Universal enzyme marker diluent and sample diluent combination for enzyme-linked immunosorbent assay kit, kit containing the same and preparation method thereof

Abstract

The invention provides a universal enzyme marker diluent and sample diluent combination of an enzyme-linked immunosorbent assay kit, wherein the enzyme marker diluent is a 0.02M PBS solution containing 5% V / V fetal bovine serum, 1%-3% W / V mannitol, 2% W / V sucrose, 0.05% V / V Proclin300, and pH 7.4, and the sample diluent is a 0.02M PBS solution containing 0.4%-0.8% W / V gelatin, 0.1% V / V glycerol, 0.1% W / V EDTA, 0.05% V / V Proclin300, and pH 7.4. The enzyme marker diluent and sample diluent combination of the invention can be used for the preparation of animal disease ELISA antibody detection kits for various animal diseases and various different detection types (including competitive method, indirect method, blocking method, sandwich method), and the antibody sensitivity of each prepared kit for detecting various animal diseases is equivalent to or even better than that of the original kit, and the specificity is good.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a universal enzyme marker diluent and sample diluent combination of an enzyme-linked immunosorbent assay kit, a kit containing the same, and a preparation method thereof. Background Art

[0002] Since its introduction in the early 1970s, the enzyme-linked immunosorbent assay (ELISA) has developed rapidly and has been widely used in many fields of biology and medical science. In the field of biological detection, it is common to use ELISA to detect antibodies. The main detection types include competitive method, blocking method, indirect method and sandwich method.

[0003] Most biotechnology companies have multiple ELISA products for clinical testing. However, due to the different detection types, enzyme marker diluents, sample diluents and other components of multiple ELISA products, the formulas of each test kit are inconsistent, resulting in cumbersome preparation of each component and complex processes during large-scale production, as well as high labor and material costs.

[0004] Therefore, there is a need in the prior art to prepare universal enzyme marker diluents and sample diluents for different ELISA kits, so as to solve the problem of separately detecting multiple components and respective contents of test enzyme marker diluents and sample diluents in different kits while ensuring detection sensitivity. Summary of the invention

[0005] Based on this, an object of the present invention is to provide a universal enzyme marker diluent and sample diluent combination for an enzyme-linked immunosorbent assay kit, wherein the enzyme marker diluent is a 0.02M PBS solution containing 5% V / V fetal bovine serum, 1% to 3% W / V mannitol, 2% W / V sucrose, 0.05% V / V Proclin300, and pH 7.4; and the sample diluent is a 0.02M PBS solution containing 0.4% to 0.8% W / V gelatin, 0.1% V / V glycerol, 0.1% W / V EDTA, 0.05% V / V Proclin300, and pH 7.4.

[0006] The enzyme marker diluent and sample diluent of the present invention can be used in combination to prepare a kit for animal disease ELISA antibody detection kits of various detection types (including competitive method, indirect method, blocking method, sandwich method), and the detection sensitivity can be higher than or equivalent to that of the original detection kit.

[0007] The animal disease ELISA antibody detection kit of the present invention can be used for the preparation of animal disease ELISA antibody detection kits for multiple animal diseases and multiple different detection types (including competitive method, indirect method, blocking method, sandwich method) by using enzyme marker diluent and sample diluent of specific composition. Moreover, the antibody sensitivity of each prepared kit for detecting each animal disease is equivalent to or even better than that of the original kit, and the specificity is good, which reduces the preparation simplicity during large-scale production and saves manpower and material costs.

[0008] The content of mannitol in the enzyme marker diluent can be 1% W / V, 1.1% W / V, 1.2% W / V, 1.3% W / V, 1.4% W / V, 1.5% W / V, 1.6% W / V, 1.7% W / V, 1.8% W / V, 1.9% W / V, 2% W / V, 2.1% W / V, 2.2% W / V, 2.3% W / V, 2.4% W / V, 2.5% W / V, 2.6% W / V, 2.7% W / V, 2.8% W / V, 2.9% W / V, or 3% W / V.

[0009] The content of gelatin in the sample diluent can be 0.4% W / V, 0.41% W / V, 0.42% W / V, 0.43% W / V, 0.44% W / V, 0.45% W / V, 0.46% W / V, 0.47% W / V, 0.48% W / V, 0.49% W / V, 0.5% W / V, 0.51% W / V, 0.52% W / V, 0.53% W / V, 0.54% W / V, 0.55% W / V, 0.56% W / V, 0.57% W / V, 0.58% W / V, 0.59% W / V, 0.6%W / V, 0.61%W / V, 0.62%W / V, 0.63%W / V, 0.64%W / V, 0.65%W / V, 0.66%W / V, 0.67%W / V, 0.68%W / V, 0.69%W / V, 0. 7%W / V, 0.71%W / V, 0.72%W / V, 0.73%W / V, 0.74%W / V, 0.75%W / V, 0.76%W / V, 0.77%W / V, 0.78%W / V, 0.79%W / V, 0.8%W / V.

[0010] As an embodiment of the present invention, the enzyme marker diluent is a 0.02M PBS solution containing 5% V / V fetal bovine serum, 1% W / V mannitol, 2% W / V sucrose, 0.05% V / V Proclin300, and pH7.4; the sample diluent is a 0.02M PBS solution containing 0.4% W / V gelatin, 0.1% V / V glycerol, 0.1% W / V EDTA, 0.05% V / V Proclin300, and pH7.4.

[0011] As an embodiment of the present invention, the enzyme marker dilution liquid is a dilution liquid of the following enzyme markers: enzyme-labeled porcine circovirus type 2 monoclonal antibody 3H11; enzyme-labeled novel coronavirus protein S1, the novel coronavirus protein S1 is shown in SEQ ID No.1; enzyme-labeled sheep anti-swine IgA antibody; enzyme-labeled foot-and-mouth disease virus type A monoclonal antibody 3B4, the foot-and-mouth disease virus type A monoclonal antibody 3B4 heavy chain variable region is shown in SEQ ID No.4, the foot-and-mouth disease virus type A monoclonal antibody 3B4 light chain variable region is shown in SEQ ID No.5; enzyme-labeled foot-and-mouth disease virus type O monoclonal antibody 7D2, the enzyme-labeled foot-and-mouth disease virus type O monoclonal antibody 7D2 heavy chain variable region is shown in SEQ ID No.8, the enzyme-labeled foot-and-mouth disease virus type O monoclonal antibody 7D2 light chain variable region is shown in SEQ ID As shown in No.9; enzyme-labeled CSFV monoclonal antibody 15A9; enzyme-labeled PRVgE protein monoclonal antibody 11H1; and enzyme-labeled sheep anti-pig IgG or enzyme-labeled rabbit anti-pig IgG.

[0012] As an embodiment of the present invention, the sample diluent is a diluent for the following samples: serum samples suspected of being infected with porcine circovirus type 2, serum samples suspected of being infected with the new coronavirus, milk samples, serum samples, saliva samples, anal swab samples, and fecal samples suspected of being infected with PEDV, serum samples suspected of being infected with foot-and-mouth disease virus type A, serum samples suspected of being infected with foot-and-mouth disease virus type O, serum samples suspected of being infected with CSFV, serum samples suspected of being infected with pseudorabies virus, and serum samples suspected of being immunized with pseudorabies virus antigen.

[0013] The combined use of the enzyme marker diluent and the sample diluent of the present invention can ensure higher sensitivity, which can not only meet the detection of IgG antibodies in serum, but also ensure the detection of low-content IgA and early-produced IgM antibodies, realize the early and timely detection of pathogen infection and the detection of different test samples, and can meet various clinical detection conditions and needs.

[0014] As an embodiment of the present invention, the enzyme-linked immunosorbent assay kit is a porcine circovirus type 2 competitive ELISA antibody detection kit, a new coronavirus double antigen sandwich ELISA antibody detection kit, a porcine epidemic diarrhea virus indirect ELISA antibody detection kit, a foot-and-mouth disease virus type A competitive ELISA antibody detection kit, a foot-and-mouth disease virus type O competitive ELISA antibody detection kit, a classical swine fever virus blocking ELISA antibody detection kit, a pseudorabies virus gE protein blocking ELISA antibody detection kit, and a pseudorabies virus gD protein indirect ELISA antibody detection kit.

[0015] As an embodiment of the present invention, the new coronavirus double antigen sandwich ELISA antibody detection kit is an IgM antibody detection kit or an IgG antibody detection kit, and the porcine epidemic diarrhea virus indirect ELISA antibody detection kit is an IgA antibody detection kit or an IgG antibody detection kit.

[0016] The present invention also relates to an enzyme-linked immunosorbent assay kit, wherein the kit comprises: an antigen-coated plate coated with an animal disease antigen; an enzyme-labeled reagent, wherein the enzyme-labeled reagent is prepared by diluting the enzyme-labeled animal disease antibody or antigen with the enzyme-labeled diluent, filtering and then packaging; the sample diluent; a washing solution; a color developing solution; and a stop solution.

[0017] As an embodiment of the present invention, the kit further includes a negative control and a positive control.

[0018] As an embodiment of the present invention, when the enzyme-linked immunosorbent assay kit is a porcine circovirus type 2 competitive ELISA antibody detection kit, the animal disease antigen is porcine circovirus type 2 Cap protein; when the enzyme-linked immunosorbent assay kit is a new coronavirus double antigen sandwich ELISA antibody detection kit, the animal disease antigen is the new coronavirus protein S1, and the new coronavirus protein S1 is as shown in SEQ ID No.1; when the enzyme-linked immunosorbent assay kit is a porcine epidemic diarrhea virus indirect ELISA antibody detection kit, the animal disease antigen is an antigen-antibody complex of porcine epidemic diarrhea virus monoclonal antibody 8A3A10-porcine epidemic diarrhea virus HN1301 strain inactivated antigen, the porcine epidemic diarrhea virus monoclonal antibody 8A3A10 is secreted by hybridoma cell 8A3A10 strain, the hybridoma cell 8A3A10 has a preservation number of CCTCC No.C2014197, and the porcine epidemic diarrhea virus HN1301 strain has a preservation number of CCTCC No: V201514; when the enzyme-linked immunosorbent assay kit is a competitive ELISA antibody detection kit for foot-and-mouth disease virus type A, the animal disease antigen is an antigen-antibody complex of foot-and-mouth disease virus type A monoclonal antibody 2E11-foot-and-mouth disease virus type A virus-like particle protein, the heavy chain variable region of the foot-and-mouth disease virus type A monoclonal antibody 2E11 is shown as SEQ ID No.2, and the light chain variable region is shown as SEQ ID No.3; when the enzyme-linked immunosorbent assay kit is a competitive ELISA antibody detection kit for foot-and-mouth disease virus type O, the animal disease antigen is an antigen-antibody complex of foot-and-mouth disease virus type O monoclonal antibody 6G6-foot-and-mouth disease virus type O virus-like particle protein, the heavy chain variable region of the foot-and-mouth disease virus type O monoclonal antibody 6G6 is shown as SEQ ID No.6, and the light chain variable region is shown as SEQ ID As shown in No.7; when the enzyme-linked immunosorbent assay kit is a classical swine fever virus blocking ELISA antibody detection kit, the animal disease antigen is classical swine fever virus E2 protein; when the enzyme-linked immunosorbent assay kit is a pseudorabies virus gE protein blocking ELISA antibody detection kit, the animal disease antigen is a mixture of inactivated antigens of the pseudorabies virus HN1201 strain and the Fa strain inactivated antigen, and the pseudorabies virus HN1201 strain preservation number is CCTCC No: V201311; and when the enzyme-linked immunosorbent assay kit is a pseudorabies virus gD protein indirect ELISA antibody detection kit, the animal disease antigen is pseudorabies virus gD protein.

[0019] The present invention also relates to a method for preparing an enzyme-linked immunosorbent assay kit, wherein the method comprises: step (1) diluting and coating an animal disease coating material with a pH 9.6, 0.05 mol / L carbonate buffer to prepare an antigen-coated plate; step (2) diluting the enzyme-labeled animal disease antibody or antigen with the enzyme labeling diluent, filtering and packaging to prepare an enzyme-labeled reagent; and step (3) preparing the sample diluent, washing solution, color developing solution, and stop solution, and assembling the sample diluent, washing solution, color developing solution, and stop solution together with the antigen-coated plate prepared in step (1) and the enzyme-labeled reagent prepared in step (2) to form a kit.

[0020] As an embodiment of the present invention, the animal disease coating source in step (1) of the preparation method includes porcine circovirus type 2 PCV2 Cap protein, new coronavirus recombinant protein S1, antigen-antibody complex of porcine epidemic diarrhea virus monoclonal antibody 8A3A10 and inactivated antigen of porcine epidemic diarrhea virus HN1301 strain, antigen-antibody complex of type A foot-and-mouth disease virus monoclonal antibody 2E11 and type A foot-and-mouth disease virus-like particle protein, antigen-antibody complex of type O foot-and-mouth disease virus monoclonal antibody 6G6 and type O foot-and-mouth disease virus-like particle protein, classical swine fever virus E2 protein, pseudorabies virus, and pseudorabies virus gD protein; wherein the new coronavirus recombinant protein S1 is encoded by the nucleotide sequence shown in SEQ ID No.1, the heavy chain variable region of type A foot-and-mouth disease virus monoclonal antibody 2E11 is encoded by the nucleotide sequence shown in SEQ ID No.2, the light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No.3, the heavy chain variable region of type O foot-and-mouth disease virus monoclonal antibody 6G6 is encoded by the nucleotide sequence shown in SEQ ID The light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No.6 and the light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No.7.

[0021] As an embodiment of the present invention, the enzyme-labeled animal disease antibody or antigen in step (2) of the preparation method correspondingly includes porcine circovirus type 2 monoclonal antibody 3H11, new coronavirus recombinant protein S1, sheep anti-swine IgA antibody, type A foot-and-mouth disease virus monoclonal antibody 3B4, type O foot-and-mouth disease virus monoclonal antibody 7D2, classical swine fever virus monoclonal antibody 15A9, pseudorabies virus monoclonal antibody 11H1, sheep anti-swine IgG-HRP or rabbit anti-swine IgG-HRP; wherein the new coronavirus recombinant protein S1 is encoded by the nucleotide sequence shown in SEQ ID No.1, the heavy chain variable region of the type A foot-and-mouth disease virus monoclonal antibody 3B4 is encoded by the nucleotide sequence shown in SEQ ID No.4, and the light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No.5, and the heavy chain variable region of the type O foot-and-mouth disease virus monoclonal antibody 7D2 is encoded by the nucleotide sequence shown in SEQ ID No.8, and the light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No.9.

[0022] As an embodiment of the present invention, the enzyme marker diluent in step (2) is a 0.02M PBS solution containing 5% V / V fetal bovine serum, 1% W / V to 3% W / V mannitol, 2% W / V sucrose, 0.05% V / V Proclin300, and pH 7.4; preferably, the enzyme marker diluent is a 0.02M PBS solution containing 5% V / V fetal bovine serum, 1% W / V mannitol, 2% W / V sucrose, 0.05% V / V Proclin300, and pH 7.4.

[0023] As an embodiment of the present invention, the sample diluent in step (3) is a 0.02M PBS solution with a pH of 7.4 containing 0.4% W / V to 0.8% W / V gelatin, 0.1% V / V glycerol, 0.1% W / V EDTA, and 0.05% V / V Proclin300; preferably, the sample diluent is a 0.02M PBS solution with a pH of 7.4 containing 0.4% W / V gelatin, 0.1% V / V glycerol, 0.1% W / V EDTA, and 0.05% V / V Proclin300.

[0024] As an embodiment of the present invention, the color developing solution in step (3) includes color developing solution A and color developing solution B, wherein the color developing solution A is a solution containing 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid, and 0.03% w / v urea peroxide, and the color developing solution B is a solution containing 0.02% w / v tetramethylbenzenediamine and 10% v / v anhydrous ethanol; and the stop solution is a 2MH2SO4 solution.

[0025] The invention also relates to the use of the enzyme marker diluent and the sample diluent in combination to prepare an enzyme-linked immunosorbent assay kit.

[0026] The enzyme marker diluent and the sample diluent prepared by the present invention are combined to prepare an animal disease ELISA antibody detection kit, which can be used for the preparation of animal disease ELISA antibody detection kits of multiple animal diseases and multiple different detection types (including competitive method, indirect method, blocking method, sandwich method) at the same time, and the antibody sensitivity of each prepared kit for detecting each animal disease is equivalent to or even better than that of the original kit, and the specificity is good, which reduces the preparation simplicity during large-scale production and saves manpower and material costs. DETAILED DESCRIPTION

[0027] Hereinafter, some exemplary embodiments of the present invention are described in detail so that the present invention can be more easily understood by those skilled in the art.

[0028] The term "enzyme-linked immunosorbent assay kit" is also known as ELISA kit, ELISA test kit, ELISA Kit, enzyme-linked immunosorbent assay kit, enzyme-linked immunosorbent assay kit, enzyme-linked immunosorbent assay kit, enzyme-linked immunosorbent assay kit, etc.

[0029] The term "animal disease envelope antigen" refers to antigens or proteins related to animal diseases, including but not limited to whole animal disease viruses, animal disease virus proteins, animal disease virus monoclonal antibodies-whole animal disease viruses complexes, and animal disease virus monoclonal antibodies-animal disease virus protein complexes.

[0030] In the present invention, "enzyme-labeled antibody" can be used interchangeably with "labeled antibody", which is a monoclonal antibody mainly labeled with an enzyme. The term "enzyme" includes but is not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, and calf intestinal alkaline phosphatase. Among them, the substrate used by horseradish peroxidase HRP is o-phenylenediamine (OPD), tetramethylbenzidine (TMB), aminosalicylic acid, o-benzylamine or 2,2'-amino-2 (3-ethyl-thiazolinesulfonic acid-6) ammonium salt, preferably tetramethylbenzidine (TMB); the substrate used by alkaline phosphatase is p-nitrophenyl phosphate (p-NPP) or naphthol-AS-Mx phosphate + diazonium salt; the substrate used by glucose oxidase is ABTS + HRP + glucose or glucose + methylthiophenol + thiazolyl blue; the substrate used by β-galactosidase is 4-methylumbelliferyl-β-D galactoside (4MUG) or nitrophenol galactoside (ONPG).

[0031] The term "phosphate buffer" refers to a solution containing phosphoric acid or its salt and adjusted to an ideal pH value, and is the most widely used buffer in biochemical research. Generally, phosphate buffer is prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are already known in the art, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate. It is known that phosphates exist in the form of salt hydrates. Due to the secondary dissociation of the buffer, the pH range of the buffer is very wide, such as the range of about pH 4 to about pH 10, preferably the range of about pH 5 to pH 9, more preferably the range of about pH 7 to about pH 8, and most preferably about pH 7.4. Further preferably, the phosphate buffer is a phosphate buffer containing sodium chloride and potassium chloride. In the present invention, "phosphate buffer" and PBS are used interchangeably.

[0032] The term "preservative" includes, but is not limited to, gentamicin, kanamycin sulfate, sodium azide, thimerosal and its salts, and Proclin300.

[0033] The terms "enzyme marker diluent" and "enzyme label diluent" are used interchangeably.

[0034] The term "pH 9.6, 0.05 mol / L carbonate buffer" refers to a buffer composed of sodium carbonate and sodium bicarbonate, which is a conventional buffer used for ELISA coated plates.

[0035] The porcine monoclonal antibodies involved in the present invention mainly include:

[0036] (1) Porcine circovirus type 2 monoclonal antibody 3H11, see Chinese patent CN105542000A.

[0037] (2) porcine epidemic diarrhea virus monoclonal antibody 8A3A10, which is secreted and expressed by mouse myeloma hybridoma cell 8A3A10, wherein the hybridoma cell strain 8A3A10 has a deposit number of CCTCC No. C2014197, and is deposited with China Center for Type Culture Collection, Wuhan University, Wuhan, China, on November 3, 2014, see Chinese patents CN104694480A and CN110967480A.

[0038] (3) The heavy chain variable region of monoclonal antibody 2E11 against type A foot-and-mouth disease virus is encoded by SEQ ID No. 2, and the light chain variable region is encoded by SEQ ID No. 3; the heavy chain variable region of monoclonal antibody 3B4 is encoded by SEQ ID No. 4, and the light chain variable region is encoded by SEQ ID No. 5.

[0039] (4) The heavy chain variable region of monoclonal antibody 6G6 against type O foot-and-mouth disease virus is encoded by SEQ ID No.6, and the light chain variable region is encoded by SEQ ID No.7; the heavy chain variable region of monoclonal antibody 7D2 is encoded by SEQ ID No.8, and the light chain variable region is encoded by SEQ ID No.9.

[0040] (5) Classical swine fever virus monoclonal antibody 15A9, see Chinese patent CN105837686A.

[0041] (6) Monoclonal antibody 11H1 against gE protein of pseudorabies virus, see Chinese patent CN109959788A.

[0042] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. These embodiments are exemplary only and are not the only form of implementing or applying the specific embodiments of the present invention, and therefore do not constitute any limitation to the scope of the present invention. The following description of the embodiments covers the features of multiple specific embodiments and the method steps and their sequences for constructing and operating these specific embodiments. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0043] The PBS buffer used in the embodiments of the present invention is prepared as follows: 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, adjusted to pH 7.4, and fixed to 1 L, including but not limited to this formula; chemical reagents are all analytically pure and purchased from Sinopharm Group.

[0044] The experimental methods described in the present invention are all conventional methods unless otherwise specified; the biological materials described can be obtained from commercial channels unless otherwise specified.

[0045] Example 1 Preparation of enzyme marker diluent

[0046] Prepare enzyme marker dilution solutions A1 to A5 according to Table 1.

[0047] Table 1 Summary of enzyme marker diluent components

[0048]

[0049] In the preparation process of the kit of the present invention, the detection effect is equivalent when the content of mannitol in the enzyme marker diluent is 1% W / V to 3% W / V. For the convenience of briefly describing the embodiment of the present invention, only the content of mannitol in the enzyme marker diluent is 1% W / V as an example.

[0050] Example 2 Preparation of sample diluent

[0051] Prepare sample diluents B1 to B6 according to Table 2.

[0052] Table 2 Summary of components contained in sample diluent

[0053]

[0054] In the preparation process of the kit of the present invention, the detection effect is equivalent when the gelatin content in the sample diluent is 0.4% W / V to 0.8% W / V. For the convenience of briefly describing the embodiment of the present invention, only the gelatin content in the sample diluent is 0.4% W / V as an example.

[0055] Example 3 Porcine circovirus type 2 ELISA antibody detection kit (competitive method)

[0056] 3.1 Preparation of the kit

[0057] The enzyme marker diluents A1 to A5 prepared in Example 1 were used respectively to prepare the kits PCV2A1 to PCV2A5 according to the preparation method of the kit in patent CN105542000A (wherein the coating material is porcine circovirus type 2 Cap protein, see accession number KF524259.1, and the enzyme marker is enzyme-labeled porcine circovirus type 2 monoclonal antibody 3H11), and the detection was carried out according to the detection method, and the optimal enzyme marker dilution was selected.

[0058] 3.2 Evaluation of kits PCV2A1 to PCV2A5

[0059] Sensitivity test: The porcine circovirus type 2 positive serum with a titer of 1:3200 in the immunoperoxidase monolayer cell test IPMA was diluted in multiple proportions, and the sensitivity test was evaluated using kits PCV2A1 to PCV2A5, and the kit prepared in the CN105542000A method was used as the PCV2 original kit for comparison; the results are shown in Table 3: The sensitivity of kits PCV2A1 to PCV2A4 was weaker than that of the PCV2 original kit, and only the sensitivity of kit PCV2A5 was higher than that of the PCV2 original kit.

[0060] Table 3 Summary of test results of kit PCV2A1~PCV2A5

[0061]

[0062] Specificity test: The kits PCV2A1~PCV2A5 and the original PCV2 kit were used to perform specificity tests on other swine virus positive sera (including porcine circovirus type 1 PCV1 positive serum, porcine circovirus type 3 PCV3 positive serum, classical swine fever virus CSFV positive serum, porcine reproductive and respiratory syndrome virus PRRSV positive serum, pseudorabies virus PRV positive serum, porcine parvovirus PPV positive serum, foot-and-mouth disease virus type A (A FMDV) positive serum, and foot-and-mouth disease virus type O (O FMDV) positive serum). The results were all negative, indicating good specificity.

[0063] Clinical application: Kits PCV2A1~PCV2A5 and the original PCV2 kit were used to detect 30 samples of porcine circovirus type 2 positive sera and 30 samples of negative sera (including 10 samples of porcine circovirus type 1 positive sera) identified by IPMA, respectively. The results are shown in Table 3: All negative samples detected by kits PCV2A1~PCV2A5 were negative, and there was no non-specific phenomenon; however, the positive compliance rate of the kit PCV2A1~PCV2A4 was significantly lower than that of the kit PCV2A5 and the original PCV2 kit.

[0064] The results showed that the enzyme marker diluent A5 can be used to prepare porcine circovirus type 2 ELISA antibody detection kit, and the kit has high sensitivity, good sensitivity and good specificity when detecting samples.

[0065] Example 4 Novel Coronavirus Double Antigen Sandwich ELISA Antibody Detection Kit (Sandwich Method)

[0066] 4.1 Preparation of the kit

[0067] The preparation method of the novel coronavirus double antigen sandwich ELISA antibody detection kit (sandwich method) is disclosed in patent application CN202010167856.3, as follows:

[0068] Antigen coated plate: Dilute the novel coronavirus recombinant protein S1 (its nucleotide sequence is shown in SEQ ID No. 1) prepared by genetic engineering means to 0.5 μg / ml with carbonate buffer (pH 9.6, 0.05 mol / L) for coating, 100 μl / well, 2-8°C for 16-24 hours, wash with washing solution, add blocking solution (phosphate buffer containing 5% M / V sucrose, 20% V / V newborn calf serum, 0.05% V / V ProClin300) at 150 μl / well, block at 2-8°C for 16-24 hours or 37°C for 2 hours, discard the blocking solution and dry at 18-26°C and relative humidity not higher than 30% for 3-6 hours, seal and store at 2-8°C for later use.

[0069] Enzyme labeling reagent: The enzyme-labeled novel coronavirus recombinant protein S1 was diluted with the enzyme labeling diluents A1 to A5 in Example 1 at 1:8000 as an enzyme labeling reagent, and then filtered and packaged aseptically.

[0070] 20× concentrated washing solution: Take 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800mL ultrapure water, and 10mL Tween 20. After complete dissolution, add ultrapure water to make up to 1000mL, filter with a 0.22μm filter membrane, and dispense aseptically. Dilute 20 times with distilled water before use.

[0071] Positive control: The new coronavirus recombinant protein S1 prepared in Example 1 was subcutaneously injected into healthy rabbits (500 μg / rabbit), and then immunized again after an interval of 14 days. Blood was collected and serum was separated after 14 days. The collected serum was the positive serum. 200 ml of newborn calf serum, 10 ml of positive serum, and 0.5 ml of ProClin-300 were mixed and supplemented with phosphate buffer to make the volume 1000 ml, and then filtered and aseptically packaged.

[0072] Negative control: Mix 200 ml of newborn calf serum and 0.5 ml of ProClin-300, and add phosphate buffer to make the volume 1000 ml. After filtering, sterile aliquot.

[0073] Color developing solution: Disodium hydrogen phosphate 14.7g, citric acid 9.3g, urea peroxide 0.3g, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution A. Tetramethylbenzidine (TMB) 0.2g, anhydrous ethanol 100ml, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution B.

[0074] Stop solution: 2M H2SO4 solution.

[0075] The above components are assembled into a kit, which is divided into kits nCoVA1 to nCoVA5 according to the different enzyme marker diluents A1 to A5.

[0076] 4.2 Establishment of detection method

[0077] The steps are as follows:

[0078] (1) Sample addition: Add 100 μl of the sample to be tested to the corresponding wells, set up 2 wells for positive control and 2 wells for negative control (both with 100 μl), seal the plate and incubate at 37°C for 30 min.

[0079] (2) Washing: Wash 5 times with detergent and dry for the last time.

[0080] (3) Add enzyme labeling reagent: Add 100 μl of enzyme labeling reagent diluent to each well, seal the plate and incubate at 37°C for 30 min.

[0081] (4) Washing: Wash 5 times with detergent and dry for the last time.

[0082] (5) Color development: Add 50 μl of color development solution A and 50 μl of color development solution B to each well, mix well, and develop color at 37°C or room temperature in the dark for 15 min.

[0083] (6) Stop and detect: Add 50 μl of stop solution to each well and measure the OD of each well using an ELISA reader within 10 minutes. 450nm The value is determined according to the following result judgment criteria.

[0084] When the positive control well OD 450nm When the value is ≥0.3 and the OD value of the negative control well is <0.1, the test is established, otherwise it is invalid. When the test is established, the critical value (CUT OFF) is calculated as: critical value = 0.26 + the average OD value of the negative control well (if the average OD value of the negative control well is lower than 0.03, it is calculated as 0.03). Result judgment:

[0085] Samples with an OD value ≥ the critical value (CUT OFF) are positive for novel coronavirus antibodies.

[0086] If the sample OD value is less than the critical value (CUT OFF), it is negative for the new coronavirus antibody. If the result is positive, on-site investigation and sampling are required for etiological verification.

[0087] 4.3 Evaluation of the kit

[0088] 4.3.1 Specificity test

[0089] The kits nCoVA1 to nCoVA5 were used to perform specific tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, CSFV positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, A FMDV positive serum, and OFMDV positive serum). The results were all negative, indicating good specificity.

[0090] 4.3.2 Preparation of positive sera from different animal sources

[0091] The novel coronavirus recombinant protein S1 (500 μg / mouse) was injected into 5 SPF mice and 5 healthy rabbits, and 5 mice were set up as non-immunized control groups. Blood was collected on days 0, 1, 3, 5, 7, 9, 10, and 14 after immunization, and serum was collected.

[0092] 4.3.3 Preparation of IgM antibody detection kit by indirect ELISA method

[0093] Take the antigen-coated plate with the coating concentration of the recombinant protein S1 of the new coronavirus at 0.5 μg / ml, and use the commercial enzyme-labeled goat anti-mouse IgM antibody and the enzyme-labeled goat anti-rabbit IgM antibody at a dilution of 1:10000 as the secondary antibody, together with the washing solution, the color developing solution, the stop solution, and the stop solution to prepare the indirect ELISA IgM antibody kit, referred to as the mouse IgM kit and the rabbit IgM kit.

[0094] 4.3.4 Preparation of IgG antibody detection kit by indirect ELISA method

[0095] Take the antigen-coated plate coated with the recombinant protein S1 of the new coronavirus at a concentration of 0.5 μg / ml, and use the commercial enzyme-labeled goat anti-mouse IgG antibody and the enzyme-labeled goat anti-rabbit IgG antibody at a dilution of 1:10000 as the secondary antibody, together with the washing solution, the color developing solution, the sample diluent, and the stop solution to prepare the indirect ELISA IgG antibody kit, referred to as the mouse IgG kit and the rabbit IgG kit.

[0096] 4.3.5 Comparison of different kits for detecting mouse IgM and IgG antibodies

[0097] The kits nCoVA1~nCoVA5, mouse IgM kit and mouse IgG kit were used to detect the sera of mice on days 0, 1, 3, 5, 7, 9, 10 and 14, respectively. The results are shown in Table 4: The serum tests of the control group were all negative, and the experiment was established. The mouse IgM kit could only detect antibodies on days 3 to 9 after immunization, and the mouse IgG kit could only detect antibodies on days 7 to 14, while the kits nCoVA1~nCoVA5 could detect antibodies on days 3 to 14 after immunization. However, only the antibodies detected by the kits nCoVA3 and nCoVA5 were equivalent to the sum of IgM and IgG antibodies, and the detection effects of the two were equivalent.

[0098] Table 4 Results of testing immunized mice on different days

[0099]

[0100]

[0101] 4.3.6 Comparison of different kits for detecting rabbit IgM and IgG antibodies

[0102] The rabbit sera on days 0, 1, 3, 5, 7, 9, 10 and 14 were tested with the nCoVA1~nCoVA5 kits, as well as the rabbit IgM kit and the rabbit IgG kit. The results are shown in Table 5: The sera of the control group were all negative, and the experiment was established. The rabbit IgM kit could only detect antibodies on days 3 to 9 after immunization, and the rabbit IgG kit could only detect antibodies on days 7 to 14, while the nCoVA1~nCoVA5 kits could detect antibodies on days 3 to 14 after immunization. However, only the antibodies detected by the nCoVA3 and nCoVA5 kits were equivalent to the sum of IgM and IgG antibodies, and the detection effects were equivalent.

[0103] Table 5 Results of testing immunized rabbits on different days

[0104]

[0105] This indicates that the kits nCoVA3 (containing enzyme-labeled diluent A3) and nCoVA5 (containing enzyme-labeled diluent A5) can be used for the detection of different animal sera. As long as there are antibodies to the new coronavirus, whether they are IgM or IgG antibodies, specific reactions can occur with good specificity.

[0106] Example 5 Porcine epidemic diarrhea virus ELISA antibody (IgA) detection kit (indirect method)

[0107] 5.1 Preparation of kits PEDVA1 to PEDVA5

[0108] Using the enzyme marker diluents A1 to A5 prepared in Example 1 and the sample diluent B6 prepared in Example 2, the kits PEDVA1 to PEDVA5 were prepared according to the preparation method of the kit in patent CN110967480A (wherein the coating was originally an antigen-antibody complex of porcine epidemic diarrhea virus monoclonal antibody 8A3A10-porcine epidemic diarrhea virus HN1301 strain inactivated antigen, and the HN1301 strain preservation number is CCTCC No: V201514; the enzyme labeling reagent was an enzyme-labeled sheep anti-swine IgA secondary antibody) and the detection was carried out according to the detection method thereof.

[0109] 5.2 Evaluation of Kits PEDVA1 to PEDVA5

[0110] Sensitivity test: The milk samples and serum samples identified by IPMA were tested with the kits PEDVA1~PEDVA5, respectively, and compared with the original PEDV kit (see patent CN110967480A for details, wherein the coating was originally the porcine epidemic diarrhea virus monoclonal antibody 8A3A10-porcine epidemic diarrhea virus HN1301 strain inactivated antigen complex, and the enzyme labeled reagent was enzyme-labeled sheep anti-swine IgA secondary antibody). The results are shown in Table 6. When the milk samples and serum samples were negative, the kits PEDVA1~PEDVA5 were also negative; when the milk samples and serum samples were positive, the kits PEDVA1~PEDVA5 were also positive, but only the kits PEDVA3~PEDVA5 had the same detection effect as the original PEDV kit.

[0111] Table 6 Sensitivity test results of kits PEDVA1 to PEDVA5

[0112]

[0113]

[0114] Specificity test: The kits PEDVA3 to PEDVA5 were used to detect other pig-originated virus positive sera (including porcine transmissible gastroenteritis virus TGEV positive serum, porcine deltacoronavirus PDCoV positive serum, porcine rotavirus PoRV positive serum, PRV positive serum, porcine parvovirus positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, CSFV positive serum, PPV positive serum, A FMDV positive serum, and O FMDV positive serum). The results were all negative, indicating that the kits PEDVA3 to PEDVA5 had good specificity.

[0115] Clinical application: A total of 400 clinical samples of multiple targets were detected using the original PEDV kit and kits PEDVA3~PEDVA5, including 100 breast milk (40 of which were colostrum), 100 serum, 75 saliva, 50 anal swabs, and 75 stool samples (all identified by IPMA, 240 positive and 160 negative). The coincidence rate of the samples with IPMA was calculated, and the results are shown in Table 7. The positive coincidence rate and negative coincidence rate of kits PEDVA3~PEDVA5 were both ≥81%, among which the positive coincidence rate of kits PEDVA4~PEDVA5 was ≥96%, and the negative coincidence rate was ≥98%, which was equivalent to the detection effect of the original PEDV kit.

[0116] Table 7 Clinical application test results of kits PEDVA3~PEDVA5

[0117]

[0118] 5.3 Preparation of kits PEDVB1 to PEDVB6

[0119] The enzyme marker diluent A5 prepared in Example 1 and the sample diluents B1 to B6 prepared in Example 2 were used to prepare the kits PEDVB1 to PEDVB6 according to the preparation method of the kit in patent CN110967480A and the detection was performed according to the detection method.

[0120] 5.4 Evaluation of Kits PEDVB1 to PEDVB6

[0121] Sensitivity test: The milk samples and serum samples identified by IPMA were tested with the PEDVB1~PEDVB6 kits, respectively, and compared with the original PEDV kit (see patent CN110967480A for details). The results are shown in Table 8. When the milk samples and serum samples were negative, the PEDVB1~PEDVB6 kits were also negative; when the milk samples and serum samples were positive, the PEDVB1~PEDVB6 kits were also positive. However, only the PEDVB1 and PEDVB6 kits had equivalent detection effects to the original PEDV kit.

[0122] Table 8 Test kit PEDVB1~PEDVB6 sensitivity test results

[0123]

[0124] Table 8 (Continued) Test kit PEDVB1 to PEDVB6 sensitivity test results

[0125]

[0126] Specificity test: The kits PEDVB1 and PEDVB6 were used to detect other pig virus positive sera (including porcine transmissible gastroenteritis virus positive serum, porcine deltacoronavirus positive serum, porcine rotavirus positive serum, porcine pseudorabies virus positive serum, porcine parvovirus positive serum, porcine circovirus type 2 positive serum, porcine circovirus type 3 positive serum, porcine reproductive and respiratory syndrome virus positive serum, classical swine fever virus positive serum, and porcine Japanese encephalitis virus positive serum). The results were all negative, indicating that the kits PEDVB1 and PEDVB6 have good specificity.

[0127] Clinical application: The 400 clinical samples used in Example 5.2 were used to calculate the coincidence rate of the samples with IPMA using the original kit, kits PEDVB1 and PEDVB6. The results are shown in Table 9. The positive coincidence rate and negative coincidence rate of the kits PEDVB1 and PEDVB6 were both ≥94%, among which the positive coincidence rate of the kit PEDVB6 was ≥96% and the negative coincidence rate was ≥98%, indicating that the sensitivity of the kit was equivalent to that of the original kit.

[0128] Table 9 Clinical application test results of PEDVB1 and PEDVB6 kits

[0129]

[0130] Example 6 Foot-and-mouth disease virus type A ELISA antibody detection kit (competitive method)

[0131] 6.1 Preparation of the kit

[0132] The preparation method of the foot-and-mouth disease virus type A ELISA antibody detection kit (competitive method) is disclosed in patent application CN201911203052.8, as follows:

[0133] Antigen coated plate: Use carbonate buffer (pH 9.6, 0.05 mol / L) to coat the foot-and-mouth disease virus type A monoclonal antibody 2E11 (the heavy chain variable region is shown in SEQ ID No. 2, and the light chain variable region is shown in SEQ ID No. 3) at 0.5 μg / well and 50 μl / well, gently shake and mix, and coat at 2-8°C for 16-24 hours; discard the liquid in the plate, wash with washing solution for 3-5 times, and then add blocking solution (containing 1.5% W / V BSA phosphate buffer), gently shake to mix, seal the plate with a sealing film and block at 37°C for 2 hours; discard the blocking solution, wash with a washing solution for 3 to 5 times, pat dry, add 10 μg / well, 50 μl / well of type A foot-and-mouth disease virus-like particle protein (according to CN105566449A), seal the plate with a sealing film and incubate at 37°C for 1 hour; discard the liquid in the plate, wash with a washing solution for 3 to 5 times, dry at 18°C ​​to 26°C and relative humidity not higher than 30% for 3 to 6 hours, seal and store at 2 to 8°C for use.

[0134] Enzyme labeling reagent: Foot-and-mouth disease virus type A monoclonal antibody 3B4 (the heavy chain variable region protein sequence is shown in SEQ ID No. 4, and the light chain variable region protein sequence is shown in SEQ ID No. 5) is diluted 5000 times with enzyme labeling diluents A1 to A5 and packaged aseptically.

[0135] Positive control: Highly immune serum against type A foot-and-mouth disease virus antigen, sterilely packaged.

[0136] Negative control: pig serum without foot-and-mouth disease antibodies, sterile aliquot.

[0137] Sample diluent: refer to Example 2.

[0138] 20× concentrated washing solution: Take 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800mL ultrapure water, and 10mL Tween 20. After complete dissolution, add ultrapure water to make up to 1000mL, filter with a 0.22μm filter membrane, and dispense aseptically. Dilute 20 times with distilled water before use.

[0139] Color developing solution: Disodium hydrogen phosphate 14.7g, citric acid 9.3g, urea peroxide 0.3g, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution A. Tetramethylbenzidine (TMB) 0.2g, anhydrous ethanol 100ml, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution B.

[0140] Stop solution: 2M H2SO4 solution.

[0141] The above kits were assembled into a kit. According to the different enzyme marker diluents (the sample diluent is B6), they were labeled as kits A_FMDVA1 to A_FMDVA5; according to the different sample diluents (the enzyme marker diluent is A5), they were labeled as kits A_FMDVB1 to A_FMDVB6.

[0142] 6.2 Establishment of detection method

[0143] All reagents should be equilibrated to room temperature (about 30 minutes) before use. Liquid reagents should be gently shaken to mix before the experiment and immediately sealed and stored at 2-8°C after use.

[0144] The steps are as follows:

[0145] (1) Sample addition: Dilute the sample to be tested 5-fold with sample diluent and add 50 μl to the corresponding wells of the microplate. Set up negative control wells (add 50 μl of negative control per well) and positive control wells (add 50 μl of positive control per well). Seal the plate and incubate at 37°C for 1 hour.

[0146] (2) Add enzyme labeling reagent: Add 50 μl of enzyme labeling reagent to each well, seal the plate and incubate at 37°C for 1 hour.

[0147] (3) Washing: Wash 5 times with detergent and dry for the last time.

[0148] (4) Color development: Add 50 μl of color development solution A and 50 μl of color development solution B to each well, mix well, and develop color at 37°C or room temperature in the dark for 15 min.

[0149] (5) Stop and detect: Add 50 μl of stop solution to each well, mix well, and measure the A value of each well with an ELISA reader within 10 minutes: OD 450nm The value is determined according to the following result judgment criteria.

[0150] When the negative control well A value OD 450nm The test is established when the value is ≥1.5 and the A value of the positive control well is ≤0.5, otherwise it is invalid. When the test is established, the blocking rate (PI) = (1-sample OD 450nm / average OD of negative control 450nm )×100%. Result determination: When the blocking rate is ≥50%, it is antibody positive; when the blocking rate is <50%, it is antibody negative.

[0151] 6.3 Detection of Kits A_FMDVA1 to A_FMDVA5

[0152] 6.3.1 Sensitivity evaluation

[0153] The confirmed type A foot-and-mouth disease virus positive serum was diluted 2-fold and tested using kits A_FMDVA1 to A_FMDVA5, respectively. The results are shown in Table 10. It was found that only kit A_FMDVA5 had a high sensitivity.

[0154] Table 10 Sensitivity test results of kits A_FMDVA1 to A_FMDVA5

[0155]

[0156] 6.3.2 Specificity test

[0157] The kit A_FMDVA5 was used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, CSFV positive serum, and OFMDV positive serum). The results were all negative, indicating that the kit A_FMDVA5 had good specificity.

[0158] 6.3.3 Clinical application

[0159] 170 clinical A FMDV positive sera were collected and compared with the commercial liquid phase blocking kit. The results showed that all 170 samples detected by the kit A_FMDVA5 were positive, which was better than the commercial liquid phase blocking kit (the detection rate was only 96.5%). This showed that the sensitivity of the kit A_FMDVA5 was good.

[0160] 6.4 Detection of Kits A_FMDVB1 to A_FMDVB6

[0161] 6.4.1 Sensitivity evaluation

[0162] The confirmed type A foot-and-mouth disease virus positive serum was diluted 2-fold and tested with kits A_FMDVB1 to A_FMDVB6 respectively. The results are shown in Table 11. It was found that only kit A_FMDVB6 had a high sensitivity.

[0163] Table 11 Sensitivity test results of kit A_FMDVB1~A_FMDVB6

[0164]

[0165] 6.4.2 Specificity test

[0166] The kit A_FMDVB6 was used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, CSFV positive serum, and OFMDV positive serum). The results were all negative, indicating that the kit A_FMDVB6 has good specificity.

[0167] 6.4.3 Clinical application

[0168] 170 clinical A FMDV positive sera were collected and compared with the commercial liquid phase blocking kit. The results showed that all 170 samples detected by the kit A_FMDVB6 were positive, which was better than the commercial liquid phase blocking kit (the detection rate was only 96.5%). This shows that the sensitivity of the kit A_FMDVB6 is good.

[0169] Example 7 Foot-and-mouth disease virus O type ELISA antibody detection kit (competitive method)

[0170] 7.1 Preparation of the kit

[0171] The preparation method of the foot-and-mouth disease virus O type ELISA antibody detection kit (competitive method) is disclosed in patent application CN201910881627.5, as follows:

[0172] Antigen coated plate: Use carbonate buffer (pH 9.6, 0.05 mol / L) to coat the foot-and-mouth disease virus type O monoclonal antibody 6G6 (the heavy chain variable region is shown in SEQ ID No.6, and the light chain variable region is shown in SEQ ID No.7) at 0.08 μg / well and 50 μl / well, gently shake and mix, and coat at 2-8°C for 16-24 hours; discard the liquid in the plate, wash with washing solution for 3-5 times, and add blocking solution (containing 1.5% W / V BSA phosphate buffer), gently shake to mix, seal the plate with a sealing film and block at 37°C for 2 hours; discard the blocking solution, wash with a washing solution for 3 to 5 times, pat dry, add 10 μg / well, 50 μl / well of O-type foot-and-mouth disease virus-like particle protein (according to CN105566449A), seal the plate with a sealing film and incubate at 37°C for 1 hour; discard the liquid in the plate, wash with a washing solution for 3 to 5 times, dry at 18°C ​​to 26°C and relative humidity not higher than 30% for 3 to 6 hours, seal and store at 2 to 8°C for use.

[0173] Enzyme labeling reagent: Foot-and-mouth disease virus type O monoclonal antibody 7D2 (the heavy chain variable region protein sequence is shown in SEQ ID No. 8, and the light chain variable region protein sequence is shown in SEQ ID No. 9) was diluted 5000 times with enzyme labeling diluents A1 to A5 and packaged aseptically.

[0174] Positive control: high immune serum against type O foot-and-mouth disease virus antigen, sterilely packaged.

[0175] Negative control: pig serum without foot-and-mouth disease antibodies, sterile aliquot.

[0176] Sample diluent: refer to Example 2.

[0177] 20× concentrated washing solution: Take 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800mL ultrapure water, and 10mL Tween 20. After complete dissolution, add ultrapure water to make up to 1000mL, filter with a 0.22μm filter membrane, and dispense aseptically. Dilute 20 times with distilled water before use.

[0178] Color developing solution: Disodium hydrogen phosphate 14.7g, citric acid 9.3g, urea peroxide 0.3g, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution A. Tetramethylbenzidine (TMB) 0.2g, anhydrous ethanol 100ml, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution B.

[0179] Stop solution: 2M H2SO4 solution.

[0180] The above kits are assembled into a kit. According to the different enzyme marker diluents (the sample diluent is B6), they are labeled as kits O_FMDVA1 to O_FMDVA5; according to the different sample diluents (the enzyme marker diluent is A5), they are labeled as kits O_FMDVB1 to O_FMDVB6.

[0181] 7.2 Establishment of detection method

[0182] All reagents should be equilibrated to room temperature (about 30 minutes) before use. Liquid reagents should be gently shaken to mix before the experiment and immediately sealed and stored at 2-8°C after use.

[0183] The steps are as follows:

[0184] (1) Sample addition: Dilute the sample to be tested 5-fold with sample diluent and add 50 μl to the corresponding wells of the microplate. Set up negative control wells (add 50 μl of negative control per well) and positive control wells (add 50 μl of positive control per well). Seal the plate and incubate at 37°C for 1 hour.

[0185] (2) Add enzyme labeling reagent: Add 50 μl of enzyme labeling reagent to each well, seal the plate and incubate at 37°C for 1 hour.

[0186] (3) Washing: Wash 5 times with detergent and dry for the last time.

[0187] (4) Color development: Add 50 μl of color development solution A and 50 μl of color development solution B to each well, mix well, and develop color at 37°C or room temperature in the dark for 15 min.

[0188] (5) Stop and detect: Add 50 μl of stop solution to each well, mix well, and measure the A value of each well with an ELISA reader within 10 minutes: OD 450nm The value is determined according to the following result judgment criteria.

[0189] When the negative control well A value OD 450nm The test is established when the value is ≥1.5 and the A value of the positive control well is ≤0.5, otherwise it is invalid. When the test is established, the blocking rate (PI) = (1-sample OD 450nm / average OD of negative control 450nm )×100%. Result determination: When the blocking rate is ≥50%, it is antibody positive; when the blocking rate is <50%, it is antibody negative.

[0190] 7.3 Detection of kits O_FMDVA1 to O_FMDVA5

[0191] 7.3.1 Sensitivity evaluation

[0192] The confirmed Southeast Asian type O-type foot-and-mouth disease virus-positive serum and the Chinese type O-type foot-and-mouth disease virus-positive serum were diluted 2-fold and tested using kits O_FMDVA1 to O_FMDVA5, respectively. The results are shown in Table 12. It was found that only kit O_FMDVA5 had a high sensitivity.

[0193] Table 12 Sensitivity test results of kits O_FMDVA1 to O_FMDVA5

[0194]

[0195] 7.3.2 Specificity test

[0196] The kit O_FMDVA5 was used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, CSFV positive serum, and O FMDV positive serum). The results were all negative, indicating that the kit O_FMDVA5 has good specificity.

[0197] 7.3.3 Clinical application

[0198] 186 clinical Southeast Asian type O FMDV positive sera were collected and compared with the commercial liquid phase blocking kit for detection. The results showed that all 186 samples detected by the kit O_FMDVA5 were positive, which was better than the commercial liquid phase blocking kit (the detection rate was only 95.7%).

[0199] 194 clinical Chinese type O FMDV positive sera were collected and compared with the commercial liquid phase blocking kit. The results showed that the kit O_FMDVA5 detected 194 positive sera, which was better than the commercial liquid phase blocking kit (the detection rate was only 92.7%). This shows that the kit O_FMDVA5 has good sensitivity.

[0200] 7.4 Detection of kits O_FMDVB1 to O_FMDVB6

[0201] 7.4.1 Sensitivity evaluation

[0202] The confirmed Southeast Asian type O-type foot-and-mouth disease virus-positive serum and the Chinese type O-type foot-and-mouth disease virus-positive serum were diluted 2-fold and tested using kits O_FMDVB1 to O_FMDVB6 respectively. The results are shown in Table 13. It was found that only kit O_FMDVB6 had a high sensitivity.

[0203] Table 13 Sensitivity test results of kits O_FMDVB1 to O_FMDVB6

[0204]

[0205] Table 13 (Continued) Sensitivity test results of kits O_FMDVB1 to O_FMDVB6

[0206]

[0207] 7.4.2 Specificity test

[0208] The kit O_FMDVB6 was used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, CSFV positive serum, and O FMDV positive serum). The results were all negative, indicating that the kit O_FMDVB6 has good specificity.

[0209] 7.4.3 Clinical application

[0210] 186 clinical Southeast Asian type O FMDV positive sera were collected and compared with the commercial liquid phase blocking kit for detection. The results showed that all 186 samples detected by the kit O_FMDVB6 were positive, which was better than the commercial liquid phase blocking kit (the detection rate was only 95.7%).

[0211] 194 clinical Chinese type O FMDV positive sera were collected and compared with the commercial liquid phase blocking kit. The results showed that all 194 samples were positive when tested by the O_FMDVB6 kit, which was better than the commercial liquid phase blocking kit (the detection rate was only 92.7%). This showed that the O_FMDVB6 kit had good sensitivity.

[0212] Example 8 Classical Swine Fever Virus Blocking ELISA Antibody Detection Kit (Blocking Method)

[0213] 8.1 Preparation of the kit

[0214] The enzyme marker diluents A1 to A5 prepared in Example 1 and the sample diluent B6 prepared in Example 2 were used respectively to prepare the kits CSFVA1 to CSFVA5 according to the preparation method of the kit in patent CN105837686A (wherein the coating material is classical swine fever virus E2 protein and the enzyme marker is enzyme-labeled classical swine fever virus monoclonal antibody 15A9), and the kits were tested according to the detection method to select the optimal enzyme marker diluent.

[0215] 8.2 Evaluation of Kits CSFVA1 to CSFVA5

[0216] Sensitivity test: The clinical swine fever virus positive serum with an IPMA titer of 1:12800 was diluted in multiple proportions, and the sensitivity was evaluated using the kits CSFVA1 to CSFVA5. The kit prepared in the CN105837686A method (wherein the enzyme marker was enzyme-labeled swine fever virus monoclonal antibody 15A9) was used as the CSFV original kit for comparison; the results are shown in Table 14: The sensitivity of the kits CSFVA1 to CSFVA4 was weaker than that of the CSFV original kit, and only the sensitivity of the kit CSFVA5 was higher than that of the CSFV original kit.

[0217] Table 14 Summary of test results of kit CSFVA1 to CSFVA5

[0218]

[0219] Specificity test: The kits CSFVA1~CSFVA5 and the original CSFV kit were used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, A FMDV positive serum, and O FMDV positive serum). The results were all negative, indicating good specificity.

[0220] Clinical application: 324 clinical samples of classical swine fever were collected, and 128 samples were positive and 173 samples were negative by the CSFV original kit and IDEXX classical swine fever antibody test kit. The remaining 23 samples were positive by the original kit and negative by the IDEXX kit, and were verified to be positive by the neutralization test, that is, there were 151 positive samples and 173 negative samples. The results of the test by the kits CSFVA1 to CSFVA5 are shown in Table 14. The positive and negative coincidence rates of the kits CSFVA1 to CSFVA4 were significantly lower than those of the kit CSFVA5, indicating that the enzyme marker diluent A5 can be used to prepare the classical swine fever virus ELISA antibody test kit with good sensitivity.

[0221] 8.3 Preparation of kits CSFVB1 to CSFVB6

[0222] Use the enzyme marker diluent A5 prepared in Example 1 and the sample diluents B1 to B6 prepared in Example 2 to prepare the kit CSFVB1 to CSFVB6 according to the preparation method of the kit in patent CN105837686A (wherein the enzyme marker is enzyme-labeled classical swine fever virus monoclonal antibody 15A9) and perform detection according to its detection method.

[0223] 8.4 Evaluation of Kits CSFVB1 to CSFVB6

[0224] Sensitivity test: The clinical swine fever virus positive serum with an IPMA titer of 1:12800 was diluted in multiple proportions, and the sensitivity was evaluated using the kits CSFVB1 to CSFVB6. The kit prepared in the CN105837686A method (wherein the enzyme marker was enzyme-labeled swine fever virus monoclonal antibody 15A9) was used as the CSFV original kit for comparison; the results are shown in Table 15: The sensitivity of the kits CSFVB1 to CSFVB6 was weaker than that of the CSFV original kit, and only the sensitivity of the kit CSFVB6 was higher than that of the CSFV original kit.

[0225] Table 15 Summary of test results of kit CSFVB1 to CSFVB6

[0226]

[0227] Specificity test: The kits CSFVB1~CSFVB6 and the original CSFV kit were used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, PRV positive serum, and PPV positive serum). The results were all negative, indicating good specificity.

[0228] Clinical application: The 324 clinical samples in Example 8.2 were tested with kits CSFVB1 to CSFVB6. The results are shown in Table 16. The positive and negative coincidence rates of kits CSFVB1 to CSFVB5 were significantly lower than those of kit CSFVB6, indicating that sample diluent B6 can be used to prepare a classical swine fever virus ELISA antibody detection kit with good sensitivity.

[0229] Example 9 ELISA Antibody Detection Kit for Pseudorabies Virus gE Protein (Blocking Method)

[0230] 9.1 Preparation of the kit

[0231] The enzyme marker diluents A1 to A5 prepared in Example 1 and the sample diluent B6 prepared in Example 2 were used respectively to prepare the kits PRVgEA1 to PRVgEA5 according to the preparation method of the kit in patent CN109959788A (wherein the coating was originally a mixture of inactivated antigens of porcine pseudorabies virus HN1201 strain and inactivated antigens of Fa strain, the deposit number of porcine pseudorabies virus HN1201 strain is CCTCC No: V201311, the Fa strain was purchased from the China Veterinary Drug Administration, and the enzyme marker was enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1 disclosed in CN109959788A), and the detection was carried out according to the detection method, and the optimal enzyme marker diluent was selected.

[0232] 9.2 Evaluation of Kits PRVgEA1 to PRVgEA5

[0233] Sensitivity test: The classical strain Fa and variant strain HN1201 of pseudorabies virus of porcine were divided into 2 groups, each with 5 pigs for challenge test. 80 sera collected from 10 pigs at different days after challenge, i.e. 0, 3, 5, 7, 9, 11, 13 and 15 days, were used for sensitivity evaluation using kits PRVgEA1 to PRVgEA5. Kit 4# prepared in the method of CN109959788A was used as the original PRVgE kit for comparison. Results: The sensitivities of kits PRVgEA1 to PRVgEA4 were 48% (38 / 80), 45% (36 / 80), 44% (35 / 80) and 50% (40 / 80), respectively, which were weaker than 62.5% (50 / 80) of the original PRVgE kit. Only the sensitivity of kit PRVgEA5, 62.5% (50 / 80), was equivalent to that of the original PRVgE kit.

[0234] Specificity test: The kits PRVgEA1~PRVgEA5 and the original PRVgE kit were used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, CSFV positive serum, PPV positive serum, A FMDV positive serum, and O FMDV positive serum). The results were all negative, indicating good specificity.

[0235] Clinical application: 200 sera (including 100 positive and 100 negative) with clear backgrounds, originating from farms in different regions and with symptoms of pseudorabies infection in pigs, confirmed by serum neutralization test method were collected and tested by PRVgE original kit and kit PRVgEA5. The results are shown in Table 16. The kit PRVgEA5 detected 86 positive samples, 109 negative samples and 5 suspicious samples, which is equivalent to the detection effect of the original PRVgE kit and has good sensitivity.

[0236] Table 16 Summary of test results of PRVgEA5, PRVgEB6 and original PRVgE kits

[0237]

[0238] 9.3 Preparation of kits PRVgEB1 to PRVgEB6

[0239] The enzyme marker diluent A5 prepared in Example 1 and the sample diluents B1 to B6 prepared in Example 2 were used to prepare the kits PRVgEB1 to PRVgEB6 according to the preparation method of the kit in patent CN109959788A (wherein the enzyme marker is enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H1) and the detection was performed according to the detection method thereof.

[0240] 9.4 Evaluation of Kits PRVgEB1 to PRVgEB6

[0241] Sensitivity test: The sensitivity of 80 pig sera collected in Example 9.2 was evaluated using kits PRVgEB1 to PRVgEB6, and compared with the original PRVgE kit; Results: The sensitivities of kits PRVgEB1 to PRVgEB5 were 44% (35 / 80), 40% (32 / 80), 50% (40 / 80), and 45% (36 / 80), respectively, which were weaker than 62.5% (50 / 80) of the original PRVgE kit. Only the sensitivity of kit PRVgEB6, 62.5% (50 / 80), was equivalent to that of the original PRVgE kit.

[0242] Specificity test: The kits PRVgEB1~PRVgEB6 and the original PRVgE kit were used to perform specificity tests on other swine virus positive sera (including PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, CSFV positive serum, and PPV positive serum). The results were all negative, indicating good specificity.

[0243] Clinical application: 200 sera (including 100 positive and 100 negative) with clear backgrounds, originating from farms in different regions, with symptoms of pseudorabies infection in pigs, and confirmed by the serum neutralization test method were collected and tested by the PRVgE original kit and the kit PRVgEB6. The results are shown in Table 17. The kit PRVgEB6 detected 86 positive samples, 109 negative samples, and 5 suspicious samples, which is equivalent to the detection effect of the original PRVgE kit and has good sensitivity.

[0244] Example 10 ELISA Antibody Detection Kit for Pseudorabies Virus gD Protein (Indirect Method)

[0245] 10.1 Preparation of the kit

[0246] Antigen coated plate: Dilute the pseudorabies virus gD protein prepared according to patent CN105251000A to 0.5 μg / ml with carbonate buffer (pH 9.6, 0.05 mol / L) for coating, 100 μl / well, 2-8°C for 16-24 hours, wash with detergent, add blocking solution (phosphate buffer containing 5% W / V sucrose, 20% V / V newborn calf serum, 0.05% V / V ProClin300) at 150 μl / well, block at 2-8°C for 16-24 hours or 37°C for 2 hours, discard the blocking solution and dry at 18-26°C, relative humidity not higher than 30% for 3-6 hours, seal and store at 2-8°C for standby use.

[0247] Enzyme labeling reagent: Dilute the commercial goat anti-swine IgG-HRP or rabbit anti-swine IgG-HRP with the enzyme labeling diluents A1 to A5 prepared in Example 1 to a final volume of 0.05% V / V, mix well, filter through 0.22 μm, and package aseptically.

[0248] Positive control: Variant strain of pseudorabies inactivated vaccine (HN1201-ΔgE strain, from Pleco Biotech Co., Ltd.) was used to immunize healthy susceptible pigs with negative PRV antigen and antibody in the neck muscle (2ml / head). After 28 days, blood was collected and serum was separated. The collected serum was the positive serum. 200ml of newborn calf serum, 10ml of positive serum, and 0.5ml of ProClin-300 were mixed and phosphate buffer was added to make the volume 1000mL. After filtering, the mixture was sterilely packaged.

[0249] Negative control: Select healthy susceptible pigs with negative PRV antigen and antibody, collect blood from the carotid artery and separate serum. The collected serum is negative serum. Mix 200ml of newborn calf serum, 10ml of negative serum, and 0.5ml of ProClin-300, add phosphate buffer to make the volume 1000mL, filter and package aseptically.

[0250] Sample diluent: refer to Example 2.

[0251] 20× concentrated washing solution: Take 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800mL ultrapure water, and 10mL Tween 20. After complete dissolution, add ultrapure water to make up to 1000mL, filter with a 0.22μm filter membrane, and dispense aseptically. Dilute 20 times with distilled water before use.

[0252] Color developing solution: Disodium hydrogen phosphate 14.7g, citric acid 9.3g, urea peroxide 0.3g, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution A. Tetramethylbenzidine (TMB) 0.2g, anhydrous ethanol 100ml, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution B.

[0253] Stop solution: 2M H2SO4 solution.

[0254] The above kits were assembled into a kit. According to the different enzyme marker diluents (the sample diluent is B6), they were labeled as kits PRVgDA1 to PRVgDA5; according to the different sample diluents (the enzyme marker diluent is A5), they were labeled as kits PRVgDB1 to PRVgDB6.

[0255] 10.2 Establishment of detection method

[0256] All reagents should be equilibrated to room temperature (about 30 minutes) before use. Liquid reagents should be gently shaken to mix before the experiment and immediately sealed and stored at 2-8°C after use.

[0257] The steps are as follows:

[0258] (1) Add sample diluent: Add 100 μl of sample diluent to the corresponding wells of the microtiter plate. At the same time, set up negative control wells (add 100 μl of negative control / well), positive control wells (add 100 μl of positive control / well), and blank control wells (add 100 μl of PBS buffer).

[0259] (2) Add the sample to be tested: Add 10 μl of the sample to be tested to the corresponding wells except the negative control well, positive control well, and blank control well. Seal the plate and incubate at 37°C for 30 minutes.

[0260] (3) Washing: Wash 5 times with detergent and dry for the last time.

[0261] (4) Add enzyme-labeled reagent: Add 100 μl of enzyme-labeled reagent to each well except the blank control well, seal the plate and incubate at 37°C for 30 min.

[0262] (5) Washing: Wash 5 times with detergent and dry for the last time.

[0263] (6) Color development: Add 50 μl of color development solution A and 50 μl of color development solution B to each well, mix well, and develop color at 37°C or room temperature for 15 min in the dark.

[0264] (7) Stop and detect: Add 50 μl of stop solution to each well and measure the A value of each well using an ELISA reader within 10 minutes: OD 450nm Value (measured after adjusting the blank control well to zero), or OD 450nm -OD 600nm～650nm The value is determined according to the following result judgment criteria.

[0265] When the positive control well A value OD 450nm The test is established when the A value of the negative control well is ≥0.2 and the A value of the negative control well is ≤0.1, otherwise it is invalid. When the test is established, the S / P value is calculated as: sample A value / positive control A value average. Result judgment:

[0266] Those with an S / P value ≥ 0.5 are positive for pseudorabies virus antibodies.

[0267] Those with an S / P value of <0.5 were negative for pseudorabies virus antibodies.

[0268] 10.3 Detection of kits PRVgD A1 to PRVgD A5

[0269] 10.3.1 Sensitivity

[0270] Pig sera with clear background were collected, including 15 sera from screened pigs with negative pseudorabies virus antigen and antibody, 60 sera from experimental animals immunized with pseudorabies virus vaccine, and 15 sera from newborn piglets that did not eat colostrum, totaling 90 sera. All sera were tested using kits PRVgD A1 to PRVgD A5, and the neutralizing antibody was determined using the variant strain HN1201 with reference to the serum neutralization test method according to GB / T18641-2002 (neutralization titer determination standard: neutralization titer <1:2 is negative, ≥1:2 is positive). Results (see Table 17): In the laboratory application test, there were 38 samples with neutralization titer <1:2, and among the negative coincidence rate of the test kits PRVgD A1 to PRVgD A5, only PRVgD A5 was 100%; there were 52 samples with neutralization titer ≥1:2, and among the positive coincidence rate of the test kits PRVgD A1 to PRVgD A5, only PRVgD A5 was >90% (94%).

[0271] Table 17 Summary of test results of kit PRVgD A1~PRVgD A5

[0272]

[0273] 10.3.2 Specificity

[0274] Five confirmed pseudorabies virus antibody negative sera, one PCV2 positive serum, one PPV positive serum, one PRRSV positive serum, one CSFV positive serum, one JEV positive serum, one Haemophilus parasuis (HPs) positive serum, one porcine atrophic rhinitis (Bb) positive serum, one Escherichia coli (BL21) positive serum, one Mycoplasma hyopneumoniae (Mhp) positive serum, one PEDV positive serum, one TGEV positive serum, one PRoV positive serum, one A FMDV positive serum, and one OFMDV positive serum were tested using the kit PRVgDA5 prepared in Example 3, a neutralization test, and a commercial kit BioChek PRVgB antibody kit, respectively. The results showed that the test results of the kit PRVgDA5 were consistent with the neutralization test and the commercial kit test results, all of which were negative, indicating that the specificity of the kit PRVgDA5 was good.

[0275] 10.3.3 Clinical Application

[0276] 70 samples of clinical pig sera were collected and tested using kits PRVgD A1 to PRVgD A5 and a commercialized Biochek pseudorabies virus antibody detection kit and compared. The results are shown in Table 17: Among the kits PRVgD A1 to PRVgD A5, the positive coincidence rate was greater than 90% (92%) only for PRVgD A5, and the negative coincidence rate was 100% only for PRVgD A5. The overall coincidence rate was 96%, indicating that the PRVgD A5 kit had good sensitivity.

[0277] 10.4 Detection of kits PRVgD B1 to PRVgD B6

[0278] 10.4.1 Sensitivity

[0279] The 90 sera of Example 10.3.1 were tested with the kits PRVgD B1 to PRVgD B6, and the neutralization test was used as a comparison. Results: In the laboratory application test, there were 38 samples with a neutralization titer of <1:2, and among the negative coincidence rates of the kits PRVgD B1 to PRVgD B6, only PRVgD B6 was 100%; there were 52 samples with a neutralization titer of ≥1:2, and among the positive coincidence rates of the kits PRVgD B1 to PRVgD B5, only PRVgD B6 was >90% (94%).

[0280] Table 18 Summary of test results of kit PRVgD B1 to PRVgD B6

[0281]

[0282] 10.4.2 Specificity

[0283] The specificity of Example 10.3.2 was tested with the serum samples using the PRVgDB6 kit prepared in Example 10.4.1, the neutralization test, and the commercialized BioChek PRVgB antibody kit. The results showed that the PRVgDB6 test results were consistent with those of the neutralization test and the commercialized kit, all of which were negative, indicating that the PRVgDB6 kit had good specificity.

[0284] 10.4.3 Clinical Application

[0285] 70 samples of clinical pig sera were collected and tested using kits PRVgD B1 to PRVgD B6 and a commercialized Biochek pseudorabies virus antibody detection kit and compared. The results are shown in Table 18: Among the positive coincidence rates of kits PRVgD B1 to PRVgD B6, only PRVgD B6 was >90% (92%), and among the negative coincidence rates, only PRVgD B6 was 100%. The overall coincidence rate was 96%, indicating that the sensitivity of the PRVgD B6 kit was good.

[0286] In summary, the composition of the animal disease ELISA antibody detection kit prepared by the present invention can be used simultaneously for the preparation of animal disease ELISA antibody detection kits for multiple animal diseases and multiple different detection types (including competitive method, indirect method, blocking method, sandwich method), and the antibody sensitivity of each prepared kit for detecting each animal disease is equivalent to or even better than that of the original kit, and the specificity is good, which reduces the preparation simplicity during large-scale production and saves manpower and material costs.

[0287] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. SEQUENCE LISTING <110> Luoyang Putai Biotechnology Co., Ltd. <120> Universal enzyme marker diluent and sample diluent combination for enzyme-linked immunosorbent assay kit, and reagent containing the same Box and method for making the same <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 313 <212> PRT <213> Novel coronavirus COVID-19 <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr 305 310 <210> 2 <211> 115 <212> PRT <213> Foot-and-mouth disease Virus Type A Monoclonal cell Strain 2E11 <400> 2 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Ser Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Phe Pro Thr Ser Asp Ser Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Leu Ser Val Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 3 <211> 113 <212> PRT <213> Foot-and-mouth disease Virus Type A Monoclonal cell Strain 2E11 <400> 3 Asp Ile Val Met Thr Gln Phe Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Ser Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Asn Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 4 <211> 118 <212> PRT <213> Foot-and-mouth disease Virus Type A Monoclonal cell Strain 3B4 <400> 4 Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Asp Ser Gly Ser Phe Asn Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Val His Tyr Arg His Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 5 <211> 102 <212> PRT <213> Foot-and-mouth disease Virus Type A Monoclonal cell Strain 3B4 <400> 5 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Tyr Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Glu 100 <210> 6 <211> 126 <212> PRT <213> Foot-and-mouth disease Virus Type O Monoclonal cell Strain 6G6 <400> 6 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Val Pro Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Pro Tyr 65 70 75 80 Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Arg Lys Gly Tyr Tyr Arg Tyr Asp Val Glu Thr Ser Gly Phe Ala 100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 114 <212> PRT <213> Foot-and-mouth disease Virus TypeO Monoclonal cell Strain 6G6 <400> 7 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Val Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 8 <211> 118 <212> PRT <213> Foot-and-mouth disease Virus TypeO Monoclonal cell Strain 7D2 <400> 8 Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Asp Ser Gly Ser Phe Asn Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Val His Tyr Arg His Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 9 <211> 107 <212> PRT <213> Foot-and-mouth disease Virus Type O Monoclonal cell Strain 7D2 <400> 9 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Val Trp Tyr Gln Glu Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105

Claims

1. A combination of a universal enzyme marker diluent and a sample diluent for an ELISA kit, wherein: The components of the enzyme marker diluent are 5% V / V fetal bovine serum, 1% W / V mannitol, 2% W / V sucrose, 0.05% V / V Proclin 300, and a 0.02M PBS solution at pH 7.4; the components of the sample diluent are 0.4% W / V gelatin, 0.1% V / V glycerol, 0.1% W / V EDTA, 0.05% V / V Proclin 300, and a 0.02M PBS solution at pH 7.4; The enzyme marker diluent is a diluent of the following enzyme markers: enzyme-labeled porcine circovirus type 2 monoclonal antibody 3H11; enzyme-labeled novel coronavirus protein S1, the sequence of which is shown in SEQ ID No.1; enzyme-labeled sheep anti-swine IgA antibody; enzyme-labeled foot-and-mouth disease virus type A monoclonal antibody 3B4, the heavy chain variable region sequence of which is shown in SEQ ID No.4, and the light chain variable region sequence of which is shown in SEQ ID No.5; enzyme-labeled foot-and-mouth disease virus type O monoclonal antibody 7D2, the heavy chain variable region sequence of which is shown in SEQ ID No.8, and the light chain variable region sequence of which is shown in SEQ ID As shown in No.9; enzyme-labeled CSFV monoclonal antibody 15A9; enzyme-labeled PRVgE protein monoclonal antibody 11H1; and enzyme-labeled goat anti-swine IgG or enzyme-labeled rabbit anti-swine IgG; The sample diluent is a diluent for the following samples: serum samples suspected of being infected with porcine circovirus type 2, serum samples suspected of being infected with the new coronavirus, milk samples, serum samples, saliva samples, anal swab samples, and fecal samples suspected of being infected with PEDV, serum samples suspected of being infected with foot-and-mouth disease virus type A, serum samples suspected of being infected with foot-and-mouth disease virus type O, serum samples suspected of being infected with CSFV, serum samples suspected of being infected with pseudorabies virus, and serum samples suspected of being immunized with pseudorabies virus antigen.

2. The combination of enzyme marker diluent and sample diluent according to claim 1, wherein: The enzyme-linked immunosorbent assay kit is a porcine circovirus type 2 competitive ELISA antibody detection kit, a new coronavirus double antigen sandwich ELISA antibody detection kit, a porcine epidemic diarrhea virus indirect ELISA antibody detection kit, a foot-and-mouth disease virus type A competitive ELISA antibody detection kit, a foot-and-mouth disease virus type O competitive ELISA antibody detection kit, a classical swine fever virus blocking ELISA antibody detection kit, a pseudorabies virus gE protein blocking ELISA antibody detection kit, or a pseudorabies virus gD protein indirect ELISA antibody detection kit.

3. The enzyme marker diluent and sample diluent combination according to claim 2, wherein: The new coronavirus double antigen sandwich ELISA antibody detection kit is an IgM antibody detection kit or an IgG antibody detection kit, and the porcine epidemic diarrhea virus indirect ELISA antibody detection kit is an IgA antibody detection kit or an IgG antibody detection kit.

4. Enzyme-linked immunosorbent assay kit, wherein: The kit comprises: an antigen-coated plate coated with an animal disease antigen; an enzyme-labeled reagent, wherein the enzyme-labeled reagent is prepared by diluting an enzyme-labeled animal disease antibody or an enzyme-labeled animal disease antigen with the enzyme marker diluent described in any one of claims 1 to 3, filtering and packaging; the sample diluent described in any one of claims 1 to 3; a washing solution; a color developing solution; and a stop solution; the kit also comprises a negative control and a positive control.

5. The ELISA kit according to claim 4, wherein When the enzyme-linked immunosorbent assay kit is a porcine circovirus type 2 competitive ELISA antibody detection kit, the animal disease antigen is porcine circovirus type 2 Cap protein; When the enzyme-linked immunosorbent assay kit is a novel coronavirus double antigen sandwich ELISA antibody detection kit, the animal disease antigen is novel coronavirus protein S1, and the sequence of the novel coronavirus protein S1 is shown in SEQ ID No. 1; When the enzyme-linked immunosorbent assay kit is an indirect ELISA antibody detection kit for porcine epidemic diarrhea virus, the animal disease antigen is an antigen-antibody complex of porcine epidemic diarrhea virus monoclonal antibody 8A3A10 and porcine epidemic diarrhea virus HN1301 strain inactivated antigen, the porcine epidemic diarrhea virus monoclonal antibody 8A3A10 is secreted by hybridoma cell 8A3A10 strain, the hybridoma cell 8A3A10 has a preservation number of CCTCC No.C2014197, and the porcine epidemic diarrhea virus HN1301 strain has a preservation number of CCTCC No: V201514; When the enzyme-linked immunosorbent assay kit is a foot-and-mouth disease virus type A competitive ELISA antibody detection kit, the animal disease antigen is an antigen-antibody complex of foot-and-mouth disease virus type A monoclonal antibody 2E11-foot-and-mouth disease virus type A virus-like particle protein, and the heavy chain variable region sequence of the foot-and-mouth disease virus type A monoclonal antibody 2E11 is shown in SEQ ID No. 2, and the light chain variable region sequence is shown in SEQ ID No. 3; When the enzyme-linked immunosorbent assay kit is a foot-and-mouth disease virus type O competitive ELISA antibody detection kit, the animal disease antigen is an antigen-antibody complex of foot-and-mouth disease virus type O monoclonal antibody 6G6 and foot-and-mouth disease virus type O virus-like particle protein, and the heavy chain variable region sequence of the foot-and-mouth disease virus type O monoclonal antibody 6G6 is shown in SEQ ID No.6, and the light chain variable region sequence is shown in SEQ ID No.7; When the enzyme-linked immunosorbent assay kit is a classical swine fever virus blocking ELISA antibody detection kit, the animal disease antigen is classical swine fever virus E2 protein; When the enzyme-linked immunosorbent assay kit is a pseudorabies virus gE protein blocking ELISA antibody detection kit, the animal disease antigen is a mixture of an inactivated antigen of the pseudorabies virus HN1201 strain and an inactivated antigen of the Fa strain, and the pseudorabies virus HN1201 strain has a preservation number of CCTCC No: V201311; and When the enzyme-linked immunosorbent assay kit is an indirect ELISA antibody detection kit for pseudorabies virus gD protein, the animal disease antigen is pseudorabies virus gD protein.

6. A method for preparing an enzyme-linked immunosorbent assay kit, wherein: The method comprises: Step (1) diluting and coating the animal disease coating material with a pH 9.6, 0.05 mol / L carbonate buffer to prepare an antigen-coated plate; Step (2) Enzyme-labeled animal disease antibodies or antigens are diluted with the enzyme labeling diluent described in any one of claims 1 to 3, filtered and packaged to prepare enzyme labeling reagents; Step (3) assembling the sample diluent, washing solution, color developing solution, and stop solution described in any one of claims 1 to 3, together with the antigen-coated plate prepared in step (1) and the enzyme-labeled reagent prepared in step (2) into a kit.

7. Use of the combination of the enzyme marker diluent and the sample diluent according to any one of claims 1 to 3 in the preparation of an enzyme-linked immunosorbent assay kit.

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