseudorabies virus gD and gE protein antibody detection chip is coated with 4.0 ng/point porcine pseudorabies virus gD protein, 1.0 ng/point porcine pseudorabies virus gE protein and 6.4 ng/point CSFV E2 protein.

The invention also provides a method for simultaneously detecting the porcine pseudorabies virus antibody and the CSFV antibody in the serum, and the high-sensitivity detection is ensured by the optimized selection of the CSFV E2 protein coating amount and the titer and the content of the enzyme-labeled CSFV monoclonal antibody.

In a preferred embodiment of the present invention, the coating amounts of the goat anti-mouse polyclonal antibody of the quality control point (1), the quality control point (2) and the quality control point (3) in the detection chip are 1 ng/point, 2 ng/point, 4 ng/point and a blank control point (4) as background detection points, the porcine pseudorabies virus gD protein is spotted on the point (5), the porcine pseudorabies virus gE protein is spotted on the point (6), and the CSFV E2 protein is spotted on the point (7); the sampling points of the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) are respectively fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points (5), (6) and (7) can be respectively sampled at other positions of the detection chip, the distance between the points and the edge of any one of the other points is more than or equal to 700 mu m, and the shortest distance between the middle point and the point edge of the sampling points (1) to (7) is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank reference point sample point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point to the left edge and the right edge of the detection chip subunit are not less than 5 mm; the antibody joint inspection kit comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chip subunits, an independent detection hole is formed by a bottom end, an upper grid and a detection chip which is arranged between the bottom end and the upper grid and clamped by the bottom end and the upper grid, the detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip.

The quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank control sample point (4) on the detection chip of the joint detection kit are fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the porcine pseudorabies virus gD protein sample point (5), the porcine pseudorabies virus gE protein sample point (6) and the CSFV E2 protein sample point (7) can be respectively positioned at any other position on the detection chip, the distance between the edges of any other point is more than or equal to 700 mu m, and the distance between the middle points of the sample points (1) to (7) is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank reference point sample point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point to the left edge and the right edge of the detection chip subunit are not less than 5 mm.

The joint inspection kit can comprise one or more porcine pseudorabies virus gD and gE protein antibody detection chip subunits, and when the joint inspection kit comprises a plurality of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip, so that the detection of a plurality of samples can be realized at the same time, and the efficiency is increased.

The joint inspection kit prepared by the invention can obtain data of two antibodies gD and gE by detecting 1 sample, thereby not only monitoring the wild virus infection state of the porcine pseudorabies virus in the swinery, providing safety measures for the purification of the porcine pseudorabies virus, but also evaluating the vaccine immunization program and the immunization effect in the swinery.

Particularly, the detection result of the PRVgD antibody in the joint detection kit can realize semi-quantitative detection of the built-in label, has strong applicability and is more convenient for clinical analysis.

The gD antibody detected by the joint detection kit can evaluate the serum antibody level of the vaccine of different strains of the porcine pseudorabies after immunization, and the detection result detects a positive result earlier than a virus neutralization test; the gD and gE antibodies can simultaneously carry out quantitative detection on early infection serum of different types of strains (classical strains and variant strains) of the porcine pseudorabies virus, have high sensitivity and good specificity, and are beneficial to the purification of the pseudorabies. Meanwhile, the antibody titer can be presented in a datamation manner, so that the method has scientific guiding effect on clinical analysis.

The invention also provides a method for preparing the joint inspection kit, wherein the method comprises the following steps: respectively preparing porcine pseudorabies virus gD protein and gE protein or porcine pseudorabies virus gD protein, gE protein and CSFV E2 protein, respectively diluting the proteins together with an goat anti-mouse polyclonal antibody by using a sample solution, and spotting the diluted solutions on a membrane to prepare an antibody detection chip of the porcine pseudorabies virus gD and gE protein or an antibody detection chip of the porcine pseudorabies virus gD, gE protein and CSFV E2 protein; step (2) forming independent detection holes by a bottom end, an upper grid and the antibody detection chip in the step (1) arranged between the bottom end and the upper grid, wherein the antibody detection chip is clamped by the bottom end and the upper grid, one detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip; respectively marking the porcine pseudorabies virus gD protein monoclonal antibody and the gE protein monoclonal antibody or the porcine pseudorabies virus gD protein monoclonal antibody, the gE protein monoclonal antibody and the CSFV E2 protein monoclonal antibody by using enzyme, diluting the mixture by using an enzyme-labeled diluent according to the final volume of 0.05 percent V/V, and uniformly mixing the diluted mixture to be used as an enzyme-labeled reagent, wherein the enzyme-labeled diluent contains a PBS (phosphate buffer solution) solution of fetal bovine serum with the final volume of 20 percent V/V; preparing a sample diluent, a washing solution and a substrate solution; and (5) assembling the subunit of the detection chip for the antibodies against the porcine pseudorabies virus gD and gE protein prepared in the step (2) or the subunit microarray of the detection chip for the antibodies against the porcine pseudorabies virus gD, gE protein and CSFV E2 protein or the subunit thereof, the enzyme labeling reagent prepared in the step (3), and the sample diluent, the washing solution and the substrate solution prepared in the step (4) into the antibody joint inspection kit.

As an embodiment of the present invention, the porcine pseudorabies virus gD protein, gE protein and classical swine fever virus E2 protein in the step (1) can be prepared by molecular biology means, including but not limited to eukaryotic expression system expression (such as baculovirus expression and CHO expression), prokaryotic expression system expression (such as E.coli expression) and yeast expression system expression.

As an embodiment of the present invention, the labeled enzyme in the step (3) is horseradish peroxidase, alkaline phosphatase or beta-D-galactosidase.

In one embodiment of the present invention, the spotting fluid in the step (1) is a 5% glycerol solution, a 5% sorbitol solution, a 0.05% triton solution, a DMSO solution, and a PBS (ph6.8) solution, which are mixed uniformly in a volume ratio of 10: 15: 0.1: 50: 100; diluting the goat anti-mouse polyclonal antibody to 50 mug/ml, 100 mug/ml and 200 mug/ml respectively by using the sample solution, and using the diluted goat anti-mouse polyclonal antibody as quality control point sample solution to be respectively spotted on the points (1), (2) and (3); using the sample solution as a blank control sample solution to sample a spot (4), using PRVgD sample solution to sample a spot (5), using PRVgE sample solution to sample a spot (6), using CSFV E2 sample solution to sample a spot (7); the sampling points of the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) are respectively fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points (5), (6) and (7) are respectively sampled on the detection chip and have a distance of more than or equal to 700 mu m from the edge of any one point, the shortest distance between the middle point and the point edge of the sampling points (1) to (7) is also more than or equal to 700 mu m, the distances from the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) to the upper edge and the lower edge of a detection chip subunit are more than or equal to 8mm, and the distances from the left edge and the right edge of the detection chip subunit are more than or equal to 5 mm; in the step (3), the porcine pseudorabies virus gD protein monoclonal antibody is porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6, the porcine pseudorabies virus gE protein monoclonal antibody is porcine pseudorabies virus monoclonal antibody 11H1, the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6 and the enzyme-labeled porcine pseudorabies virus monoclonal antibody 11H1 are diluted by the enzyme-labeled diluent according to the final volume of 0.05 percent V/V and then mixed uniformly, and the mixture is filtered by 0.22 mu m and packaged in an aseptic manner; or in the step (3), the porcine pseudorabies virus gD protein monoclonal antibody is porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6, the porcine pseudorabies virus gE protein monoclonal antibody is porcine pseudorabies virus monoclonal antibody 11H1, the classical swine fever virus E2 protein monoclonal antibody is porcine pestivirus E2 protein monoclonal antibody 15A9, the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and/or 3B6, the enzyme-labeled porcine pseudorabies virus monoclonal antibody 11H1 and the enzyme-labeled porcine pestivirus E2 protein monoclonal antibody 15A9 are diluted by the enzyme-labeled diluent and mixed uniformly according to the final antibody volume of 0.05% V/V, and the mixture is filtered by 0.22 mu m and packaged in an aseptic mode; when the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody is enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 and 3B6, the content ratio of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 to the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 3B6 is 1: 1; and the sample diluent in the step (4) is PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/V Proclin300, and the PBS solution is filtered by 0.22 mu m and is subjected to aseptic subpackage; the washing solution is PBS solution containing 1% V/V Tween 20, and is filtered by 0.22 μm, aseptically subpackaged, and diluted by 20 times with purified water during detection; the substrate solution is TMB (3,3',5,5' -tetramethylbenzidine) solution and is packaged in an aseptic mode.

The invention also provides the application of the joint inspection kit in preparing non-immune diagnosis; preferably, the applications in the non-immune diagnosis comprise epidemiological analysis, qualitative and quantitative porcine pseudorabies virus antibody detection on isolated tissues, international live pig trade, import and export inspection and quarantine, and porcine pseudorabies vaccine quality control.

The invention also provides an application method of the joint inspection kit, which comprises the following steps:

numbering, namely numbering chip holes according to the sequence of samples;

soaking in the step (2), adding 300 mu L/hole of washing liquid, and after soaking for 3 minutes, discarding the liquid and patting dry;

adding sample, namely adding 50 mu L/hole of sample to be detected after 50 mu L/hole of sample diluent, and incubating for 30 minutes at 37 ℃ at 500 rpm in a constant-temperature oscillation incubator; discarding the liquid, adding 300 mu L/hole of washing liquid, soaking for 30-60 seconds, discarding the liquid, repeatedly washing for 5 times, and finally drying by beating for the last time;

adding enzyme standard reagent, adding 100 mul/hole enzyme standard reagent, and incubating for 30min at 37 deg.C and 500 rpm in a constant temperature shaking incubator; discarding the liquid, adding 300 mu L/hole of washing liquid, soaking for 30-60 seconds, discarding the liquid, repeatedly washing for 5 times, and finally drying by beating for the last time;

developing, adding 100 mu L/hole of substrate solution, and incubating for 15 minutes at 37 ℃;

step (6), after the upper cover of the chip is removed, the dust-free paper is placed on the detection chip, the detection chip is lightly pressed, and the result is measured by a micropore disk chip imager within 10 minutes, so that an S/N value (sample S value/quality control point N value) is derived;

and (7) testing validity judgment: the S value of the quality control point is not less than 6000, the S value of the blank control point is not more than 3000, otherwise, the test is invalid, the judgment is automatically completed by an internal data processing and analyzing system, and the result judgment standard is as follows:

calculation of the S/N ratio: S/N is sample A value/negative quality control product A value (wherein the sample A value is a sample detection gray value; the negative quality control product A value is a certain quality control point detection gray value, the quality control point is preferably selected according to the condition that the analysis value of the calibrated known negative and positive sample is in accordance with the standard value when the batch is debugged, the preferred standard is the quality control product point corresponding to the condition that the gray value ratio of the calibrated known negative and positive sample to the three quality control product points 1, 2 and 3 is calculated and the standard value and the coincidence degree of the judgment result are highest),

when S/N is judged to be not more than 0.600, PRV gD and gE antibodies are positive, when S/N is judged to be more than 0.700, PRV gD and gE antibodies are negative, when S/N is more than 0.700, PRV gD and gE antibodies are suspicious, when S/N is more than 0.600 and not more than 0.700, resampling is needed for detection, and if the detection is still suspicious, the PRV gD and gE antibodies are judged to be negative.

The invention has the advantages that:

the combined detection kit and the traditional ELISA kit both have negative control, positive control or standard substance as quality control substances in the kit, and the kit is technically improved, the quality control substances are arranged on reaction carriers, the quality control substances are commercialized goat anti-mouse polyclonal antibodies, and the negative control substances do not need to be arranged in a kit, namely, complex preparation steps of immunization or toxicity attack of animals, serum collection and the like, and the cost of manpower and animal test sites are not needed to be screened in advance, and the convenience and the accuracy of the operation of the kit are greatly improved.

The joint inspection kit prepared by the invention has the advantages of high integration of detection items, rapid detection, simple and convenient operation, one-key intelligent data processing, elimination of complex data processing and mass data analysis after detection by a conventional method, easier control of the health condition of the swinery and convenience for intensive management of the breeding industry.

The joint inspection kit prepared by the invention has accurate and reliable results, high coincidence rate of the result of detecting the gD antibody and the virus neutralization test result, and is suitable for evaluating the vaccine immunization effect and immunization program scientificity prepared by different strains of the porcine pseudorabies virus; the sensitivity for detecting the gE antibody is high, the antibody positive can be detected 5-7 days after artificial infection, the method is suitable for early warning of wild virus (classical strains and variant strains) infection, and the early purification of pseudorabies is easy. The joint inspection kit prepared by the invention can be used for detecting the infection state of the porcine pseudorabies virus in the swinery, evaluating the immune effect of the porcine pseudorabies virus vaccine in the swinery and identifying the infection condition of the newly introduced porcine pseudorabies virus.

Drawings

FIG. 1 is a schematic diagram showing the spotting pattern of each chip well on a porcine pseudorabies virus gD and gE protein antibody duplex detection chip, wherein A, B, C, D, E, F, G, H, I, J in FIG. 1 shows different spotting patterns respectively, and 5 and 6 in the same figure can be interchanged in specific positions on the detection chip;

FIG. 2 is a schematic diagram showing the spotting pattern of each chip well on the PRRSV gD, gE protein, CSFV E2 protein antibody triple detection chip, wherein K, L, M in FIG. 2 shows different spotting patterns, and the specific positions of 5, 6, and 7 on the detection chip can be interchanged in the same figure;

FIG. 3 is a diagram showing three kits with different amounts of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, wherein a partial enlarged view shows a detection chip with porcine pseudorabies virus gD and gE protein antibody detection chip subunits, and O, P, Q in FIG. 3 is a diagram showing a kit with one, three, or multiple antibody detection chip subunits respectively.

Reference numerals

1 is a quality control point 1, 2 is a quality control point 2, 3 is a quality control point 3, 4 is a blank control point, 5 is a PRVgD detection point, 6 is a PRVgE detection point, and 7 is a CSFV E2 protein detection point.

Detailed Description

The term "goat anti-mouse polyclonal antibody" is used interchangeably with goat anti-mouse secondary antibody, goat anti-mouse IgG antibody.

The term "enzyme" includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase. Wherein, the substrates used by the horseradish peroxidase are o-phenylenediamine (OPD) and Tetramethylbenzidine (TMB), preferably Tetramethylbenzidine (TMB); the substrate used for alkaline phosphatase is p-nitrophenylphosphate (p-NPP); the substrate used for beta-D-galactosidase was 4-methylumbelliferyl-beta-D-galactoside (4 MUG).

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The phosphate buffer used in the examples of the present invention was PBS with pH 7.4, and its formulation volume of 1L was: NaCl 8.0g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.24g, but this embodiment is not intended to limit the present invention in any way. Goat anti-mouse polyclonal antibodies are commercially available or can be prepared by conventional methods.

The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

EXAMPLE 1 preparation of raw materials for porcine pseudorabies virus gD and gE protein antibody joint detection kit

1.1 preparation and identification of porcine pseudorabies virus gD protein

Preparing PRVgD protein according to CN105251000A patent, purifying with Ni affinity chromatography column to obtain recombinant PRVgD protein, identifying with SDS-PAGE that a protein band is visible at about 50KDa, and analyzing to obtain 94% purity; the protein content was determined using the BCA kit and found to be 5.6 mg/ml.

1.2 preparation and identification of porcine pseudorabies virus gE protein

1.2.1 amplification of porcine pseudorabies Virus gE Gene

PRV HN1201 strain virus (porcine Pseudorabies virus strain HN1201 strain, strain HN1201) is inoculated on well-grown PK15 cells, the preservation number is CCTCC NO. V201311, the PRV is preserved in China center for type culture collection, the preservation address is Wuhan university in Wuhan, the preservation date is 2013, 5, 20 days and is disclosed in CN105251000A), PRV genome DNA is extracted by using a TAKARA MiniBEST Viral RNA/DNA Extraction Kit Ver.3.0 Kit after the virus is harvested. Mu.l of genomic DNA (see PRV HN1201 accession number: KP722022.1 for gE genomic sequence) was used as a template with the following gE-specific primers:

gESF5′-GTGATGGTGATGGTGATGGTGATGGAGGCCCCGAGCCTCTCCGCCG-3′

gESR5′-CGCCAGCACAAACAGCCGCCCG-3′

by usingHS DNA Polymerase and designed specific primer are used for carrying out PCR amplification on the gE fragment, and the PCR reaction program is as follows: 2min at 98 ℃; 10s at 98 ℃, 5s at 60 ℃, 1min at 72 ℃ and 30s for 30 cycles; 10min at 72 ℃. The PCR product was named PRVgE.

1.2.2 acquisition and identification of recombinant Bacmid

The PCR product PRVgE obtained by high fidelity enzymatic amplification was cloned into pFastBac/HBM-TOPO vector (purchased from Invitrogen corporation) in the following cloning system: PCR product PRVgE 4. mu.l, saline solution 1. mu.l, TOPO vector 1. mu.l, total 6. mu.l. Mixing uniformly, incubating at room temperature for 5 minutes, transforming One shotR Mach1TMT1R competent cells, coating an ampicillin resistance plate, selecting a single clone to identify the insertion direction of the gE gene, sending the plasmid with the correct insertion direction to Invitrogen company for sequencing, identifying the correctness of the gE sequence, and naming the plasmid with the correct sequencing as pFastBac/HBM-TOPO-gE.

The Plasmid pFastBac/HBM-TOPO-gE was transformed into DH10Bac competent cells (purchased from Invitrogen), pFastBac/HBM-TOPO-gE and shuttle Plasmid Bacmid in the competent cells were transposed, the obtained recombinant Plasmid was extracted with PureLink HiPure Plasmid DNA Miniprep Kit of Invitrogen, and insertion of gE was identified with pUCM13Forward/Pucm13 Reverse primer, and positive Bacmid was named Bacmid-gE.

1.2.3 transfection to obtain recombinant baculovirus

The procedure was carried out according to the method provided in the instruction manual of Bac-to-Bac HBM TOPO Secreted Expression System of Invitrogen. 6 pore plate 8X 10 layers per hole⁵Sf9 cells, which were transfected after cell attachment according to instructions for Cellfectin II transfection reagent: mu.l Cellffectin II and 1. mu.g Bacmid-gE DNA were diluted to 100. mu.l SF-90, respectivelyIn a 0II culture medium, Vortex is uniformly mixed, the diluted DNA and the diluted Cellffectin II (the total volume is 210 mu l) are mixed uniformly, the mixture is incubated for 15 to 30 minutes at room temperature, and the mixture is dripped into cells. After cytopathic effect occurred 72 hours after transfection, cell culture supernatant was collected and recorded as recombinant virus vBac-gE of P0 generation. P0 generation recombinant virus vBac-gE infects Sf9 cells, and after 3 generation amplification culture, P3 generation vBac-gE is obtained for recombinant protein expression.

1.2.4 infection of High-five cells by recombinant baculovirus to obtain recombinant protein

The P3 generation recombinant baculovirus vBac-gE was inoculated into High-five cells (purchased from Invitrogen). Suspension culture of High-five cells in 500ml Erlenmeyer flask until the cell density reaches 7.0X 10⁵After cell/ml, the virus was inoculated at 1MOI and cell culture supernatants were harvested 72 hours after infection. Purifying the recombinant PRVgE protein by using a Ni affinity chromatographic column, identifying a protein band which is obvious at a position of about 62KDa by SDS-PAGE, and analyzing the purity to 93%; the protein content was determined by BCA kit and found to be 12 mg/ml.

1.3 preparation and identification of classical swine fever virus E2 protein

Preparing hog cholera virus E2 protein according to CN105527442A patent, purifying with Ni affinity chromatography column to obtain recombinant CSFV E2 protein with analytical purity of 90%; the protein content was determined using the BCA kit and found to be 2.5 mg/ml.

1.4 preparation and identification of enzyme-labeled antibody

1.4.1 preparation

And (3) respectively labeling the porcine pseudorabies virus gD protein monoclonal antibodies 5G7 and 3B6, the porcine pseudorabies virus gE monoclonal antibody 11H1 and the classical swine fever virus E2 protein monoclonal antibody 15A9 by using an improved sodium periodate method. 20mg of horseradish peroxidase (HRP) was weighed out and dissolved in 1ml of ultrapure water, and 1ml of freshly prepared NaIO was added₄Solution (30mg NaIO)₄Dissolving in 1ml of ultrapure water), mixing, and keeping away from light at 4 ℃ for 30 minutes; adding 40 mul of ethylene glycol into the solution, and keeping away from light at 4 ℃ for 30 minutes; mixing the purified monoclonal antibody 1mg with the mixture 100 μ l, adding into dialysis bag, mixing, and dialyzing with CB buffer solutionFor 6 hours. The whole operation needs to be carried out in a dark place; the dialyzed mixture was transferred to a 1.5ml EP tube and 10. mu.l of freshly prepared NaBH was added₄Solution (20mg NaBH)₄Dissolved in 1ml of ultrapure water), acted for 2 hours at room temperature, and uniformly mixed once every 30 minutes; adding saturated ammonium sulfate with the same volume, mixing uniformly, and acting for 15 minutes at 4 ℃. Centrifuge at 12000 rpm for 10 minutes and discard the supernatant. The pellet was suspended in a volume of PBS mixed with glycerol equal to the volume of purified antibody (V: V ═ 1: 1).

1.4.2 identification

Appearance: at room temperature, the liquid is reddish brown liquid, and no floccule precipitate is seen.

And (3) quality evaluation: and detecting the absorbance value A of the enzyme-labeled antibody at 403nm and 280nm by using an ultraviolet spectrophotometer. The corresponding enzyme parameters were calculated according to the formula:

enzyme amount (mg/ml) ═ A_403nmX 0.4 x dilution factor.

IgG amount (mg/ml) ═ A_280nm-A_403nm0.3). times.0.62 times the dilution factor.

Gram-molecule ratio (E/P) ═ enzyme amount × 4/IgG amount.

Mark rate is A_403nm/A_280nm。

Through the detection and calculation of light absorption values, the specific results are shown in table 1:

TABLE 1 quality evaluation of different enzyme-labeled antibodies

Titer detection of enzyme-labeled antibodies 5G7, 3B6 and 11H 1: after the enzyme-labeled antibodies 5G7, 3B6 and 11H1 were diluted with PBS, the IFA titer of the enzyme-labeled antibodies was measured by the indirect immunofluorescence IFA method. The IFA detection method comprises the following steps: culturing adherent PK-15 cells, inoculating the virus seeds containing PRV HN1201 strain, Fa strain and MA strain at a dose of 0.005-0.1 MOI, respectively, setting healthy cell control holes at 37 deg.C and 5% CO₂Culturing for 48-72 hours under the condition, and then removing the supernatant; fixing with 80% acetone solution at 2-8 deg.C for 30min, washing with PBS 3 times, adding 100 μ l of 1: 1 into virus inoculation hole and cell control hole respectivelyDiluting with 10 times of the original sample, adding PRV positive serum into the virus inoculation hole as positive control, and reacting at 37 deg.C for 60 min; washing with PBS for 3 times and drying; adding FITC-labeled goat anti-mouse IgG diluted at a ratio of 1: 500, and allowing the mixture to act at 37 ℃ for 60 minutes; washing with PBS for 3 times and drying; after addition of 50. mu.l PBS, observation was carried out under a fluorescence microscope. And (3) determining IFA titer: the maximum dilution of the antibody corresponding to the well of the cytotoxic cell plate in which yellow-green fluorescence was observed was used as the IFA titer of the antibody. Results (see table 2): after the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibodies 5G7 and 3B6 are labeled, the IFA titer to different strains is 1: 3200-1: 6400, and part of the IFA titer is slightly reduced compared with the IFA titer before labeling; after the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 is labeled, the IFA titer to different strains is 1: 3200-1: 6400.

TABLE 2 IFA titre determination of different enzyme-labeled antibodies

Enzyme-labeled antibody 15A9 titer detection: after the enzyme-labeled antibody 15A9 was diluted with PBS, the IFA titer of the enzyme-labeled antibody was measured by the indirect immunofluorescence IFA method (classical swine fever virus CH-1R strain was prepared as an IFA antigen plate by the method described above). As a result: the IFA titer of the enzyme-labeled antibody 15A9 is 1: 6400-1: 25600.

Example 2 preparation of a bivalent detection kit for gD and gE protein antibodies of porcine pseudorabies virus

2.1 preparation of spotting fluid

Preparation of 5% glycerol solution: precisely weighing 5.00g of glycerol, placing the glycerol in a 100ml volumetric flask, adding a small amount of purified water, slightly rotating the glycerol to fully dissolve the glycerol so as to avoid generating excessive bubbles, adding the purified water to scale marks, and turning and shaking the glycerol up and down for 10 times for later use;

preparation of 5% sorbitol solution: precisely weighing 5.00g of sorbitol, putting the sorbitol into a 250ml beaker, adding a proper amount of purified water, stirring to completely dissolve the sorbitol, completely transferring the sorbitol into a 100ml volumetric flask, adding the purified water to set a scale mark, and turning and shaking up and down for 10 times for later use;

preparation of 0.05% triton solution: measuring 50 μ l of triton with a pipette, placing in a 100ml volumetric flask, adding appropriate amount of purified water to dissolve completely, adding purified water to set scale mark, and shaking for 10 times;

DMSO solution: directly adopting a DMSO reagent;

PBS (pH6.8) solution preparation: preparing 0.2mol/L disodium hydrogen phosphate solution and 0.3mol/L sodium dihydrogen phosphate solution, and mixing the two solutions according to a volume ratio of 49.5:51 to obtain a phosphate buffer solution with the pH value of 6.8;

the solutions are uniformly mixed according to the volume ratio of 10: 15: 0.1: 50: 100 to be used as a sampling solution.

2.2 preparation of a two-way test kit

The detection chip for the porcine pseudorabies virus gD and gE protein dual-detection antibody comprises: diluting the porcine pseudorabies virus gD protein prepared in example 1 to a proper concentration by using the sampling solution prepared in 2.1 to serve as a detection point PRVgD sampling solution; diluting the porcine pseudorabies virus gE protein prepared in example 1 to a proper concentration by using a sampling solution to serve as a detection point PRVgE sampling solution; respectively diluting the goat anti-mouse polyclonal antibody to 50 mug/ml, 100 mug/ml and 200 mug/ml by using a sample application liquid as quality control point sample application liquids, respectively applying samples to points 1, 2 and 3 in the figure 1, setting 3 concentrations, and conveniently selecting corresponding quality control points according to debugging results to carry out data statistical analysis; the spotting fluid prepared in 2.1 was spotted on spot 4 as a blank control spotting fluid. Starting a sample applicator, setting a program and sample application parameters, and applying sample application of a detection point PRVgD sample solution to a point 5, applying sample application of a detection point PRVgE sample solution to a point 6, applying sample application of a quality control point sample solution to points 1, 2 and 3 in the figure 1, and applying sample application of a blank control point sample solution to a point 4 in the figure 1 to a specified position in the figure 1. And taking the spotted membrane out, placing the spotted membrane in the middle of a bottom plate of the chip, adding a cover plate for pressing, fixing edgings on two side cards, assembling the detection chip of the porcine pseudorabies virus gD and gE protein dual-detection antibody, packaging, and storing at 2-8 ℃. Except that the points 1, 2, 3 and 4 are respectively fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points 5 and 6 can be respectively spotted at any other positions of the detection chip, the distance between the points and the edges of other points is only required to be more than or equal to 700 mu m, and the shortest distance between any point and the point is also more than or equal to 700 mu m. The distances from the quality control point 1, the quality control point 2, the quality control point 3 and the blank reference point sample point 4 to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point 4 to the left edge and the right edge of the detection chip subunit are not less than 5 mm.

The antibody joint inspection kit comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chip subunits, an independent detection hole is formed by a bottom end, an upper grid and a detection chip which is arranged between the bottom end and the upper grid and clamped by the bottom end and the upper grid, the detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of porcine pseudorabies virus gD and gE protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip. FIG. 3 shows the kit with different amounts of porcine pseudorabies virus gD, gE protein antibody detection chip subunits, O, P, Q in FIG. 3 is the kit with one, three, multiple antibody detection chip subunits respectively, and its partial enlarged view shows the sampling points on the detection chip in one porcine pseudorabies virus gD, gE protein antibody detection chip subunit.

Enzyme labeling reagent: and (3) uniformly mixing the PBS solution and 200ml of fetal calf serum, and adding PBS to a constant volume of 1L to be used as an enzyme-labeled diluent. The enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibodies 5G7 and 3B6 and the enzyme-labeled porcine pseudorabies virus monoclonal antibody 11H1 prepared in example 1 were mixed with the above solution at a final volume of 0.05% V/V, filtered at 0.22 μm, and aseptically dispensed.

Sample diluent: PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05% -0.5% W/V Casein and 1% W/V Proclin300, filtering at 0.22 μm, and aseptically packaging.

Washing liquid: PBS solution containing 1% V/V Tween 20 was filtered at 0.22 μm and aseptically dispensed. The test was performed by 20-fold dilution with purified water.

Substrate solution: TMB (3,3',5,5' -tetramethylbenzidine) solution, commercial product, sterile packaging.

2.3 detection method establishment

The operation steps are as follows:

(1) and numbering, namely numbering the chip holes according to the sequence of the samples.

(2) Soaking, adding 300 μ L of washing solution per hole, soaking for 3 min, discarding solution, and patting dry.

(3) Adding sample, namely adding 50 mu L/hole of sample to be detected after 50 mu L/hole of sample diluent, and incubating for 30 minutes at the temperature of 37 ℃ and at the speed of 500 r/min in a constant-temperature oscillation incubator; and (4) discarding the liquid, adding 300 mu L/hole of washing liquid, and soaking for 30-60 seconds to discard the liquid. Washing repeatedly for 5 times, and drying for the last time.

(4) Adding enzyme standard reagent, adding 100 μ l/well enzyme standard reagent, and incubating at 37 deg.C and 500 rpm for 30 min; and (4) discarding the liquid, adding 300 mu L/hole of washing liquid, and soaking for 30-60 seconds to discard the liquid. Washing repeatedly for 5 times, and drying for the last time.

(5) Color development, adding substrate solution 100 u L/hole, placed at 37 degrees C temperature 15 minutes incubation.

(6) And (3) measuring, namely after the upper cover of the chip is removed, placing dust-free paper on the detection chip, slightly pressing, measuring the result by using a micropore disk chip imager within 10 minutes, and deriving the S/N value (the S value of the sample/the N value of the quality control point).

And (3) testing validity judgment: the S value of the quality control point is not less than 6000, the S value of the blank control point is not more than 3000, otherwise, the test is invalid, and the judgment is automatically completed by an internal data processing and analyzing system. The criteria for result determination (which is also done by the internal one-touch intelligent data processing and analyzing system, and does not require the technician to perform data calculation and statistical analysis) are as follows:

calculation of the S/N ratio: and S/N is the sample A value/negative quality control product A value (wherein the sample A value is a sample detection gray value; the negative quality control product A value is a detection gray value of a certain quality control point, and the quality control point is preferably selected according to the condition that the analysis value of the calibrated known negative and positive sample is in accordance with the standard value when the batch is debugged, and the preferred standard is the quality control product point corresponding to the condition that the gray value ratio of the calibrated known negative and positive sample to the three quality control product points 1, 2 and 3 is calculated and the standard value and the goodness of fit of the judgment result are the highest).

When the S/N is judged to be less than or equal to 0.600 positively, the PRV gD and gE antibodies are positive;

when the S/N is more than 0.700, the PRV gD and gE antibodies are negative;

when S/N is more than 0.600 and less than or equal to 0.700, PRV gD and gE antibodies are suspicious, and re-sampling is needed for detection, and if the detection is still suspicious, the result is negative.

2.4 determination of protein coating amount of porcine pseudorabies virus gD and gE protein dual-detection antibody detection chip

PRVgD positive, PRVgE negative, CSFV negative pig serum (abbreviation P1): the neutralization potency is 1: 51.3 through the neutralization test of the porcine pseudorabies virus; the detection result of the Biochek PRVgB kit is positive, and the S/P value is 2.393; the result of the test by an IDEXX PRVgE kit is negative, and the S/N value is 0.989; the result of the detection by an IDEXX CSFV antibody detection kit is negative, and the blocking rate is 11 percent.

PRVgE-positive and PRVgD-positive CSFV-negative pig serum (abbreviated as P2): the neutralization potency is 1: 11.2 by the neutralization test of the porcine pseudorabies virus; the detection result of the Biochek PRVgB kit is positive, and the S/P value is 1.152; the result is positive by IDEXX PRVgE kit determination, and the S/N value is 0.196; the result of the detection by an IDEXX CSFV antibody detection kit is negative, and the blocking rate is 8 percent.

PRVgD negative, PRVgE negative, CSFV positive pig serum (abbreviation P3): the neutralizing titer is less than 1: 2 by the neutralization test of the porcine pseudorabies virus; the detection result of the Biochek PRVgB kit is negative, and the S/P value is 0.126; the result is negative by IDEXX PRVgE kit determination, and the S/N value is 0.912; the result is positive by IDEXX CSFV antibody detection kit, and the blocking rate is 74%.

PRVgD negative, PRVgE negative, CSFV negative pig serum (abbreviated as N): the neutralizing potency is less than 1: 2 by the neutralization test of the porcine pseudorabies virus; the detection result of the Biochek PRVgB kit is negative, and the S/P value is 0.191; the result is negative by IDEXX PRVgE kit, and the S/N value is 0.958; the result of the detection by an IDEXX CSFV antibody detection kit is negative, and the blocking rate is 3 percent.

The serum is used for selecting porcine pseudorabies virus gD and gE protein dual-detection antibodies to detect the protein antigen coating amount of the chip, and the principle of the method is an enzyme-linked immunosorbent assay, so that the protein antigen coating condition corresponding to the maximum N/P value is selected as the optimum protein antigen solid phase condition for screening by calculating the N/P value, and the details are as follows:

2.4.1 selection of porcine pseudorabies Virus gD protein coating

The porcine pseudorabies virus gD protein of example 1 was diluted with a spotting solution to an appropriate concentration as shown in example 2.1, and the spotting volume was 20nl, so that the final content of PRVgD protein spotted on the membrane was as shown in Table 3, respectively, and none of the others was changed. The assay was carried out using P1, N serum according to the method of example 2.2, with the results (see Table 3): the detection is negative under the condition that the coating amount of PRVgE is not changed (not shown in Table 3); the numerical value of N/P1 is small when the PRVgD coating amount is low, and the data is obviously improved when the PRVgD coating amount reaches above 0.25 ng/point, the size of the N/P1 value corresponds to the negative and positive distinguishing capability of the method, and the larger the data is, the more suitable the detection condition is; the detection can be satisfied when the N/P1 value is 2.28-3.07 when the coating amount of the PRVgD protein is 0.05-10 ng/point, but the N/P1 value is slightly better (3.27-3.74) when the coating amount is 0.25-8 ng/point, and the N/P1 value is optimal (3.74) when the coating amount is 4.0 ng/point.

TABLE 3 porcine pseudorabies virus gD protein coating optimization results

2.4.2 selection of porcine pseudorabies Virus gE protein coating

The porcine pseudorabies virus gE protein of example 1 was diluted with a sample solution to an appropriate concentration as shown in example 2.1 in a sample application volume of 20nl so that the final content of PRVgE protein applied to the membrane was as shown in Table 4, respectively, and was not changed otherwise. The assay was carried out using P2, N serum according to the method of example 2.2, with the results (see Table 4): the detection result of the N/P2 value has no significant difference under the condition that the PRVgD coating amount is unchanged (not shown in Table 4); the value of N/P2 is small when the PRVgE coating amount is low, the data is obviously improved when the PRVgE coating amount reaches above 0.1 ng/point, the size of the N/P2 value corresponds to the negative and positive distinguishing capability of the method, and the larger the data is, the more suitable the detection condition is. The detection can be satisfied when the coating amount of the PRVgE protein is 0.02-8 ng/point, the N/P2 value is 1.98-2.18, but the N/P2 value is slightly better (3.50-4.56) when the coating amount is 0.1-4 ng/point, and the N/P2 value is optimal (5.90) when the coating amount is 1.0 ng/point.

TABLE 4 porcine pseudorabies virus gE protein coating optimization results

2.4.3 verification of porcine pseudorabies virus gD and gE protein coating amount

In order to verify whether cross reaction exists when PRVgD and gE proteins are detected from the corresponding antibodies, a sample with clear background and detected by a commercial kit is selected to further verify the coating amount of gD and gE proteins of the porcine pseudorabies virus. According to the above test results, the porcine pseudorabies virus gD protein was orthogonally spotted at 4 ng/spot and 2 ng/spot and the porcine pseudorabies virus gE protein at 2 ng/spot and 1 ng/spot to prepare protein antigen plates 1-4 (see Table 5 for details). And 3 parts of serum after immunization of a porcine pseudorabies live vaccine (Bartha strain), 3 parts of PRVgDgE antibody after challenge of a porcine pseudorabies virus Fa strain, 3 parts of positive serum after challenge of a porcine pseudorabies virus HN1201 strain, and 7 parts of specific serum (comprising 1 part of PCV2 positive serum, 1 part of CSFV positive serum, 1 part of PPV positive serum, 1 part of PRRSV positive serum, 1 part of healthy pig serum and 2 parts of PRVgDgE negative serum), wherein 16 parts are respectively detected, and the corresponding coating amount is the optimal coating amount when the result is combined with the standard symbol 16/16, namely the coincidence rate is 100%. Results (see table 5): the results of the detection of 16 samples by the kit prepared from the protein antigen plates 1-4 are consistent with those of a commercial kit or a neutralization test method, and the coincidence rate is 100%.

TABLE 5 evaluation results of preparation of kits for different types of protein antigens with different coating amounts

2.5 selection of enzyme-labeled porcine pseudorabies Virus gD protein monoclonal antibody

The chip for detecting the porcine pseudorabies virus gD and gE protein dual-detection antibody prepared by the kit 2 is diluted by 1: 500 of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody 5G7 or 3B6 or 5G7+3B6 (shown in Table 6) in the example 1 according to the example 2.1, and is mixed with the diluent of the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H 11: 1000 in equal volume. The assay was carried out using P1, N serum according to the method of example 2.2, with the results (see Table 6): compared with the N/P1 result detected by 3B6, the working solution of the 3 enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody is optimal, which shows that the epitope of the porcine pseudorabies virus gD protein recognized by the monoclonal antibody 3B6 is more advantageous than the epitope recognized by 5G 7. In the subsequent test, diluted mixed liquor of 1: 2000 of the enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody and the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 is used as an enzyme-labeled reagent.

TABLE 6 results of selection of enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody

Labeled monoclonal antibody	5G7	3B6	5G7+3B6 (equal volume mixing)
				N	37896	41238	39926
P1	8989	5669	7564
				N/P1	4.22	7.27	5.28

2.6 selection of sample dilutions

Preparing a sample diluent by using the protein antigen plate 2 and the enzyme labeling reagent together with the reagent in the table 7, and detecting the masculine property of N, P1 and P2, wherein the result is shown in the table 7: after 5 sample processing liquids are used for processing samples, compared with the sample processing liquid 5 for processing samples, the numerical values of N/P1 and N/P2 are the maximum, and the effect is the best.

TABLE 7 test results for different sample dilutions

To evaluate the content of Casein in the sample dilution, the other components were kept unchanged, and the content of Casein was adjusted to 0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 1.0% W/V to prepare a kit and to perform detection, as a result: when the Casein contained in the sample treatment solution is 0.01%, the sensitivity of the prepared kit for detecting the PRVgD antibody is reduced by 10-50 times; when the Casein contained in the sample treatment solution is 1.0%, the sensitivity of the prepared kit for detecting the PRVgE antibody is reduced by 20 times; the detection sensitivity of the kit prepared when the content of Casein in the sample treatment solution was 0.05%, 0.25% or 0.5% was comparable to that of the sample diluent 5, and therefore the content of Casein in the sample diluent was set to 0.05% to 0.5% W/V.

2.7 quality control Point

The kit adopts a blocking method for detection, the blocking method is to use the dominant epitope corresponding to an antibody marker to carry out the reaction of blocking antigen and antibody, and the level of the antibody to be detected is evaluated through the blocking effect. The invention adopts the specific reaction between the proper concentration of the goat anti-mouse polyclonal antibody in a single detection hole and the monoclonal antibody marker, thereby obtaining the reaction result in the single hole.

Through screening a large amount of goat anti-mouse IgG with different concentrations, the optimal quality control point sample amount is screened so as to match the detection results of the porcine pseudorabies virus gD and gE protein antibodies. For convenience of description, the porcine pseudorabies virus gD and gE protein antibodies are selected as positive and negative samples respectively for detection, the negative and positive of the antibodies are confirmed by commercial kits and neutralization tests, the positive and negative samples are judged by calculating the ratio of detection points/quality control points (namely S/N value) under each condition, and the condition with correct screening result can be used as the selection of the optimal condition. Meanwhile, the ratios (namely N/P values) of the S/N values of the negative samples and the S/N values of the positive samples of the porcine pseudorabies virus gD and gE protein antibodies are respectively calculated, and the condition with the maximum screening value is taken as the optimal condition. And selecting the sample application quantity of the optimal quality control point when the two conditions are met. Test results (see table 8): when the kit detects the PRVgD antibody, the detection result is matched with the detection standard when the goat anti-mouse polyclonal antibody is 0.25-4 ng/point, and when the goat anti-mouse polyclonal antibody is 0.5-4 ng/point, the detection result is matched with the detection standard. When the kit detects the PRVgD antibody, the result of calculating the N/P value is 12.0-12.5 when the goat anti-mouse polyclonal antibody is 1-4 ng/point, and the value is the largest; when the PRVgE antibody is detected, the result of calculating the N/P value is 10.8-10.9 when the goat anti-mouse polyclonal antibody is 1-4 ng/point, and the value is the maximum. Namely, when the goat anti-mouse polyclonal antibody is 1-4 ng/point, the detection result of the antibody matched with the porcine pseudorabies virus gD and gE protein is optimal.

According to the matching degree of the detection result, the optimal condition of the goat anti-mouse polyclonal antibody is finally selected to be 1-4 ng/point, the better control quality control condition is considered, the difference between sample application amount batches is avoided, the sample application amount of the sample application points is respectively selected to be 1 ng/point, 2 ng/point and 4 ng/point to be used as the sample application amount of the quality control points, and the optimal quality control point is selected to be used for the confirmation of the final determination method of the kit.

TABLE 8 comparison of the coating amounts of different goat anti-mouse polyclonal antibodies

Note: when the S/N is judged to be less than or equal to 0.600 positively, the PRV gD and gE antibodies are positive; when the S/N is more than 0.700, the PRV gD and gE antibodies are negative; when S/N is more than 0.600 and less than or equal to 0.700, PRV gD and gE antibodies are suspicious, and re-sampling is needed for detection, and if the detection is still suspicious, the result is negative. The larger the N/P value in the blocking method is, the more accurate the negative, positive and positive samples can be distinguished.

For subsequent evaluation, a protein antigen plate 2(PRVgD protein 4 ng/point, PRVgE protein 1 ng/point), an enzyme labeling reagent (enzyme-labeled porcine pseudorabies virus gD protein monoclonal antibody, and the final volume of enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 are mixed solution diluted by 1: 2000), a sample diluent 5 (PBS solution containing 10% V/V fetal bovine serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.1% W/V Casein and 1% W/V Proclin 300) are prepared into a kit A together with a substrate solution and a washing solution for subsequent detection.

Example 3 preparation of triple detection kit for gD, gE protein antibodies and E2 protein antibodies of porcine pseudorabies virus

3.1 selection of the coating amount of classical swine fever virus E2 protein

The CSFV E2 protein of example 1 was diluted with a sample solution to a suitable concentration as shown in example 2.1 and the sample volume was 20nl, so that the final content of CSFV E2 protein spotted on the membrane was as shown in Table 3, respectively, but not otherwise. The assay was carried out using P3, N serum according to the method of example 2.2, with the results (see Table 9): the detection is negative under the condition that the coating amount of PRVgD and PRVgE is not changed (not shown in Table 9); the value of N/P3 is small when the coating amount of CSFV E2 is low, and the data is obviously improved when the coating amount of CSFV E2 is above 0.4 ng/point, the size of the N/P3 value corresponds to the negative and positive distinguishing capability of the method, and the larger the data is, the more suitable the detection condition is; the detection can be satisfied when the coating amount of CSFV E2 protein is 0.05-12.8 ng/point, the N/P3 value is 2.59-2.70, but the N/P3 value is slightly better (3.13-3.68) when the coating amount is 0.2-6.4 ng/point, and the N/P3 value is optimal (3.74) when the coating amount is 6.4 ng/point.

TABLE 9 optimization of classical swine fever virus E2 protein coating amount

3.2 preparation of triple detection kit

The swine fever virus E2 protein was added to the swine fever virus gD, gE protein dual detection antibody detection chip of example 2.2, the swine fever virus E2 protein prepared in example 1 was diluted to an appropriate concentration with a spotting solution to obtain a swine fever virus E2 protein spotting solution (spotted at 7 in FIG. 2). Triple detection antibody detection chips were prepared as described in example 2.2 (see FIG. 2 for spotting format). Likewise, the triple assay kit may be assembled in a different manner as shown in FIG. 3.

The enzyme-labeled classical swine fever virus E2 protein monoclonal antibody 15A9 is added to the enzyme-labeled reagent in example 2.2: the enzyme-labeled antibody 3B6, the enzyme-labeled antibody 11H1, and the enzyme-labeled antibody 15A9 prepared in example 1 were mixed with an enzyme-labeled diluent at a final volume of 0.05% V/V, filtered at 0.22 μm, and aseptically dispensed.

A method for judging the swine fever antibody is added on the basis of the method for judging the swine fever, and the method for judging the swine fever is as follows:

PI (1-sample a value/negative quality control a value) × 100%.

Judging the antibody CSFV to be positive when the PI is more than or equal to 40 percent;

judging the CSFV antibody to be suspicious when PI is more than 30% and less than 40%;

and judging that the antibody is CSFV antibody negative when the PI is less than or equal to 30 percent.

For subsequent evaluation, a protein antigen plate (coated with PRVgD protein, PRVgE protein and CSFV E2 protein in sequence of 4.0 ng/point, 1.0 ng/point and 6.4 ng/point), an enzyme labeling reagent, a sample diluent 5, a substrate solution and a washing solution are prepared into a kit B for subsequent detection.

Example 4 application of Combined detection kit

4.1 sensitivity

4.1.1 porcine pseudorabies virus gD, gE protein antibody

105 piglets, all of which were negative for 21-day-old PRV antigen antibody, were randomly divided into 10 groups, 5 groups/group.

Group 1 cervical muscle immunization pig pseudorabies live vaccine (Bartha strain), 1ml (containing 10)^6.0TCID₅₀Per ml); group 2 cervical muscle immunization pig pseudorabies live vaccine (Bartha strain), 1ml (containing 10)^5.0TCID₅₀Per ml); group 3 cervical muscle immunization porcine pseudorabies live vaccine (Bartha strain), 1ml (containing 10)^4.0TCID₅₀Per ml); group 4 cervical muscle immunization pig pseudorabies live vaccine (Bartha strain), 1ml (containing 10)^3.0TCID₅₀Per ml); group 5 was not immunized and not challenged as a control. Groups 1-5 were immunized at 0 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months.

Group 6 cervical muscle immunization pig pseudorabies live vaccine (Bartha strain), 1ml (containing 10)^5.0TCID₅₀Ml), 0 days after immunization 1ml (containing 10) was nasally instilled with HN1201 strain vaccine^7.0TCID₅₀Per ml) counteracting toxic substance; group 7 is not immunized and only detoxified, and 1ml (containing 10) of HN1201 strain is dripped into nose at 49 days of piglet day age^6.0TCID₅₀Per ml) counteracting toxic substance; group 8 is not immunized and only detoxified, and the nose is dripped with Fa strain 1ml (containing 10) when piglets are 49 days old^6.0TCID₅₀Per ml) for counteracting toxic substance. Blood was collected at 0 week, 1 week, 2 weeks, 3 weeks, and 4 weeks (i.e., 0 day after challenge) after immunization in group 6 and at 0 day, 3 days, 5 days, 6 days, 7 days, 9 days, 11 days, and 14 days after challenge in groups 6, 7, and 8. At the same time, blood was collected from group 5 pigs for comparison.

Group 9 was immunized with a commercial porcine pseudorabies vaccine (Eye A strain); group 10 immunization with porcine pseudorabies vaccine (strain HB-98); group 11 cervical muscle immunized porcine pseudorabies virus vaccine (HN1201 strain). Blood was collected 35 days after immunization in groups 9-11.

The above serums are all detected by a kit A and a kit B, the antibody immunity effect is detected by a commercial Biochek porcine pseudorabies gB antibody detection kit, the result of the attacking antibody is detected by a commercial IDEXX porcine pseudorabies virus gE antibody detection kit, and the neutralizing antibody is determined by referring to the method of the GB/T18641-2002 serum neutralization test.

According to the detection results, the detection results of the immune samples of different porcine pseudorabies virus vaccines of groups 1-5 are shown in the table 10: the detection results of the kit A and the kit B for detecting the PRVgD antibody are basically consistent with those of a BioChek commercial kit, are superior to a virus neutralization test, and accord with the development trend of PRVgD antibody increase; the detection results of the kit A and the kit B for detecting the PRVgE antibody are consistent with those of the IDEXX PRVgE antibody detection kit, and both are negative.

TABLE 10 summary of the results of the detection of various doses of porcine pseudorabies virus vaccine in the immunized samples

The porcine pseudorabies virus gD protein monoclonal antibody 3B6 is used as an antibody to be detected, the kit A is used for repeatedly detecting for multiple times (as y), an equation is fitted according to a detection result and is divided into 3 sections, each section can correspond to one equation (see table 11), and the detection result has the same trend with the background result. For further verification, the detection results of the samples are converted into antibody titer values (as x), and the results are found to be consistent with those in table 10, which shows that the kit A and the kit B can realize the semi-quantitative detection of the PRVgD antibody in-vivo built-in standard curve through the antibody level in numerical value, are convenient for clinical analysis and are more beneficial to clinical application.

TABLE 11 equations corresponding to the PRVgD antibody in-vivo labeling and decision results

PRVgDgE antibody detection is carried out on three groups of porcine pseudorabies virus challenge serial samples of 5-7 groups, and the result (see table 12) is as follows: the results of the kit A and the kit B for detecting different types of strains (classical strains and variant strains) are consistent, the detection time is earlier than that of a PRVgE antibody IDEXX commercial kit, the sensitivity is high, the results are superior to the results of virus neutralization tests, and the development trend of antibody growth is met; and the kit A and the kit B are simple to operate and short in reaction time, so that the virus can be purified more conveniently, and the health condition of the swinery can be better controlled. And the PRVgD antibody is detected, the result kit A and the result kit B are consistent with the BioChek kit and the neutralization test result, and the detection result is exact.

TABLE 12 summary of results of series of samples tested by different methods for detecting porcine pseudorabies virus

The sera of groups 4, 1, 2, 6, 7 were tested in different ways to compare the difference in the levels of the two antibodies of PRVgDgE, and the results (see table 13): the 1 st group and the 2 nd group are vaccine immunization groups, and the S/N values of the 1 st group and the 2 nd group are obviously reduced when the kit A and the kit B detect the PRVgE antibodies which are negative and the PRVgD antibodies, namely the PRVgD antibodies show an obviously increased trend and the antibody level is gradually improved, which is consistent with the detection result trends of commercial kits and pilot test tests. Compared with the groups 1 and 2, the pre-toxicity-attacking kit A, the kit B, the commercial kit and the neutralization test method are negative when the group 6 is an immune toxicity-attacking group and the group 7 is an toxicity-attacking control group, PRVgE antibodies in each group are detected by the kit A and the kit B before toxicity attacking, the trend of detecting the PRVgD antibodies in the group 6 is consistent with that of the groups 1 and 2, and the antibody level of the group 7 is low because the group 7 is not immunized.

After the challenge, the kit A and the kit B detect that the PRVgD antibodies in the 6 th and 7 th groups rapidly rise and rise to very high titer (up to 4.0 after 1 week of challenge), and the PRVgD antibodies still maintain high titer (up to more than 3.5) after 2 weeks of challenge, which is equivalent to the detection trend of commercial kits and neutralization tests; the PRVgE antibodies of the 6 th and 7 th groups after virus attack are obviously increased and detected to be positive by the kit a and the kit B, the PRVgE antibodies of the 2 th groups after virus attack are further obviously increased, while the PRVgE antibodies of the 1 st groups after virus attack are obviously increased but still negative or suspicious (with the sign of turning positive), the PRVgE antibodies of the 2 th groups after virus attack are turned positive by the commercial IDEXX kit, and the PRV antibodies of the 1 st groups after virus attack are negative and the PRV antibodies of the 2 th groups after virus attack are positive by the neutralization test. In addition, PRVgD antibody levels after challenge in groups 6 and 7 were significantly higher than PRVgD antibody levels after immunization in groups 1 and 2 and before challenge after immunization in group 6. According to the data and the detection condition of clinical tests, if PRV antibodies are detected to be positive by using a commercial BioChek PRVgB kit, whether the pig or a pig herd is infected with the porcine pseudorabies virus cannot be directly judged, and further, the commercial IDEXX PRVgE kit or a neutralization test is used for confirmation. The kit A can identify and diagnose PRV immunity or infection conditions of the pigs or the swinery and infection processes of the pigs or the swinery through one-time detection, namely, the health state or early, middle or late infection of the pigs or the swinery can be comprehensively judged through the PRVgD antibody level and the PRVgE antibody level, so that the clinical establishment of an immunity strategy and a purification measure can be guided.

TABLE 13 summary of the results of the detection of the porcine pseudorabies virus immune and challenge series of samples by different methods

Note: the BioChek PRVgB kit judges that the standard S/P is more than or equal to 0.5 and is positive, and the S/P is less than 0.5 and is negative; the IDEXX PRVgE kit judges that the standard S/N is less than or equal to 0.6 and is positive, the S/N is more than 0.7 and is negative, and the S/N is more than 0.6 and less than or equal to 0.7 and is suspicious; the virus neutralization test has the judgment standard that the virus neutralization test is positive when the ratio is more than or equal to 1: 2 and negative when the ratio is less than 1: 2.

And (3) detecting serum samples 28 days and 35 days after the 21-day-old piglets immunized by the 4 commercial porcine pseudorabies virus vaccines in the groups 2 and 9-11, wherein the results are as follows: the results of the PRVgD antibody detected by the kit A and the kit B are consistent with the results of the commercial BioChek PRVgB kit and the neutralization test for 28 days and 35 days, and are positive, so that the kit A and the kit B meet the growth trend of the antibody; the PRVgE antibodies were all negative and the herd was healthy.

4.1.2 classical swine fever virus antibodies

2 parts of swine fever virus antibody positive serum (the neutralization titer is 1: 5120 determined by swine fever neutralization immunofluorescence test operating protocol (SN/T1379.3-2006)) are diluted by 10 times respectively and diluted by 2 times, and are detected by a kit B and a commercial IDEXX swine fever virus antibody detection kit respectively.

From the results, 2 sets of samples positive for gradient dilution were tested, and the results are shown in table 14: the detection result of the reagent kit B for detecting the antibody of the classical swine fever virus is consistent with that of the IDEXX commercial reagent kit, the antibodies of the classical swine fever virus detected by 10-160 times dilution of 2 serum samples are positive, and the antibodies of the classical swine fever virus detected by 320-640 times dilution are negative. The sensitivity of the positive sample detected by the kit B is equivalent to that of a commercial kit, and the coincidence condition is better.

TABLE 14 summary of the test results of gradient dilution of swine fever virus antibody positive serum samples by different methods

4.2 specificity

Detecting the porcine pseudorabies virus antibody negative serum 5 parts, porcine circovirus type 2 (PCV2) positive serum 1 part, Porcine Parvovirus (PPV) positive serum 1 part, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum 1 part, Classical Swine Fever Virus (CSFV) positive serum 1 part, porcine Japanese Encephalitis Virus (JEV) positive serum 1 part, Haemophilus Parasuis (HPs) positive serum 1 part, porcine atrophic rhinitis (Bb) positive serum 1 part, Escherichia coli (BL21) positive serum 1 part, mycoplasma hyopneumoniae (Mhp) positive serum 1 part, Porcine Epidemic Diarrhea Virus (PEDV) positive serum 1 part, porcine Transmissible Gastroenteritis (TGEV) positive serum 1 part, porcine rotavirus (PRoV) positive serum 1 part by respectively using the kit A, the kit B and the neutralization test, the commercialized BioCheVgB antibody kit and the EXVX PRgE antibody detection kit prepared in the embodiment 2, as a result: the three kits are negative in detection, which shows that the kit A has no cross reaction with positive serum of other viruses, and the kit B has no cross reaction with the positive serum of other viruses except the positive serum of the hog cholera virus, and has good specificity.

4.3 clinical applications

4.3.1 collect 2099 samples of 32 provinces and 76 pig farms in Henan, Shanxi, Jiangxi, Liaoning, Shandong, Zhejiang and Sichuan. And respectively detecting by using a kit A and a kit B, and simultaneously rechecking by using a BioChek PRVgB antibody detection kit, an IDEXX PRVgE antibody detection kit and an IDEXX CSFV antibody detection kit. Results (see table 15): the coincidence rate of the result of detecting the PRVgD antibody by the kit A and the kit B with the BioChek kit is 96.82 percent (1429/1476), and the coincidence rate of the result of detecting the PRVgE antibody with the IDEXX kit is 98.22 percent (1929/1964). The result of the CSFV antibody detection by the kit B corresponds to 97.63% (1851/1896) of IDEXX kit.

Table 15A clinical trial results

Table 15B clinical trial results

4.3.2 selecting a certain pig farm and a certain piglet farm with clear immune background (a temporary non-immune swine fever vaccine), and collecting serum of the immune porcine pseudorabies live vaccine after 28 days, wherein the serum comprises a breeding boar, a breeding sow and a piglet. Test results and analytical results (see table 16): the kit B detects that the CSFV antibodies are all negative (not shown in Table 16), the kit A and the kit B detect that the PRVgD antibodies and the PRVgE antibodies have consistent results, the kit A, B detects that the PRVgD antibodies and the PRVgE antibodies have the same results with the commercial kit and virus neutralization test results, particularly, the PRVgD antibodies of the pigs with good immune effect are high and the PRVgE antibodies are negative, the eliminated PRVgD antibodies and PRVgE antibodies of the pigs are higher and positive, and the PRVgD antibodies and the PRVgE antibodies of the pigs needing PRV vaccine immunization are negative. The kit A, B is shown to be capable of effectively evaluating the immune effect of the porcine pseudorabies vaccine and effectively judging whether the porcine pseudorabies vaccine is infected by wild viruses or not, and is more favorable for controlling the health condition of the swinery at any time through one-time detection in clinic.

TABLE 16 pig farm test results and analysis

Note: the BioChek PRVgB kit judges that the standard S/P is more than or equal to 0.5 and is positive, and the S/P is less than 0.5 and is negative; the IDEXX PRVgE kit judges that the standard S/N is less than or equal to 0.6 and is positive, the S/N is more than 0.7 and is negative, and the S/N is more than 0.6 and less than or equal to 0.7 and is suspicious; the virus neutralization test has the judgment standard that the virus neutralization test is positive when the ratio is more than or equal to 1: 2 and negative when the ratio is less than 1: 2.

The 11# to 20# piglets are all attacked by the virus with the attacking dose of 10 porcine pseudorabies virus HN1201 strain^6.0TCID₅₀First, the clinical symptoms were observed after challenge and the results are shown in Table 17. And PCR identification is carried out on No. 13 piglets and No. 16 piglets, and results show that the piglets are infected by wild strains of the porcine pseudorabies virus. The 13# piglet was found by kit A, B to be suspicious by PRVgD antibody and positive by PRVgE antibody, while the commercial Biochek PRVgB antibody was detected to be negative, IDEXX PRVgE antibody was negative, and neutralizing antibody was detected to be negativeThe positive result shows that the sensitivity of the kit A, B is higher than that of a commercial kit, and whether wild virus is infected or not can be found earlier.

TABLE 17 pig farm test results and analysis

The data analysis in table 16 and table 17 shows that: the kit A and the kit B can monitor the health conditions of the breeding pigs and the piglets by simultaneously detecting the PRVgD antibody and the PRVgE antibody, so that an immune program or a purification measure can be accurately formulated; furthermore, the piglet toxicity attacking test and the PCR detection result prove that the detection result of the kit A is accurate, and corresponding measures can be accurately guided to be clinically taken.

In conclusion, the kit prepared by the invention can be used for simultaneously detecting the PRVgD antibody, the PRVgE antibody and even the hog cholera virus antibody, has accurate and reliable results, and can be used for detecting the infection state of the porcine pseudorabies virus in a swinery, evaluating the immune effect of the porcine pseudorabies virus vaccine in the swinery and identifying the infection condition of newly introduced porcine pseudorabies virus.

The kit prepared by the invention has higher coincidence rate of the detection result of the PRVgD antibody than the virus neutralization test result, is suitable for evaluating the vaccine immunization effect and immunization program scientificity prepared by different strains of the porcine pseudorabies virus, particularly reflects the antibody level by numerical value and realizes the semi-quantitative detection of the PRVgD antibody in-vivo labeled koji, is convenient for clinical analysis and is more beneficial to clinical application; the sensitivity for detecting the PRVgE antibody is high, the method is suitable for early warning of wild virus (classical strains and variant strains) infection, and the early purification of pseudorabies is easy.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.