CN114252624A

CN114252624A - Universal enzyme marker diluent and sample diluent combination of enzyme linked immunosorbent assay kit, kit containing same and preparation method thereof

Info

Publication number: CN114252624A
Application number: CN202011010411.0A
Authority: CN
Inventors: 田克恭; 邓均华
Original assignee: Luoyang Pu Tai Biotechnology Co ltd
Current assignee: Luoyang Pu Tai Biotechnology Co ltd
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2022-03-29

Abstract

The invention provides a general enzyme marker diluent and a sample diluent combination of an enzyme-linked immunoassay kit, wherein the enzyme marker diluent is a 0.02M PBS solution containing 5% V/V fetal calf serum, 1-3% W/V mannitol, 2% W/V sucrose, 0.05% V/V Proclin300 and pH7.4, 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 pH7.4. The enzyme marker diluent and the sample diluent can be used for preparing animal epidemic disease ELISA antibody detection kits for various animal epidemic diseases and various detection types (including a competition method, an indirect method, a blocking method and a sandwich method), and the sensitivity of antibodies for detecting various animal epidemic diseases of each prepared kit is equivalent to or even better than that of the original kit, and the specificity is good.

Description

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

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a combination of a universal enzyme marker diluent and a sample diluent of an enzyme-linked immunosorbent assay kit, a kit containing the same and a preparation method thereof.

Background

Enzyme-linked immunosorbent assay (ELISA) has been developed rapidly since the first 70 th century, and is now widely used in many fields of biology and medical science. In the field of biological detection, it is common to detect antibodies by ELISA, and the main detection types include competition methods, blocking methods, indirect methods, and sandwich methods.

Most biotechnology companies have a plurality of ELISA products for clinical detection, but the detection types, enzyme marker diluent, sample diluent and other components of the ELISA products are different, so that the formulas of the kits are different, and the components are complex to prepare, complex in process and high in labor and material cost during large-scale production.

Therefore, the prior art needs to prepare universal enzyme marker diluent and sample diluent for different enzyme-linked immunoassay kits, so as to solve the problem of detecting various components and contents of the enzyme marker diluent and the sample diluent in different kits respectively and ensure detection sensitivity.

Disclosure of Invention

Based on the above, one objective of the present invention is to provide a combination of a universal enzyme marker diluent and a sample diluent of an enzyme-linked immunoassay kit, wherein the enzyme marker diluent is a 0.02M PBS solution containing 5% V/V fetal calf serum, 1-3% W/V mannitol, 2% W/V sucrose, 0.05% V/V Proclin300 and pH7.4, 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 pH7.4.

When the enzyme marker diluent and the sample diluent are combined for preparing the kit, the enzyme marker diluent and the sample diluent can be applied to animal epidemic disease ELISA antibody detection kits of various detection types (including a competition method, an indirect method, a blocking method and a sandwich method), and the detection sensitivity of the enzyme marker diluent and the sample diluent can be higher than or equal to that of the original detection kit.

The animal epidemic disease ELISA antibody detection kit of the invention can be used for preparing animal epidemic disease ELISA antibody detection kits of various animal epidemic diseases and various detection types (including competition method, indirect method, blocking method and sandwich method) by using enzyme marker diluent and sample diluent with specific compositions, and the prepared kits have the antibody sensitivity for detecting the animal epidemic diseases equivalent to or even better than that of the original kit and good specificity, thereby reducing the preparation simplicity and convenience in large-scale production and saving the labor and material costs.

The content of mannitol in the enzyme marker diluent may 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, 3% W/V.

The gelatin content of the sample diluent may 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, 0.69% W/V, 0.55% 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.

In one embodiment of the present invention, the enzyme marker diluent is a 0.02M PBS solution containing 5% V/V fetal calf serum, 1% W/V mannitol, 2% W/V sucrose, 0.05% V/V Proclin300 and pH7.4, and 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.

In one embodiment of the present invention, the enzyme label diluent is a diluent of the following enzyme labels: enzyme-labeled porcine circovirus type 2 monoclonal antibody 3H 11; enzyme-labeled novel coronavirus protein S1, wherein the novel coronavirus protein S1 is shown as SEQ ID No. 1; enzyme-labeled goat anti-pig IgA antibody; the enzyme-labeled foot-and-mouth disease virus A type monoclonal antibody 3B4, wherein the foot-and-mouth disease virus A type monoclonal antibody 3B4 heavy chain variable region is shown as SEQ ID No.4, and the foot-and-mouth disease virus A type monoclonal antibody 3B4 light chain variable region is shown as SEQ ID No. 5; the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2, wherein the variable region of the heavy chain of the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 is shown as SEQ ID No.8, and the variable region of the light chain of the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 is shown as SEQ ID No. 9; enzyme-labeled CSFV monoclonal antibody 15A 9; enzyme-labeled PRVgE protein monoclonal antibody 11H 1; and enzyme-labeled goat anti-pig lgG or enzyme-labeled rabbit anti-pig lgG.

As an embodiment of the present invention, the sample diluent is a diluent of the following samples: a serum sample suspected of porcine circovirus type 2 virus infection, a serum sample suspected of new coronavirus infection, a milk sample suspected of PEDV infection, a serum sample, a saliva sample, an anus swab sample, a stool sample, a serum sample suspected of foot-and-mouth disease virus type A infection, a serum sample suspected of foot-and-mouth disease virus type O infection, a serum sample suspected of CSFV infection, a serum sample suspected of porcine pseudorabies virus infection, and a serum sample suspected of porcine pseudorabies virus antigen immunization.

The combined use of the diluent of the enzyme marker and the sample diluent can ensure higher sensitivity, not only can meet the detection of IgG antibodies in serum, but also ensure the detection of low-content IgA and early-stage generated IgM antibodies, realize the early and timely detection of pathogenic infection and the detection of different detection samples, and can meet various detection conditions and requirements clinically.

As an implementation mode of the 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 porcine pseudorabies virus gE protein blocking ELISA antibody detection kit and a porcine pseudorabies virus gD protein indirect ELISA antibody detection kit.

As an implementation mode of the 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.

The invention also relates to an enzyme linked immunosorbent assay kit, wherein the kit comprises: an antigen coated plate coated with an animal epidemic disease coating antigen; the enzyme-labeled reagent is prepared by diluting an enzyme-labeled animal epidemic disease antibody or antigen with the enzyme-labeled diluent, filtering, subpackaging and preparing into the enzyme-labeled reagent; the sample diluent; washing liquid; a color developing solution; and a stop solution.

As an embodiment of the invention, the kit further comprises a negative control and a positive control.

As an implementation mode of the invention, when the enzyme linked immunosorbent assay kit is a porcine circovirus type 2 competitive ELISA antibody detection kit, the animal epidemic disease antigen is porcine circovirus type 2 Cap protein; when the enzyme-linked immunoassay kit is a new coronavirus double-antigen sandwich ELISA antibody detection kit, the animal epidemic disease antigen is a novel coronavirus protein S1, and the novel coronavirus protein S1 is shown as SEQ ID No. 1; when the enzyme linked immunosorbent assay kit is a swine epidemic diarrhea virus indirect ELISA antibody detection kit, the animal epidemic disease antigen is an antigen-antibody complex of a swine epidemic diarrhea virus monoclonal antibody 8A3A 10-a swine epidemic diarrhea virus HN1301 strain inactivated antigen, the swine epidemic diarrhea virus monoclonal antibody 8A3A10 is secreted by a hybridoma cell 8A3A10 strain, the hybridoma cell 8A3A10 has a preservation number of CCTCC No. C2014197, and the swine epidemic diarrhea virus HN1301 strain has a preservation number of CCTCC No: v201514; when the enzyme-linked immunoassay kit is a foot-and-mouth disease virus A type competition ELISA antibody detection kit, the animal epidemic disease antigen is an antigen-antibody complex of a foot-and-mouth disease virus A type monoclonal antibody 2E 11-foot-and-mouth disease virus A type virus-like particle protein, the foot-and-mouth disease virus A type monoclonal antibody 2E11 heavy chain variable region is shown as SEQ ID No.2, and the light chain variable region is shown as SEQ ID No. 3; when the enzyme-linked immunoassay kit is a foot-and-mouth disease virus O-type competition ELISA antibody detection kit, the animal epidemic disease antigen is an antigen-antibody complex of a foot-and-mouth disease virus O-type monoclonal antibody 6G 6-foot-and-mouth disease O-type virus-like particle protein, the foot-and-mouth disease virus O-type monoclonal antibody 6G6 heavy chain variable region is shown as SEQ ID No.6, and the light chain variable region is shown as SEQ ID No. 7; when the enzyme linked immunosorbent assay kit is a classical swine fever virus blocking ELISA antibody detection kit, the animal epidemic disease antigen is classical swine fever virus E2 protein; when the enzyme-linked immunoassay kit is a porcine pseudorabies virus gE protein blocking ELISA antibody detection kit, the animal epidemic disease antigen is a mixture of porcine pseudorabies virus HN1201 inactivated antigen and Fa inactivated antigen, and the preservation number of the porcine pseudorabies virus HN1201 is CCTCC No: V201311; and when the enzyme linked immunosorbent assay kit is a porcine pseudorabies virus gD protein indirect ELISA antibody detection kit, the animal epidemic disease antigen is porcine pseudorabies virus gD protein.

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

As an embodiment of the present invention, the animal epidemic disease coatingantigen in the step (1) in 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 porcine epidemic diarrhea virus HN1301 strain inactivated antigen, antigen-antibody complex of a type foot-and-mouth disease virus monoclonal antibody 2E11 and a type foot-and-mouth disease virus-like particle protein, antigen-antibody complex of O type foot-and-mouth disease virus monoclonal antibody 6G6 and O type foot-and-mouth disease virus-like particle protein, classical swine fever virus E2 protein, porcine pseudorabies virus gD protein; the recombinant protein S1 of the novel coronavirus is encoded by a nucleotide sequence shown by SEQ ID No.1, the heavy chain variable region of the monoclonal antibody 2E11 of the A-type foot-and-mouth disease virus is encoded by the nucleotide sequence shown by SEQ ID No.2, the light chain variable region is encoded by the nucleotide sequence shown by SEQ ID No.3, the heavy chain variable region of the monoclonal antibody 6G6 of the O-type foot-and-mouth disease virus is encoded by the nucleotide sequence shown by SEQ ID No.6, and the light chain variable region is encoded by the nucleotide sequence shown by SEQ ID No. 7.

As an embodiment of the present invention, the enzyme-labeled animal epidemic disease antibody or antigen in the step (2) in the preparation method correspondingly includes porcine circovirus type 2 monoclonal antibody 3H11, neocoronavirus recombinant protein S1, goat anti-pig IgA antibody, a type a foot-and-mouth disease virus monoclonal antibody 3B4, O type foot-and-mouth disease virus monoclonal antibody 7D2, swine fever virus monoclonal antibody 15a9, porcine pseudorabies virus monoclonal antibody 11H1, goat anti-pig lgG-HRP or rabbit anti-pig lgG-HRP; the recombinant protein S1 of the novel coronavirus is encoded by a nucleotide sequence shown by SEQ ID No.1, the heavy chain variable region of the A-type foot-and-mouth disease virus monoclonal antibody 3B4 is encoded by the nucleotide sequence shown by SEQ ID No.4, the light chain variable region is encoded by the nucleotide sequence shown by SEQ ID No.5, the heavy chain variable region of the O-type foot-and-mouth disease virus monoclonal antibody 7D2 is encoded by the nucleotide sequence shown by SEQ ID No.8, and the light chain variable region is encoded by the nucleotide sequence shown by SEQ ID No. 9.

As an embodiment of the invention, the enzyme marker diluent in the step (2) is 0.02M PBS solution containing 5% V/V fetal calf serum, 1% W/V-3% W/V mannitol, 2% W/V sucrose, 0.05% V/V Proclin300 and pH7.4; preferably, the enzyme marker diluent is 0.02M PBS solution containing 5% V/V fetal calf serum, 1% W/V mannitol, 2% W/V sucrose, 0.05% V/V Proclin300 and pH7.4.

In one embodiment of the present invention, the sample diluent in step (3) is 0.02M PBS solution containing 0.4% W/V-0.8% W/V gelatin, 0.1% V/V glycerin, 0.1% W/V EDTA, 0.05% V/V Proclin300 and having pH of 7.4; preferably, the sample diluent is 0.02M PBS containing 0.4% W/V gelatin, 0.1% V/V glycerol, 0.1% W/V EDTA, 0.05% V/V Proclin300 at pH 7.4.

AsIn one embodiment of the present invention, the color developing solution in the step (3) includes a color developing solution A and a color developing solution B, the color developing solution A is a solution containing 1.47% w/v of disodium hydrogen phosphate, 0.93% w/v of citric acid and 0.03% w/v of carbamide peroxide, and the color developing solution B is a solution containing 0.02% w/v of tetramethyldiphenyldiamine and 10% v/v of absolute ethyl alcohol; the stop solution is 2M H₂SO₄And (3) solution.

The invention also relates to the application of the enzyme marker diluent and the sample diluent in the preparation of the enzyme linked immunosorbent assay kit.

The enzyme marker diluent and the sample diluent prepared by the invention are combined to prepare the animal epidemic disease ELISA antibody detection kit, the kit can be simultaneously used for preparing animal epidemic disease ELISA antibody detection kits for various animal epidemic diseases and various detection types (including a competition method, an indirect method, a blocking method and a sandwich method), and the antibody sensitivity of each animal epidemic disease detected by each prepared kit is equivalent to or even better than that of the original kit, the specificity is good, the preparation is simple and convenient during large-scale production is reduced, and the cost of manpower and material resources is saved.

Detailed Description

Hereinafter, some exemplary embodiments of the present invention will be described in detail so that the present invention may be more easily understood by those skilled in the art.

The term "enzyme-linked immunosorbent assay Kit" is also called ELISA Kit, ELISA detection Kit, ELISA Kit, enzyme-linked immunosorbent assay Kit, enzyme immunoassay Kit, etc.

The term "animal epidemic coatingen" is animal epidemic associated antigen or protein, including but not limited to animal epidemic complete virus, animal epidemic virus protein, animal epidemic virus monoclonal antibody-animal epidemic complete virus complex, animal epidemic virus monoclonal antibody-animal epidemic virus protein complex.

The "enzyme-labeled antibody" and the "labeled antibody" in the present invention are used interchangeably, and are monoclonal antibodies that are predominantly enzyme-labeled. The term "enzyme" includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, calf intestinal alkaline phosphatase. Wherein, the substrate used by the horseradish peroxidase HRP is o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), aminosalicylic acid, o-biphenylmethylamine or 2,2' -hydrazino-2 (3-ethyl-thiazoline sulfonic acid-6) ammonium salt, preferably Tetramethylbenzidine (TMB); the substrate used by the alkaline phosphatase is p-nitrophenyl phosphate (p-NPP) or naphthol-AS-Mx phosphate + diazonium salt; the substrate used by the glucose oxidase is ABTS + HRP + glucose or glucose + methylthiophenol oral liquid + thiazole blue; the substrate used by beta-galactosidase is 4-methylumbelliferyl-beta-D galactoside (4MUG) or nitrophenol galactoside (ONPG).

The term "phosphate buffer" refers to a solution containing phosphoric acid or a salt thereof and adjusted to a desired pH, and is one of the most widely used buffers in biochemical studies. Typically, phosphate buffers are prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are known in the art, such as sodium and potassium dihydrogen phosphate, disodium and dipotassium hydrogen phosphate, sodium and potassium phosphate. Phosphate salts are known to exist as hydrates of salts. Due to the secondary dissociation of the buffer, the buffered pH ranges widely, for example, from about pH4 to about pH10, preferably from about pH5 to pH9, more preferably from about pH7 to about pH8, 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 solution" and PBS can be used interchangeably.

The term "preservative" includes, but is not limited to, gentamicin, kanamycin sulfate, sodium azide, thimerosal and its salts, Proclin 300.

The terms "enzyme label diluent", "enzyme label diluent" are used interchangeably.

The term "pH 9.6, 0.05mol/L carbonate buffer" is a buffer consisting of sodium carbonate, sodium bicarbonate, which is a conventional buffer used for ELISA-coated plates.

The swine monoclonal antibody mainly comprises the following components:

(1) the porcine circovirus type 2 monoclonal antibody 3H11 is disclosed in Chinese patent CN 105542000A.

(2) The monoclonal antibody 8A3A10 of the porcine epidemic diarrhea virus is secreted and expressed by a mouse myeloma hybridoma cell 8A3A10, wherein the preservation number of the hybridoma cell 8A3A10 strain is CCTCC No. C2014197, the preservation unit is China center for type culture Collection, the preservation address is Wuhan university in Wuhan City, China, the preservation date is 11 months and 3 days in 2014, and the Chinese patent CN104694480A and the CN110967480A are referred.

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

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

(5) The hog cholera virus monoclonal antibody 15A9, see Chinese patent CN 105837686A.

(6) The porcine pseudorabies virus gE protein monoclonal antibody 11H1 is disclosed in Chinese patent CN 109959788A.

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent as the description proceeds. These embodiments are merely exemplary, are not intended to be the only forms in which the embodiments of the invention may be practiced or carried out, and are therefore not intended to limit the scope of the invention in any way. The embodiments described below are intended to cover features of the various embodiments as well as the method steps and sequences for constructing and operating the embodiments. 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 formulation of the PBS buffer used in the examples of the present invention is exemplified below: 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of monopotassium phosphate, adjusting the pH value to 7.4, and fixing the volume to 1L, including but not limited to the formula; all chemical reagents are analytically pure and purchased from the national pharmaceutical group.

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

EXAMPLE 1 preparation of enzyme-labeled Diluent

Enzyme-labeled dilutions A1-A5 were prepared as in Table 1.

TABLE 1 dilution Components of enzyme labels

In the preparation process of the kit, the detection effect is equivalent when the content of mannitol in the enzyme marker diluent is 1-3% W/V, and the embodiment of the invention is only an example of the mannitol content in the enzyme marker diluent being 1% W/V for convenience and conciseness.

EXAMPLE 2 preparation of sample dilutions

Sample dilutions B1-B6 were prepared as in Table 2.

TABLE 2 summary of the components contained in the sample dilutions

In the preparation process of the kit, the detection effect is equivalent when the content of the gelatin in the sample diluent is 0.4% W/V-0.8% W/V, and in order to simplify and simplify the description of the embodiment of the invention, only the content of the gelatin in the sample diluent is 0.4% W/V.

Example 3 porcine circovirus type 2 ELISA antibody detection kit (competitive method)

3.1 preparation of the kit

The enzyme marker diluent a 1-a 5 prepared in example 1 is used to prepare a kit PCV2a 1-PCV 2a5 according to the preparation method of a kit in patent CN105542000A (wherein the coating source 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 is carried out according to the detection method, so that the optimal enzyme marker diluent is preferably selected.

3.2 evaluation of kit PCV2A 1-PCV 2A5

And (3) sensitivity test: diluting porcine circovirus type 2 positive serum with the IPMA titer of 1: 3200 in an immunoperoxidase monolayer cell test at a multiple ratio, carrying out sensitivity test by using a kit PCV2A 1-PCV 2A5 for evaluation, and comparing by using the kit prepared in the CN105542000A method as a PCV2 original kit; the results are shown in Table 3: the sensitivity of the kit PCV2A 1-PCV 2A4 is weaker than that of the original PCV2 kit, and only the sensitivity of the kit PCV2A5 is higher than that of the original PCV2 kit.

Table 3 summary of detection results of kit PCV2A 1-PCV 2A5

And (3) specific test: specificity test is carried out on swine other virus positive serum (including porcine circovirus type 1 PCV1 positive serum, porcine circovirus type 3 PCV3 positive serum, swine fever virus CSFV positive serum, porcine reproductive and respiratory syndrome virus PRRSV positive serum, porcine pseudorabies virus PRV positive serum, porcine parvovirus PPV positive serum, A type foot-and-mouth disease virus (A FMDV) positive serum and O type foot-and-mouth disease virus (O FMDV) positive serum) by using a PCV2A 1-PCV 2A5 and PCV2 original kit, and the result is as follows: all were negative, indicating good specificity.

The clinical application is as follows: the detection results of 30 parts of porcine circovirus type 2 positive serum and 30 parts of negative serum (containing 10 parts of porcine circovirus type 1 positive serum) identified by IPMA are shown in Table 3 by using the kits PCV2A 1-PCV 2A5 and PCV2 original kits respectively: the PCV2A 1-PCV 2A5 detection negative samples are all negative, and non-specific phenomenon does not exist; however, the positive coincidence rate of the PCV2A 1-PCV 2A4 of the kit is obviously lower than that of the original kits PCV2A5 and PCV2 of the kit.

The enzyme marker diluent A5 can be used for preparing the porcine circovirus type 2 ELISA antibody detection kit, and the kit has high sensitivity, good sensitivity and good specificity when detecting a sample.

Example 4 detection kit for double antigens sandwich ELISA antibody of novel coronavirus (sandwich method)

4.1 preparation of the kit

The preparation method of the new coronavirus double-antigen sandwich ELISA antibody detection kit (sandwich method) is disclosed in patent application CN202010167856.3, and specifically comprises the following steps:

antigen coated plate: diluting a novel coronavirus recombinant protein S1 (the nucleotide sequence of which is shown as SEQ ID No. 1) prepared by a genetic engineering means to 0.5 mu g/ml by using a carbonate buffer solution (the pH value is 9.6 and 0.05mol/L) for coating, coating for 16-24 hours at the temperature of 100 mu L/hole and 2-8 ℃, washing by using a washing solution, adding a sealing solution (a phosphate buffer solution containing 5% M/V sucrose, 20% V/V newborn bovine serum and 0.05% V/V ProClin 300) according to 150 mu L/hole, sealing for 16-24 hours at the temperature of 2-8 ℃ or sealing for 2 hours at the temperature of 37 ℃, discarding the sealing solution, drying for 3-6 hours at the temperature of 18-26 ℃ under the condition that the relative humidity is not higher than 30%, and storing for later use at the temperature of 2-8 ℃ after sealing.

Enzyme labeling reagent: the novel enzyme-labeled coronavirus recombinant protein S1 is diluted by enzyme-labeled diluent A1-A5 in example 1 according to the ratio of 1: 8000 to be used as an enzyme-labeled reagent, and sterile subpackaging is carried out after filtering.

20 × concentrated washing solution: taking 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of potassium dihydrogen phosphate, 4g of potassium chloride, 800mL of ultrapure water and 2010 mL of tween, completely dissolving, then using the ultrapure water to fix the volume to 1000mL, filtering by using a 0.22 mu m filter membrane, and carrying out sterile subpackaging. It is diluted 20 times with distilled water.

Positive control: the novel coronavirus recombinant protein S1 prepared in example 1 was injected subcutaneously into healthy rabbits (500. mu.g/rabbit), and the immunization was performed 1 time after 14 days, and blood was collected and serum was separated after 14 days, and the serum was collected as positive serum. And (3) taking 200mL of newborn bovine serum, 10mL of positive serum and 0.5mL of ProClin-300, uniformly mixing, supplementing phosphate buffer solution to a constant volume of 1000mL, filtering, and performing sterile subpackaging.

Negative control: and (3) uniformly mixing 200mL of newborn bovine serum and 0.5mL of ProClin-300, adding phosphate buffer solution to a constant volume of 1000mL, filtering, and performing sterile subpackaging.

Color development liquid: dissolving 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide in purified water, metering to 1000mL, mixing uniformly, filtering, and performing aseptic packaging to obtain color developing solution A. Taking 0.2g of Tetramethylbenzidine (TMB) and 100mL of absolute ethyl alcohol, dissolving in purified water to a constant volume of 1000mL, mixing uniformly, filtering, and packaging aseptically to obtain a color developing solution B.

Stopping liquid: 2M H₂SO₄And (3) solution.

The components are assembled into a kit, and the kit is divided into nCoVA 1-nCoVA 5 according to the difference of enzyme marker diluent A1-A5.

4.2 establishment of detection method

The operation steps are as follows:

(1) sample adding: 100 mul of sample to be detected is added into the corresponding hole respectively, 2 holes (100 mul of positive control and negative control) are arranged respectively, and the plate is sealed and then incubated at 37 ℃ for 30 minutes.

(2) Washing: washing with washing solution for 5 times, and drying.

(3) Adding an enzyme standard reagent: mu.l of enzyme-labeled reagent diluent was added to each well, and the plate was sealed and incubated at 37 ℃ for 30 minutes.

(4) Washing: washing with washing solution for 5 times, and drying.

(5) Color development: adding 50 mul of color development liquid A and color development liquid B into each hole, mixing uniformly, and developing for 15 minutes at 37 ℃ or at room temperature in a dark place.

(6) Terminating and detecting: adding 50 mul of stop solution into each hole, and measuring OD of each hole by using an enzyme-linked immunosorbent assay within 10 minutes_450nmThe values were determined according to the following result determination criteria.

When positive control well OD_450nmThe test is established when the value is more than or equal to 0.3 and the OD value of the negative control hole is less than 0.1, otherwise, the test is invalid. When the test is established, the threshold value (CUT OFF) is calculated: the cut-off value was 0.26+ the mean OD value of the negative control wells (0.03 for negative control wells with a mean OD value below 0.03). And (4) judging a result:

the sample with OD value larger than or equal to the critical value (CUT OFF) is positive for the novel coronavirus antibody.

Those with the sample OD value less than the CUT-OFF value (CUT OFF) were negative for the novel coronavirus antibody. And the result is judged to be positive, and the etiology verification is carried out by on-site investigation and sampling.

4.3 evaluation of the kit

4.3.1 specificity test

Specificity test is carried out on swine other virus positive sera (including PCV1 positive sera, PCV2 positive sera, CSFV positive sera, PRRSV positive sera, PRV positive sera, PPV positive sera, A FMDV positive sera and O FMDV positive sera) by using a kit nCoVA 1-nCoVA 5, and the result is that: all were negative, indicating good specificity.

4.3.2 preparation of Positive sera from different animal sources

The novel coronavirus recombinant protein S1 was injected into SPF mice and healthy rabbits (500. mu.g/rabbit) by 5 individuals, and 5 individuals were used as non-immunized controls. Blood was collected on days 0, 1, 3, 5, 7, 9, 10, and 14 after immunization, respectively, and serum was collected.

4.3.3 preparation of IgM antibody detection kit by Indirect ELISA method

Taking an antigen coated plate with the coating concentration of 0.5 mu g/ml of new coronavirus recombinant protein S1, respectively taking a commercial enzyme-labeled goat anti-mouse IgM antibody and an enzyme-labeled goat anti-rabbit IgM antibody diluted solution with the ratio of 1: 10000 as secondary antibodies, and preparing an indirect ELISA IgM antibody kit together with a washing solution, a developing solution, a stopping solution and a stopping solution, which are referred to as a mouse IgM kit and a rabbit IgM kit for short.

4.3.4 preparation of IgG antibody detection kit by indirect ELISA method

Taking an antigen coated plate with the coating concentration of 0.5 mu g/ml of the new coronavirus recombinant protein S1, respectively taking a commercial enzyme-labeled goat anti-mouse IgG antibody and a diluent of which the ratio is 1: 10000 for the enzyme-labeled goat anti-rabbit IgG antibody as a secondary antibody, and preparing an indirect ELISA IgG antibody kit, namely a mouse IgG kit and a rabbit IgG kit, together with a washing solution, a developing solution, a sample diluent and a stop solution.

4.3.5 comparison of detection of mouse IgM and IgG antibodies with different kits

The serum of mice on days 0, 1, 3, 5, 7, 9, 10 and 14 was detected by using the kits nCoVA 1-nCoVA 5 and the mouse IgM kit and the mouse IgG kit respectively, and the results are shown in Table 4: the detection of the control group serum is negative, the test is established, the murine IgM kit can detect the antibody only in3 to 9 days after immunization, the murine IgG kit can detect the antibody only in 7 to 14 days, the nCoVA1 to nCoVA5 can detect the antibody in3 to 14 days after immunization, but the antibodies detected by the nCoVA3 and the nCoVA5 are equivalent to the sum of the IgM and IgG antibodies and the detection effects of the IgM and IgG antibodies are equivalent.

TABLE 4 results of the test of immunized mice on different days

4.3.6 comparison of rabbit IgM and IgG antibodies detected by different kits

The serum of rabbits at 0 th, 1 th, 3 th, 5 th, 7 th, 9 th, 10 th and 14 th days was detected by using the kit nCoVA 1-nCoVA 5 and the rabbit IgM kit and the rabbit IgG kit, respectively, and the results are shown in Table 5: the detection of the control group serum is negative, the test is established, the rabbit IgM kit can detect the antibody only in3 to 9 days after immunization, the rabbit IgG kit can detect the antibody only in 7 to 14 days, the nCoVA1 to nCoVA5 can detect the antibody in3 to 14 days after immunization, but the antibodies detected by the nCoVA3 and the nCoVA5 are equivalent to the sum of the IgM and IgG antibodies, and the detection effect is equivalent.

TABLE 5 results of testing immunized rabbits on different days

The kit nCoVA3 (enzyme-containing standard diluent A3) and nCoVA5 (enzyme-containing standard diluent A5) can be used for detecting different animal serums, and specific reactions can be generated no matter IgM or IgG antibodies exist as long as novel coronavirus antibodies exist, so that the specificity is good.

Example 5 porcine epidemic diarrhea Virus ELISA antibody (IgA) detection kit (Indirect method)

5.1 preparation of the kit PEDVA 1-PEDVA 5

The enzyme-labeled diluents a1 to a5 prepared in example 1 and the sample diluent B6 prepared in example 2 were used to prepare kits PEDVA1 to PEDVA5 according to the preparation method of a kit in patent CN110967480A (wherein the coating antigen is an antigen-antibody complex of inactivated antigen of porcine epidemic diarrhea virus monoclonal antibody 8A3a 10-porcine epidemic diarrhea virus HN1301 strain, the HN1301 strain has a preservation number of CCTCC No: V201514, and the enzyme-labeled reagent is an enzyme-labeled goat anti-porcine IgA secondary antibody) and the detection is performed according to the detection method.

5.2 evaluation of the kit PEDVA 1-PEDVA 5

And (3) sensitivity test: respectively detecting a milk sample and a serum sample after IPMA identification by using kits PEDVA 1-PEDVA 5, and comparing the detected samples with a PEDV original kit (see a patent CN110967480A for details, wherein the original coating is a porcine epidemic diarrhea virus monoclonal antibody 8A3A 10-porcine epidemic diarrhea virus HN1301 strain inactivated antigen compound, and an enzyme labeling reagent is an enzyme-labeled goat anti-pig IgA secondary antibody), and the results are shown in a table 6, the milk sample and the serum sample are negative, and the detections of the kits PEDVA 1-PEDVA 5 are also negative; the milk sample and the serum sample are positive, the detection of the kit PEDVA 1-PEDVA 5 is also positive, but the detection effect of the kit PEDVA 3-PEDVA 5 is equivalent to that of the PEDV original kit.

TABLE 6 sensitivity test results of the kit PEDVA 1-PEDVA 5

And (3) specific test: the results of using the kit PEDVA 3-PEDVA 5 to detect swine other virus positive sera (including swine transmissible gastroenteritis virus TGEV positive sera, swine delta coronavirus PDCoV positive sera, swine rotavirus PoRV positive sera, PRV positive sera, swine parvovirus positive sera, PCV2 positive sera, PCV3 positive sera, PRRSV positive sera, CSFV positive sera, PPV positive sera, A FMDV positive sera and O FMDV positive sera) are negative, which indicates that the kit PEDVA 3-PEDVA 5 has good specificity.

The clinical application is as follows: the PEDV original kit and the PEDVA 3-PEDVA 5 are used for respectively detecting 400 parts of clinical samples of multiple targets, wherein the clinical samples comprise 100 parts of milk (40 parts of colostrums), 100 parts of serum, 75 parts of saliva, 50 parts of anus swab and 75 parts of excrement samples (which are identified by IPMA, 240 parts of positive and 160 parts of negative), the coincidence rate of the samples and the IPMA is calculated, the result is shown in table 7, the positive coincidence rate and the negative coincidence rate detected by the PEDVA 3-PEDVA 5 of the kit are both more than or equal to 81%, the positive coincidence rate and the negative coincidence rate of the PEDVA 4-PEDVA 5 of the kit are both more than or equal to 96%, and the negative coincidence rate is more than or equal to 98%, and the detection effect of the PEDV original kit is equivalent.

Table 7 clinical application detection results of kit PEDVA 3-PEDVA 5

5.3 preparation of kits PEDVB 1-PEDVB 6

Using the enzyme marker diluent a5 prepared in example 1 and the sample diluents B1 to B6 prepared in example 2, kits PEDVB1 to PEDVB6 were prepared according to the method for preparing the kits in patent CN110967480A, and detection was performed according to the detection method.

5.4 evaluation of the kit PEDVB 1-PEDVB 6

And (3) sensitivity test: respectively detecting the milk sample and the serum sample after IPMA identification by using the kit PEDVB 1-PEDVB 6, and comparing the results with a PEDV original kit (see a patent CN110967480A in detail), wherein the results are shown in the table 8, the milk sample and the serum sample are negative, and the detection by using the kit PEDVB 1-PEDVB 6 is also negative; the milk sample and the serum sample are positive, and the detection of the kit PEDVB 1-PEDVB 6 is also positive, but only the kit PEDVB1 and the PEDVB6 have the same detection effect as the PEDV original kit.

TABLE 8 sensitivity test results of the kit PEDVB 1-PEDVB 6

TABLE 8 (continuation) kit PEDVB 1-PEDVB 6 sensitivity test results

And (3) specific test: the results of using the kits PEDVB1 and PEDVB6 to detect swine-origin other virus positive sera (including swine transmissible gastroenteritis virus positive sera, swine delta coronavirus positive sera, swine rotavirus positive sera, swine pseudorabies virus positive sera, swine parvovirus positive sera, porcine circovirus type 2 positive sera, porcine circovirus type 3 positive sera, porcine reproductive and respiratory syndrome virus positive sera, swine fever virus positive sera and porcine encephalitis B virus positive sera) are negative, which indicates that the kits PEDVB1 and PEDVB6 have good specificity.

The clinical application is as follows: the coincidence rate of the sample and the IPMA is calculated by using 400 parts of the original kit for clinical samples, the kit PEDVB1 and the kit PEDVB6 used in the example 5.2, the results are shown in table 9, the positive coincidence rate and the negative coincidence rate detected by the kit PEDVB1 and the kit PEDVB6 are both more than or equal to 94%, wherein the positive coincidence rate of the kit PEDV B6 is more than or equal to 96%, and the negative coincidence rate is more than or equal to 98%, which indicates that the sensitivity of the kit is equivalent to the detection effect of the original kit.

TABLE 9 clinical application test results of the kit PEDVB1 and PEDVB6

Example 6 foot-and-mouth disease Virus A type ELISA antibody detection kit (Competition method)

6.1 preparation of the kit

The preparation method of the foot-and-mouth disease virus A type ELISA antibody detection kit (competition method) is disclosed in the patent application CN201911203052.8, and comprises the following specific steps:

antigen coated plate: coating the foot-and-mouth disease virus A type monoclonal antibody 2E11 (the heavy chain variable region of which is shown as SEQ ID No.2 and the light chain variable region of which is shown as SEQ ID No. 3) by using a carbonate buffer solution (pH value is 9.6 and 0.05mol/L) according to the ratio of 0.5 mu g/hole and 50 mu L/hole, lightly shaking and uniformly mixing, and coating for 16-24 hours at the temperature of 2-8 ℃; discarding the liquid in the plate, washing for 3-5 times by using a washing solution, adding a sealing solution (phosphate buffer solution containing 1.5% W/V BSA) into 150 mu l/hole, slightly shaking and uniformly mixing, sealing by using a sealing plate membrane for 2 hours at 37 ℃; discarding the confining liquid, washing for 3-5 times by using a washing liquid, then patting to dry, adding 10 mu g/hole and 50 mu l/hole of A type foot-and-mouth disease virus-like particle protein (CN 105566449A), and incubating for 1 hour at 37 ℃ after sealing by using a sealing plate membrane; and (3) discarding liquid in the plate, washing for 3-5 times by using a washing solution, drying for 3-6 hours at the temperature of 18-26 ℃ under the condition that the relative humidity is not higher than 30%, sealing, and storing at the temperature of 2-8 ℃ for later use.

Enzyme labeling reagent: foot-and-mouth disease virus A type monoclonal antibody 3B4 (the heavy chain variable region protein sequence is shown as SEQ ID No.4, and the light chain variable region protein sequence is shown as SEQ ID No. 5) is diluted 5000 times by enzyme marker diluent A1-A5, and is packaged in an aseptic manner.

Positive control: and (3) performing sterile subpackaging on the hyperimmune serum of the foot-and-mouth disease virus type A antigen.

Negative control: and (4) carrying out sterile subpackaging on the pig serum without the foot-and-mouth disease antibody.

Sample diluent: refer to example 2.

Stopping liquid: 2M H₂SO₄And (3) solution.

The above were assembled into a kit. Labeling the enzyme marker as a kit A _ FMDVA 1-A _ FMDVA5 according to different enzyme marker diluents (the sample diluent is B6); the samples were labeled as kits A _ FMDVB 1-A _ FMDVB6 according to their dilutions (enzyme label dilution A5).

6.2 establishment of detection method

All reagents should be balanced to room temperature (about 30 minutes) before use, liquid reagents are gently shaken and uniformly mixed before experiments, and the liquid reagents are immediately sealed and placed back to 2-8 ℃ for storage after use.

The operation steps are as follows:

(1) sample adding: diluting the sample to be detected by 5 times with the sample diluent, adding 50 mul into the corresponding hole of the microplate, and simultaneously setting a negative control hole (adding 50 mul/hole of the negative control) and a positive control hole (adding 50 mul/hole of the positive control). The plates were incubated at 37 ℃ for 1 hour.

(2) Adding an enzyme standard reagent: 50. mu.l of enzyme-labeled reagent was added to each well, and the plate was sealed and incubated at 37 ℃ for 1 hour.

(3) Washing: washing with washing solution for 5 times, and drying.

(4) Color development: adding 50 mul of color development liquid A and color development liquid B into each hole, mixing uniformly, and developing for 15 minutes at 37 ℃ or at room temperature in a dark place.

(5) Terminating and detecting: adding 50 mul of stop solution into each hole, mixing uniformly, and measuring the A value of each hole by using a microplate reader within 10 minutes: OD_450nmThe values were determined according to the following result determination criteria.

When the A value of the negative control well is OD_450nmThe test is established when the value is more than or equal to 1.5 and the value of A of the positive control hole is less than or equal to 0.5, otherwise, the test is invalid. When the test is established, the blocking rate (PI) ═ 1 & gt sample OD_450nmNegative control mean OD_450nm) X 100%. And (4) judging a result: the antibody is positive when the blocking rate is more than or equal to 50 percent, and the antibody is negative when the blocking rate is less than 50 percent.

6.3 detection of kits A _ FMDVA 1-A _ FMDVA5

6.3.1 sensitivity identification

The confirmed positive sera for type a foot-and-mouth disease virus were diluted 2-fold in gradient and detected with kits a _ FMDVA1 to a _ FMDVA5, respectively, and the results are shown in table 10, and it was found that only kit a _ FMDVA5 had high sensitivity.

TABLE 10 kit A _ FMDVA 1-A _ FMDVA5 sensitivity test results

6.3.2 specificity test

The specificity test of other virus positive serums (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) of pig origin is carried out by using the kit A _ FMDVA5, and the result is that: all were negative, indicating that the specificity of kit a _ FMDVA5 was good.

6.3.3 clinical applications

170 parts of clinical A FMDV positive serum is collected and is compared and detected with a commercial liquid phase blocking kit, and the result is as follows: the detection result of the kit A _ FMDVA5 is positive in 170 parts, and the detection effect is better than that of a commercial liquid phase blocking kit (the detection rate is only 96.5%). Indicating that the sensitivity of kit a _ FMDVA5 is good.

6.4 detection of kits A _ FMDVB 1-A _ FMDVB6

6.4.1 sensitivity identification

The confirmed positive serum of the type A foot-and-mouth disease virus was diluted 2-fold in gradient and detected with the kits A _ FMDVB 1-A _ FMDVB6, respectively, and the results are shown in Table 11, and only the kit A _ FMDVB6 was found to have high sensitivity.

TABLE 11 kit A _ FMDVB 1-A _ FMDVB6 sensitivity test results

6.4.2 specificity test

The specificity test of other virus positive serums (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) of swine origin is carried out by using the kit A _ FMDVB6, and the result is that: all were negative, indicating that the specificity of kit a _ FMDVB6 was good.

6.4.3 clinical applications

170 parts of clinical A FMDV positive serum is collected and is compared and detected with a commercial liquid phase blocking kit, and the result is as follows: the kit A _ FMDVB6 is positive in detection of 170 parts, and the detection effect is better than that of a commercial liquid phase blocking kit (the detection rate is only 96.5%). Indicating that the sensitivity of kit a _ FMDVB6 is good.

Example 7 foot-and-mouth disease Virus O type ELISA antibody detection kit (Competition method)

7.1 preparation of the kit

The preparation method of the foot-and-mouth disease virus O-type ELISA antibody detection kit (competition method) is disclosed in the patent application CN201910881627.5, and specifically comprises the following steps:

antigen coated plate: coating the O type monoclonal antibody 6G6 of the foot-and-mouth disease virus (the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.6, and the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 7) by using carbonate buffer solution (pH value is 9.6, 0.05mol/L) according to 0.08 mu G/hole and 50 mu L/hole, lightly shaking and uniformly mixing, and coating for 16-24 hours at the temperature of 2-8 ℃; discarding the liquid in the plate, washing for 3-5 times by using a washing solution, adding a sealing solution (phosphate buffer solution containing 1.5% W/V BSA) into 150 mu l/hole, slightly shaking and uniformly mixing, sealing for 2 hours at 37 ℃ by using a sealing plate membrane sealing plate; discarding the confining liquid, washing for 3-5 times by using a washing liquid, then patting to dry, adding 10 mu g/hole and 50 mu l/hole of O-type foot-and-mouth disease virus-like particle protein (CN 105566449A), and incubating for 1 hour at 37 ℃ after sealing by using a sealing plate membrane; and (3) discarding liquid in the plate, washing for 3-5 times by using a washing solution, drying for 3-6 hours at the temperature of 18-26 ℃ under the condition that the relative humidity is not higher than 30%, sealing, and storing at the temperature of 2-8 ℃ for later use.

Enzyme labeling reagent: the foot-and-mouth disease virus O type monoclonal antibody 7D2 (the heavy chain variable region protein sequence of which is shown in SEQ ID No.8 and the light chain variable region protein sequence of which is shown in SEQ ID No. 9) is diluted 5000 times by enzyme marker diluent A1-A5 and is packaged in an aseptic manner.

Positive control: and (3) performing sterile subpackaging on the hyperimmune serum of the O-type foot-and-mouth disease virus antigen.

Sample diluent: refer to example 2.

Stopping liquid: 2M H₂SO₄And (3) solution.

The above were assembled into a kit. Labeling the enzyme marker as a kit O _ FMDVA 1-O _ FMDVA5 according to different enzyme marker diluents (the sample diluent is B6); the samples were labeled as kits O _ FMDVB 1-O _ FMDVB6 according to their dilutions (enzyme label dilution A5).

7.2 establishment of detection method

The operation steps are as follows:

(3) Washing: washing with washing solution for 5 times, and drying.

(5) Terminating and detecting: adding 50 mul of stop solution into each hole, mixing uniformly, and measuring the A value of each hole by using a microplate reader within 10 minutes: OD_450nmValue, according to the following result judgment criteriaAnd (6) judging.

7.3 detection of O _ FMDVA 1-O _ FMDVA5 in kit

7.3.1 sensitivity identification

The confirmed southeast subtype O-type foot-and-mouth disease virus positive serum and Chinese type O-type foot-and-mouth disease virus positive serum are respectively subjected to 2-fold gradient dilution, and are respectively detected by using the kits O _ FMDVA 1-O _ FMDVA5, the results are shown in Table 12, and only the kit O _ FMDVA5 is found to have high sensitivity.

Table 12 kit O _ FMDVA 1-O _ FMDVA5 sensitivity test results

7.3.2 specificity test

The specificity test of other virus positive serums (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) of pig origin is carried out by using the kit O _ FMDVA5, and the result is that: all were negative, indicating that the specificity of the kit O _ FMDVA5 was good.

7.3.3 clinical applications

186 parts of clinical southeast Asia subtype O FMDV positive serum are collected and compared with a commercial liquid blocking kit for detection, and the result is as follows: 186 parts of the O _ FMDVA5 reagent kit are positive, and the detection effect is better than that of a commercial liquid phase blocking reagent kit (the detection rate is only 95.7%).

194 parts of clinical Chinese O FMDV positive serum are collected and compared with a commercial liquid blocking kit for detection, and the result is as follows: 194 parts of the O _ FMDVA5 reagent kit are positive, and the detection effect is better than that of a commercial liquid phase blocking reagent kit (the detection rate is only 92.7%). Indicating that the sensitivity of the kit O _ FMDVA5 is good.

7.4 detection of kit O _ FMDVB 1-O _ FMDVB6

7.4.1 sensitivity identification

The confirmed southeast subtype O-type foot-and-mouth disease virus positive serum and the Chinese O-type foot-and-mouth disease virus positive serum are respectively subjected to 2-fold gradient dilution, and are respectively detected by using the kits O _ FMDVB 1-O _ FMDVB6, the results are shown in Table 13, and only the kit O _ FMDVB6 is found to have high sensitivity.

Table 13 kit O _ FMDVB 1-O _ FMDVB6 sensitivity test results

TABLE 13 (continuation) kit O _ FMDVB 1-O _ FMDVB6 sensitivity test results

7.4.2 specificity test

The specificity test of other virus positive serums (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) of swine origin is carried out by using the kit O _ FMDVB6, and the results are as follows: all were negative, indicating that the specificity of the kit O _ FMDVB6 was good.

7.4.3 clinical applications

186 parts of clinical southeast Asia subtype O FMDV positive serum are collected and compared with a commercial liquid blocking kit for detection, and the result is as follows: 186 parts of the O _ FMDVB6 reagent kit are positive, and the detection effect is better than that of a commercial liquid phase blocking reagent kit (the detection rate is only 95.7%).

194 parts of clinical Chinese O FMDV positive serum are collected and compared with a commercial liquid blocking kit for detection, and the result is as follows: 194 parts of the kit O _ FMDVB6 are positive, and the detection effect is better than that of a commercial liquid phase blocking kit (the detection rate is only 92.7%). Indicating that the sensitivity of the kit O _ FMDVB6 is good.

Example 8 Swine fever Virus blocking ELISA antibody detection kit (blocking method)

8.1 preparation of the kit

Using the enzyme marker diluents a 1-a 5 prepared in example 1 and the sample diluent B6 prepared in example 2, respectively, the kit CSFVA 1-CSFVA 5 was prepared according to the method for preparing the kit of patent CN105837686A (in which the original coating was swine fever virus E2 protein, and the enzyme marker was the enzyme-labeled swine fever virus monoclonal antibody 15a9), and the detection was performed according to the detection method, and the optimal enzyme marker diluent was preferably selected.

8.2 evaluation of kit CSFVA 1-CSFVA 5

And (3) sensitivity test: carrying out multiple-ratio dilution on clinical swine fever virus positive serum with IPMA titer of 1: 12800, carrying out sensitivity evaluation by using a kit CSFVA 1-CSFVA 5, and comparing by using the kit prepared in the CN105837686A method (wherein an enzyme label is an enzyme-labeled swine fever virus monoclonal antibody 15A9) as a CSFV original kit; the results are shown in Table 14: the sensitivity of the kit CSFVA 1-CSFVA 4 is weaker than that of the CSFV original kit, and only the sensitivity of the kit CSFVA5 is higher than that of the CSFV original kit.

TABLE 14 summary of the results of the CSFVA 1-5 kit

And (3) specific test: specific tests are carried out on swine other virus positive sera (including PCV1 positive sera, PCV2 positive sera, PCV3 positive sera, PRRSV positive sera, PRV positive sera, PPV positive sera, A FMDV positive sera and O FMDV positive sera) by using the kit CSFVA 1-CSFVA 5 and CSFV original kit, and the results are as follows: all were negative, indicating good specificity.

The clinical application is as follows: 324 swine fever clinical samples are collected, 128 parts of positive samples and 173 parts of negative samples are detected by a CSFV original kit and an IDEXX swine fever antibody detection kit, the rest 23 parts of positive samples and the rest negative samples are detected by the original kit, the rest negative samples are detected by the IDEXX kit, and the rest positive samples are verified to be positive by a neutralization test, namely 151 parts of positive samples and 173 parts of negative samples. The results of detection by the kit CSFVA 1-CSFVA 5 are shown in Table 14, and the positive coincidence rate and the negative coincidence rate of the kit CSFVA 1-CSFVA 4 are obviously lower than those of the kit CSFVA5, which shows that the enzyme marker diluent A5 can be used for preparing the hog cholera virus ELISA antibody detection kit and has good sensitivity.

8.3 preparation of kit CSFVB 1-CSFVB 6

The enzyme-labeled diluent a5 prepared in example 1 and the sample diluents B1 to B6 prepared in example 2 were used to prepare kits CSFVB1 to CSFVB6 according to the preparation method of kits in patent CN105837686A (in which the enzyme-labeled antibody is the enzyme-labeled classical swine fever virus monoclonal antibody 15a9) and the detection was carried out according to the detection method.

8.4 evaluation of kit CSFVB 1-CSFVB 6

And (3) sensitivity test: carrying out multiple-ratio dilution on clinical swine fever virus positive serum with IPMA titer of 1: 12800, carrying out sensitivity evaluation by using a kit CSFVB 1-CSFVB 6, and comparing by using the kit prepared in the CN105837686A method (wherein an enzyme label is an enzyme-labeled swine fever virus monoclonal antibody 15A9) as a CSFV original kit; the results are shown in Table 15: the sensitivity of the kit CSFVB 1-CSFVB 6 is weaker than that of the CSFV original kit, and only the kit CSFVB6 has higher sensitivity than that of the CSFV original kit.

Table 15 summary of the results of CSFVB 1-CSFVB 6

And (3) specific test: specific tests are carried out on swine other virus positive sera (including PCV1 positive sera, PCV2 positive sera, PCV3 positive sera, PRRSV positive sera, PRV positive sera and PPV positive sera) by using the kit CSFVB 1-CSFVB 6 and CSFV original kit, and the results are as follows: all were negative, indicating good specificity.

The clinical application is as follows: 324 clinical samples in the example 8.2 are detected by using the kit CSFVB 1-CSFVB 6, the results are shown in Table 16, the positive coincidence rate and the negative coincidence rate of the kit CSFVB 1-CSFVB 5 are obviously lower than those of the kit CSFVB6, and the sample diluent B6 can be used for preparing the hog cholera virus ELISA antibody detection kit and has good sensitivity.

Example 9 detection kit for gE protein ELISA for porcine pseudorabies Virus (blocking method)

9.1 preparation of the kit

The enzyme marker diluent A1-A5 prepared in example 1 and the sample diluent B6 prepared in example 2 are respectively used for preparing a kit PRVgEA 1-PRVgEA 5 according to a preparation method of a kit in patent CN109959788A (wherein a mixture of an inactivated antigen of porcine pseudorabies virus HN1201 strain and an inactivated antigen of Fa strain is coated, the preservation number of the porcine pseudorabies virus HN1201 strain is CCTCC No: V201311, the Fa strain is purchased from a Chinese veterinary medicine inspection institute, and the enzyme marker is an enzyme marker gE protein monoclonal antibody 11H1 disclosed in CN109959788A), and the kit is detected according to a detection method, so that the optimal enzyme marker diluent is preferably selected.

9.2 evaluation of kits PRVgEA 1-PRVgEA 5

And (3) sensitivity test: the classical strain Fa and the variant strain HN1201 of the porcine pseudorabies virus are divided into 2 groups, 5 pig sera in each group are respectively subjected to a virus challenge test, 80 pig sera which are collected at different days after 10 pig virus challenges, namely 0, 3, 5, 7, 9, 11, 13 and 15 days are subjected to sensitivity evaluation through a kit PRVgEA 1-PRVgEA 5, and a kit 4# prepared in a CN109959788A method is used as a PRVgE original kit for comparison; as a result: the sensitivity of the kits PRVgEA 1-PRVgEA 4 is respectively 48% (38/80), 45% (36/80), 44% (35/80) and 50% (40/80) which are weaker than that of the original PRVgE kit 62.5% (50/80), and only the sensitivity of the kit PRVgEA5 is 62.5% (50/80) which is equivalent to that of the original PRVgE kit.

And (3) specific test: specificity test is carried out on swine other virus positive sera (including PCV1 positive sera, PCV2 positive sera, PCV3 positive sera, PRRSV positive sera, CSFV positive sera, PPV positive sera, A FMDV positive sera and O FMDV positive sera) by using the kit PRVgEA 1-PRVgEA 5 and PRVgE original kit, and the result is that: all were negative, indicating good specificity.

The clinical application is as follows: 200 parts of serum (including 100 positive parts and 100 negative parts) which is clear in background, is from different areas, has porcine pseudorabies infection symptoms and is determined by a serum neutralization test method are collected and detected by a PRVgE original kit and a PRVgEA5 kit, the result is shown in table 16, 86 positive parts, 109 negative parts and 5 suspicious parts are detected by the PRVgEA5 kit, the detection effect is equivalent to that of the PRVgE original kit, and the sensitivity is good.

TABLE 16 summary of the test results of the kits PRVgEA5, PRVgEB6 and PRVgE

9.3 preparation of kits PRVgEB 1-PRVgEB 6

Using the enzyme marker diluent a5 prepared in example 1 and the sample diluents B1 to B6 prepared in example 2, kits PRVgEB1 to PRVgEB6 were prepared and tested according to the method for preparing the kit of patent CN109959788A (in which the enzyme marker was the enzyme-labeled porcine pseudorabies virus gE protein monoclonal antibody 11H 1).

9.4 evaluation of kits PRVgEB 1-PRVgEB 6

And (3) sensitivity test: 80 parts of pig serum collected in example 9.2 are subjected to sensitivity evaluation by using a kit PRVgEB 1-PRVgEB 6, and are compared by using a PRVgE original kit; as a result: the sensitivity of the kits PRVgEB 1-PRVgEB 5 is respectively 44% (35/80), 40% (32/80), 50% (40/80) and 45% (36/80) which are weaker than that of the original PRVgE kit 62.5% (50/80), and only the sensitivity of the kit PRVgEB6 is 62.5% (50/80) which is equivalent to that of the original PRVgE kit.

And (3) specific test: the kit PRVgEB 1-PRVgEB 6 and the PRVgE original kit are used for carrying out specificity test on swine source other virus positive serum (comprising PCV1 positive serum, PCV2 positive serum, PCV3 positive serum, PRRSV positive serum, CSFV positive serum and PPV positive serum), and the result is that: all were negative, indicating good specificity.

The clinical application is as follows: 200 parts of serum (including 100 positive parts and 100 negative parts) which is clear in background, is from different areas, has porcine pseudorabies infection symptoms and is determined by a serum neutralization test method are collected and detected by a PRVgE original kit and a PRVgEB6 kit, the result is shown in table 17, 86 positive parts, 109 negative parts and 5 suspicious parts are detected by the PRVgEB6 kit, the detection effect is equivalent to that of the PRVgE original kit, and the sensitivity is good.

Example 10 porcine pseudorabies virus gD protein ELISA antibody detection kit (Indirect method)

10.1 preparation of the kit

Antigen coated plate: diluting porcine pseudorabies virus gD protein prepared according to patent CN105251000A to 0.5 mu g/ml by using carbonate buffer solution (pH value is 9.6, 0.05mol/L) for coating, coating for 16-24 hours at the temperature of 2-8 ℃ by 100 mu L/hole, washing by using washing solution, adding sealing solution (phosphate buffer solution containing 5% W/V sucrose, 20% V/V newborn bovine serum and 0.05% V/V ProClin 300) according to 150 mu L/hole, sealing for 16-24 hours at the temperature of 2-8 ℃ or sealing for 2 hours at the temperature of 37 ℃, drying for 3-6 hours at the temperature of 18-26 ℃ under the condition that the relative humidity is not higher than 30% after removing the sealing solution, and storing for later use at the temperature of 2-8 ℃ after sealing.

Enzyme labeling reagent: commercial goat-or rabbit-anti-pig lgG-HRP was diluted with the enzyme-labeled diluents A1-A5 prepared in example 1 at a final volume of 0.05% V/V, mixed well, filtered at 0.22 μm, and aseptically dispensed.

Positive control: the variant porcine pseudorabies inactivated vaccine (HN 1201-delta gE strain, from Poleco bioengineering GmbH) is a healthy susceptible pig (2 ml/head) with negative neck muscle immune PRV antigen antibodies, blood is collected 28 days later, serum is separated, and the collected serum is positive serum. And (3) taking 200mL of newborn bovine serum, 10mL of positive serum and 0.5mL of ProClin-300, uniformly mixing, supplementing phosphate buffer solution to a constant volume of 1000mL, filtering, and performing sterile subpackaging.

Negative control: selecting healthy susceptible pigs with negative PRV antigen antibodies, collecting blood from carotid artery, separating serum, and collecting serum to obtain negative serum. And (3) taking 200mL of newborn bovine serum, 10mL of negative serum and 0.5mL of ProClin-300, uniformly mixing, supplementing phosphate buffer solution to a constant volume of 1000mL, filtering, and performing sterile subpackaging.

Sample diluent: refer to example 2.

Stopping liquid: 2M H₂SO₄And (3) solution.

The above were assembled into a kit. According to the difference of enzyme marker diluent (sample diluent is B6), marking the enzyme marker as a kit PRVgDA 1-PRVgDA 5; the samples were labeled as kit PRVgDB 1-PRVgDB 6 according to the dilution (the dilution of the enzyme marker is A5).

10.2 establishment of detection method

The operation steps are as follows:

(1) adding a sample diluent: 100 mul of sample diluent is added into corresponding wells of the microplate, and a negative control well (100 mul/well of negative control), a positive control well (100 mul/well of positive control) and a blank control well (100 mul of PBS buffer) are arranged at the same time.

(2) Adding a sample to be detected: the samples to be detected are respectively added into corresponding holes except for a negative control hole, a positive control hole and a blank control hole by 10 mul. The plates were incubated at 37 ℃ for 30 minutes.

(3) Washing: washing with washing solution for 5 times, and drying.

(4) Adding an enzyme standard reagent: mu.l of enzyme-labeled reagent was added to each well except for the blank control well, and the plate was sealed and incubated at 37 ℃ for 30 minutes.

(5) Washing: washing with washing solution for 5 times, and drying.

(6) Color development: adding 50 mul of color development liquid A and color development liquid B into each hole, mixing uniformly, and developing for 15 minutes at 37 ℃ or at room temperature in a dark place.

(7) Terminating and detecting: adding 50 mul of stop solution into each well, and measuring the A value of each well by using a microplate reader within 10 minutes: OD_450nmValue (measured after zeroing blank control wells), or OD_450nm－OD_{600nm～650nm}The values were determined according to the following result determination criteria.

When the positive control well is in the A value OD_450nmThe test is established when the value is more than or equal to 0.2 and the value of the negative control hole A is less than or equal to 0.1, otherwise, the test is invalid. When the test is established, calculating an S/P value: sample a value/positive control a value mean. And (4) judging a result:

the pig pseudorabies virus antibody is positive when the S/P value is more than or equal to 0.5,

the pig pseudorabies virus antibody is negative when the S/P value is less than 0.5.

10.3 detection of the kit PRVgD A1-PRVgD A5

10.3.1 sensitivity

Collecting pig serum with clear background, comprising 15 parts of pig serum with negative screened porcine pseudorabies virus antigen antibodies, 60 parts of experimental animal serum after immunization of porcine pseudorabies virus vaccine, 15 parts of newborn piglet serum without colostrum, and 90 parts of total serum, all using a kit PRVgD A1-PRVgD A5 for detection, and determining a neutralizing antibody by using a variant HN1201 strain according to a GB/T18641-2002 method serum neutralization test method (a neutralization titer determination standard is negative if the titer is less than 1: 2, and positive if the titer is more than or equal to 1: 2). Results (see table 17): 38 parts of neutralization potency less than 1: 2 in laboratory application detection, wherein only PRVgD A5 in the negative coincidence rate of the kit PRVgD A1-PRVgD A5 is 100%; the neutralization titer is more than or equal to 1: 2, and only PRVgD A5 in the positive coincidence rate of the kit PRVgD A1-PRVgD A5 is more than 90 percent (94 percent).

Table 17 summary of the results of the PRVgD A1-PRVgD A5 test in the kit

10.3.2 specificity

The confirmed porcine pseudorabies virus antibody negative serum 5 parts, PCV2 positive serum 1 part, PPV positive serum 1 part, PRRSV positive serum 1 part, CSFV positive serum 1 part, JEV positive serum 1 part, Haemophilus Parasuis (HPs) positive serum 1 part, swine atrophic rhinitis (Bb) positive serum 1 part, Escherichia coli (BL21) positive serum 1 part, mycoplasma hyopneumoniae (Mhp) positive serum 1 part, PEDV positive serum 1 part, TGEV positive serum 1 part, PRoV positive serum 1 part, A FMDV positive serum 1 part, FMO FMDV positive serum 1 part, the kit PRVgDA5 prepared in example 3, the neutralization test and the commercialized kit BioChek PRgVB antibody kit are respectively used for detection, and the result is as follows: the detection result of the kit PRVgDA5 is consistent with the detection results of the neutralization test and the commercial kit, and the detection results are negative, which shows that the specificity of the kit PRVgDA5 is good.

10.3.3 clinical application

Clinical pig serum was collected in 70 parts, and the results of detection and comparison were carried out using kits PRVgD A1-PRVgD A5 and a commercial Biochek-based antibody detection kit for porcine pseudorabies virus, and are shown in Table 17: the positive coincidence rate of the kit PRVgD A1-PRVgD A5 is only PRVgD A5 > 90% (92%), the negative coincidence rate is only 100% of the kit PRVgD A5, the total coincidence rate is 96%, and the kit PRVgD A5 has good sensitivity.

10.4 detection of the kit PRVgD B1-PRVgD B6

10.4.1 sensitivity

90 portions of the serum from example 10.3.1 were tested using the kits PRVgD B1-PRVgD B6 and the neutralization test was used for comparison. As a result: 38 parts of neutralization titer is less than 1: 2 in laboratory application detection, and only PRVgD B6 in the negative coincidence rate of the kit PRVgD B1-PRVgD B6 is 100%; the neutralization titer is more than or equal to 1: 2, and only PRVgD B6 in the positive coincidence rate of the kit PRVgD B1-PRVgD B5 is more than 90 percent (94 percent).

Table 18 summary of the results of the detection of the kit PRVgD B1-PRVgD B6

10.4.2 specificity

The specific serum samples of example 10.3.2 were tested using the kit PRVgD B6 prepared in example 10.4.1, and the neutralization test and commercial kit BioChek PRVgB antibody kit, respectively, and as a result: the detection result of the kit PRVgD B6 is consistent with the detection results of the neutralization test and the commercial kit, and the detection results are negative, which indicates that the specificity of the kit PRVgD B6 is good.

10.4.3 clinical applications

Clinical pig serum was collected in 70 parts, and the results of detection and comparison were carried out using kits PRVgD B1-PRVgD B6 and a commercial Biochek-based antibody detection kit for porcine pseudorabies virus, and are shown in Table 18: only PRVgD B6 in the positive coincidence rate of the kit PRVgD B1-PRVgD B6 is more than 90 percent (92 percent), only the kit PRVgD B6 in the negative coincidence rate is 100 percent, and the total coincidence rate is 96 percent, which shows that the kit PRVgD B6 has good sensitivity.

In conclusion, the composition of the animal epidemic disease ELISA antibody detection kit prepared by the invention can be simultaneously used for preparing animal epidemic disease ELISA antibody detection kits for various animal epidemic diseases and various detection types (including competition method, indirect method, blocking method and sandwich method), and the prepared antibody sensitivity of each kit for detecting each animal epidemic disease is equivalent to or even better than that of the original kit, the specificity is good, the preparation is simple and convenient during large-scale production, and the cost of manpower and material resources is saved.

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

SEQUENCE LISTING

<110> Luoyang Putai Biotech Ltd

<120> enzyme-labeled diluent and sample diluent combination for enzyme-linked immunoassay kit, and reagent containing the same

Cartridge and method of making the same

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<170> PatentIn version 3.3

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<213> novel coronavirus (novel coronavirus COVID-19)

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Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

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Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

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His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

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Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

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Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

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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

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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

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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

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Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

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Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

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Ser Phe Thr Val Glu Lys Gly Ile Tyr

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Asp Ile Phe Pro Thr Ser Asp Ser Ile Asn Tyr Asn Glu Lys Phe

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Lys Thr Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Ile Leu Leu Ser Val Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr

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Val Ser Ser

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Asp Ile Val Met Thr Gln Phe Pro Ser Ser Leu Thr Val Thr Ala Gly

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Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Ser Gly Gln

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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Ile Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr

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Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

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Asp Tyr Asn Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

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Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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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

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Thr Arg Val His Tyr Arg His Gly Phe Ala Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ala

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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

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Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

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Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Tyr Tyr Pro Phe Thr

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Phe Gly Gly Gly Thr Glu

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

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Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

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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

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Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Val Arg Lys Gly Tyr Tyr Arg Tyr Asp Val Glu Thr Ser Gly Phe Ala

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Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

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Glu Lys Val Thr Val Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

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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

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Tyr Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

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Lys Arg

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Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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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

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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

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<212> PRT

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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

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Val Val Trp Tyr Gln Glu Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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Claims

1. The enzyme marker diluent is a 0.02M PBS solution containing 5% V/V fetal calf serum, 1-3% W/V mannitol, 2% W/V sucrose, 0.05% V/V Proclin300 and pH7.4, and the sample diluent is a 0.02M PBS solution containing 0.4-0.8% W/V gelatin, 0.1% V/V glycerin, 0.1% W/V EDTA, 0.05% V/V Proclin300 and pH7.4.

2. The enzyme-labeled diluent and sample diluent combination according to claim 1, wherein the enzyme-labeled 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, pH7.4, and 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, pH 7.4.

3. The enzyme-label diluent and sample diluent combination of claim 1, wherein the enzyme-label diluent is a diluent of an enzyme label selected from the group consisting of: enzyme-labeled porcine circovirus type 2 monoclonal antibody 3H 11; enzyme-labeled novel coronavirus protein S1, wherein the novel coronavirus protein S1 is shown as SEQ ID No. 1; enzyme-labeled goat anti-pig IgA antibody; the enzyme-labeled foot-and-mouth disease virus A type monoclonal antibody 3B4, wherein the foot-and-mouth disease virus A type monoclonal antibody 3B4 heavy chain variable region is shown as SEQ ID No.4, and the foot-and-mouth disease virus A type monoclonal antibody 3B4 light chain variable region is shown as SEQ ID No. 5; the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2, wherein the variable region of the heavy chain of the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 is shown as SEQ ID No.8, and the variable region of the light chain of the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 is shown as SEQ ID No. 9; enzyme-labeled CSFV monoclonal antibody 15A 9; enzyme-labeled PRVgE protein monoclonal antibody 11H 1; and enzyme-labeled goat anti-pig lgG or enzyme-labeled rabbit anti-pig lgG.

4. The enzyme-label diluent and sample diluent combination of claim 1, wherein the sample diluent is a diluent of: a serum sample suspected of porcine circovirus type 2 virus infection, a serum sample suspected of new coronavirus infection, a milk sample suspected of PEDV infection, a serum sample, a saliva sample, an anus swab sample, a stool sample, a serum sample suspected of foot-and-mouth disease virus type A infection, a serum sample suspected of foot-and-mouth disease virus type O infection, a serum sample suspected of CSFV infection, a serum sample suspected of porcine pseudorabies virus infection, and a serum sample suspected of porcine pseudorabies virus antigen immunization.

5. The enzyme-labeled diluent and sample diluent combination 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 porcine pseudorabies virus gE protein blocking ELISA antibody detection kit, and a porcine pseudorabies virus gD protein indirect ELISA antibody detection kit.

6. The enzyme marker diluent and sample diluent combination of claim 4, 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.

7. An enzyme linked immunosorbent assay kit, wherein the kit comprises: an antigen coated plate coated with an animal epidemic disease coating antigen; an enzyme labeling reagent, wherein the enzyme labeling reagent is prepared by diluting an enzyme-labeled animal epidemic disease antibody or antigen with the enzyme-labeled diluent of any one of claims 1 to 6, filtering, and subpackaging; the sample diluent of any one of claims 1 to 6; washing liquid; a color developing solution; and a stop solution; preferably, the kit further comprises a negative control and a positive control.

8. The enzyme-linked immunoassay kit according to claim 7, wherein when the enzyme-linked immunoassay kit is a porcine circovirus type 2 competitive ELISA antibody detection kit, the animal epidemic antigen is porcine circovirus type 2 Cap protein;

when the enzyme-linked immunoassay kit is a new coronavirus double-antigen sandwich ELISA antibody detection kit, the animal epidemic disease antigen is a novel coronavirus protein S1, and the novel coronavirus protein S1 is shown as SEQ ID No. 1;

when the enzyme linked immunosorbent assay kit is a swine epidemic diarrhea virus indirect ELISA antibody detection kit, the animal epidemic disease antigen is an antigen-antibody complex of a swine epidemic diarrhea virus monoclonal antibody 8A3A 10-a swine epidemic diarrhea virus HN1301 strain inactivated antigen, the swine epidemic diarrhea virus monoclonal antibody 8A3A10 is secreted by a hybridoma cell 8A3A10 strain, the hybridoma cell 8A3A10 has a preservation number of CCTCC No. C2014197, and the swine epidemic diarrhea virus HN1301 strain has a preservation number of CCTCC No: v201514;

when the enzyme-linked immunoassay kit is a foot-and-mouth disease virus A type competition ELISA antibody detection kit, the animal epidemic disease antigen is an antigen-antibody complex of a foot-and-mouth disease virus A type monoclonal antibody 2E 11-foot-and-mouth disease virus A type virus-like particle protein, the foot-and-mouth disease virus A type monoclonal antibody 2E11 heavy chain variable region is shown as SEQ ID No.2, and the light chain variable region is shown as SEQ ID No. 3;

when the enzyme-linked immunoassay kit is a foot-and-mouth disease virus O-type competition ELISA antibody detection kit, the animal epidemic disease antigen is an antigen-antibody complex of a foot-and-mouth disease virus O-type monoclonal antibody 6G 6-foot-and-mouth disease O-type virus-like particle protein, the foot-and-mouth disease virus O-type monoclonal antibody 6G6 heavy chain variable region is shown as SEQ ID No.6, and the light chain variable region is shown as SEQ ID No. 7;

when the enzyme linked immunosorbent assay kit is a classical swine fever virus blocking ELISA antibody detection kit, the animal epidemic disease antigen is classical swine fever virus E2 protein;

when the enzyme-linked immunoassay kit is a porcine pseudorabies virus gE protein blocking ELISA antibody detection kit, the animal epidemic disease antigen is a mixture of porcine pseudorabies virus HN1201 inactivated antigen and Fa inactivated antigen, and the preservation number of the porcine pseudorabies virus HN1201 is CCTCC No: V201311; and

when the enzyme-linked immunosorbent assay kit is a porcine pseudorabies virus gD protein indirect ELISA antibody detection kit, the animal epidemic disease antigen is porcine pseudorabies virus gD protein.

9. A method of making an enzyme linked immunosorbent kit, wherein the method comprises:

diluting and coating animal epidemic disease coating antigen by using a carbonate buffer solution with the pH value of 9.6 and the concentration of 0.05mol/L to prepare an antigen coated plate;

diluting an enzyme-labeled animal epidemic disease antibody or antigen with the enzyme-labeled diluent according to any one of claims 1 to 6, filtering, packaging, and preparing into an enzyme-labeled reagent;

preparing the sample diluent, the washing solution, the developing solution and the stop solution according to any one of claims 1 to 6 in step (3), and assembling the sample diluent, the washing solution, the developing solution and the stop solution into a kit together with the antigen coated plate prepared in step (1) and the enzyme labeling reagent prepared in step (2).

10. Use of the enzyme marker diluent and the sample diluent of claims 1-6 in combination for preparing an enzyme-linked immunoassay kit.