A blocking ELISA detection kit and detection method for CSFV-E2 antibodies

Abstract

The present application relates to the technical field of biological detection, and specifically discloses a blocking ELISA detection kit and detection method for CSFV-E2 antibodies. The present application discloses a blocking ELISA detection kit for CSFV-E2 antibodies, including an enzyme-labeled plate coated with a capture antibody, a standard antigen, and an enzyme-labeled antibody; the capture antibody is a monoclonal antibody of the 7C9 strain of CSFV-E2 protein; the standard antigen is a baculovirus-expressed CSFV-E2 protein antigen; the enzyme-labeled antibody is an enzyme-labeled monoclonal antibody of the 6E12 strain of CSFV-E2 protein. The present application also discloses a blocking ELISA detection method for CSFV-E2 antibodies obtained based on the above-mentioned detection kit. The blocking ELISA detection kit and detection method for CSFV-E2 antibodies provided in the present application have high sensitivity, specificity and repeatability.

Description

Technical Field

[0001] The present application relates to the technical field of biological detection, and in particular to a blocking ELISA detection kit and a detection method for CSFV-E2 antibodies. Background Art

[0002] Classical Swine Fever (CSF) is a highly contagious disease of pigs caused by Classical Swine Fever Virus (CSFV). The main characteristics of this disease are high fever and extensive bleeding throughout the body. It can cause large-scale morbidity and mortality in pigs, seriously endangering the development of my country's pig industry.

[0003] Vaccination is an important measure for the prevention and control of classical swine fever. Among them, the attenuated classical swine fever rabbit vaccine (C strain) is considered to be one of the most effective classical swine fever vaccines. The widespread use of classical swine fever C strain vaccine has played a key role in controlling the epidemic of classical swine fever. However, this type of vaccine immunization does not completely block the spread of CSFV; and in production practice, immunization failure is often caused by unreasonable immunization procedures, resulting in the coexistence of typical and atypical classical swine fever, immune tolerance and virus syndrome in the epidemic of classical swine fever. Therefore, regular monitoring and analysis of the immune level of the pig herd to eliminate pigs with low immune levels is of great significance for controlling the epidemic and purification of classical swine fever.

[0004] At present, the main methods for detecting swine fever antibodies are fluorescent antibody neutralization test (FVNT) and enzyme-linked immunosorbent assay (ELISA). Among them, FVNT can be used as the gold standard for detecting swine fever antibodies, but this detection method has high requirements for test conditions and operators, and is time-consuming. It is only suitable for professional laboratories to test a small number of samples and is not suitable for promotion and application. The ELISA method is simple to operate, has strong specificity, high sensitivity, good repeatability, can detect multiple samples at the same time, and the results are reliable. It is currently the only serological detection method recognized internationally that is suitable for large-scale application.

[0005] At present, ELISA antibody detection methods include indirect ELISA, blocking ELISA and chemiluminescent ELISA, etc., which have been widely used in clinical practice to monitor the level of swine fever antibodies. However, the above ELISA antibody detection method mainly involves directly coating the recombinant swine fever virus E2 protein (CSFV-E2) on the ELISA plate. Due to the differences in protein expression systems, the sensitivity and specificity of the detection method are poor. Summary of the invention

[0006] In order to improve the sensitivity and specificity of the CSFV-E2 antibody detection method, the present application provides a blocking ELISA detection kit and a detection method for CSFV-E2 antibodies.

[0007] In a first aspect, the present application provides a blocking ELISA detection kit for CSFV-E2 antibodies.

[0008] A blocking ELISA detection kit for CSFV-E2 antibodies, comprising an enzyme-labeled plate coated with a capture antibody, a standard antigen, and an enzyme-labeled antibody;

[0009] The capture antibody is a monoclonal antibody of the CSFV-E2 protein 7C9 strain;

[0010] The standard antigen is a CSFV-E2 protein antigen expressed by baculovirus;

[0011] The enzyme-labeled antibody is an enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody.

[0012] The present application utilizes the monoclonal antibody of CSFV-E2 protein 7C9 strain as the capture antibody coated in the ELISA plate to capture the standard antigen to form a solid phase antigen; then utilizes the enzyme-labeled monoclonal antibody of CSFV-E2 protein 6E12 strain as the competitive antibody of CSFV-E2 antibody in the serum sample to be tested, thereby obtaining a detection kit for CSFV-E2 antibody with high detection sensitivity, strong specificity and good repeatability.

[0013] The present application is based on the principle of indirect capture and coating, which moves the solid-phase antigen away from the carrier surface so that the antigenic determinant cluster can be fully exposed; at the same time, the indirectly coated antigen is subjected to affinity chromatography by the capture antibody, so that the purity of the standard antigen coated on the solid phase is greatly improved, thereby effectively improving the sensitivity and specificity of the detection method.

[0014] Preferably, the method for preparing the standard antigen is: extracting viral nucleic acid from the attenuated swine fever lapinized strain (strain C), amplifying the E2 gene by RT-PCR, and cloning it into the pFastBacHTa vector to construct a recombinant plasmid pFastBacHTa-CSFV-E2, using the Bac-to-Bac baculovirus expression system, transferring the recombinant plasmid into DH10Bac for transposition, obtaining a recombinant Bacmid containing the E2 gene, extracting the recombinant Bacmid and transfecting Sf9 insect cells to prepare a recombinant baculovirus rBV-CSFV-E2 strain expressing the E2 protein, and purifying the expressed recombinant E2 protein by Ni column affinity chromatography to obtain the CSFV-E2 protein antigen expressed by the baculovirus, which is the standard antigen used in this application.

[0015] E2 envelope glycoprotein is the main structural protein of CSFV, located on the outer surface of classical swine fever virus particles, can induce the body to produce CSFV neutralizing antibodies, has excellent activity and immunogenicity, and is the main protective antigen protein for immune pigs against CSFV attack. Therefore, E2 glycoprotein is the preferred antigen for establishing CSFV serological detection methods.

[0016] In the present application, the applicant cloned the E2 gene of CSFV into a baculovirus expression system, obtained a recombinant E2 protein expressed in vitro, and used the purified recombinant E2 protein as a standard antigen to establish a detection method for CSFV-E2 antibodies. Compared with prokaryotic expressed proteins, exogenous proteins expressed by baculovirus are closer to the spatial conformation and function of real natural proteins. Therefore, the present application uses baculovirus-expressed proteins as standard antigens, which can make the combination of antigens and antibodies in the sera of pigs to be tested have stronger specificity and sensitivity.

[0017] Preferably, the detection kit further comprises a positive control serum, a negative control serum, a sample diluent, an enzyme-labeled antibody diluent, a washing solution, a blocking solution, a TMB substrate solution, and a stop solution.

[0018] Furthermore, the coating concentration of the capture antibody is 1-4 μg / mL; the coating concentration of the standard antigen is 0.1-0.4 μg / mL.

[0019] In a specific embodiment, the coating concentration of the capture antibody can be 1 μg / mL, 2 μg / mL, or 4 μg / mL.

[0020] In some specific embodiments, the coating concentration of the capture antibody can also be 1-2 μg / mL or 2-4 μg / mL.

[0021] In a specific embodiment, the coating concentration of the standard antigen can be 0.1 μg / mL, 0.2 μg / mL, or 0.4 μg / mL.

[0022] In some specific embodiments, the coating concentration of the standard antigen may also be 0.1-0.2 μg / mL, 0.2-0.4 μg / mL.

[0023] After experimental analysis, it was found that when the coating concentration of the capture antibody reached 1-4 μg / mL, the OD of the standard antigen at the same coating concentration was 450nm The value did not change much, indicating that the capture antibody was saturated on the ELISA plate. At the same time, when the coating concentration of the standard antigen was 0.1-0.4μg / mL, the OD 450nm The value is about 1.0. Therefore, the present application controls the coating concentration of the capture antibody and the standard antigen within the above range.

[0024] Furthermore, the dilution of the enzyme-labeled antibody is 1:(6000-10000).

[0025] In a specific embodiment, the dilution of the enzyme-labeled antibody can be 1:6000, 1:8000, or 1:10000.

[0026] In some specific embodiments, the dilution of the enzyme-labeled antibody can also be 1:(6000-8000) or 1:(8000-10000).

[0027] After experimental analysis, it is known that when the dilution of the control enzyme-labeled antibody is within the above range, the negative control serum OD 450nm Value / positive control serum OD 450nm Therefore, the dilution of the enzyme-labeled antibody is controlled within the above range.

[0028] Optionally, when the serum sample to be tested is tested, the dilution ratio of the serum sample to be tested is 1:(1-3).

[0029] In a specific embodiment, the dilution ratio of the serum sample to be tested can be 1:1, 1:2, or 1:3.

[0030] In some specific embodiments, the dilution ratio of the serum sample to be tested can also be 1:(1-2) or 1:(2-3).

[0031] Through experimental analysis, it is known that when the dilution of the sample to be tested is controlled within the above range, the N / P value is larger. Therefore, the present application controls the dilution of the sample to be tested to be within the above range.

[0032] Furthermore, the reaction time of the TMB substrate solution is 10-15 min.

[0033] In a specific embodiment, the reaction time of the TMB substrate solution can be 10 min or 15 min.

[0034] Through experimental analysis, it can be known that when the reaction time of the TMB substrate solution is controlled within the above range, the N / P value is larger. Therefore, the present application controls the reaction time of the TMB substrate solution within the above range.

[0035] Furthermore, the sample diluent is 0.4-0.6wt% PBSA.

[0036] Furthermore, the enzyme-labeled antibody diluent is PBSA containing 0.18-0.22wt% sucrose and a concentration of 0.4-0.6wt%.

[0037] Furthermore, the blocking solution is 2.5-3.5wt% BSA.

[0038] Furthermore, the stop solution is 1.8-2.2 mol / L sulfuric acid.

[0039] Preferably, the detection kit further comprises an antigen protection solution;

[0040] The antigen protection solution comprises the following components in parts by weight: 90-110 parts of glycerol, 8-12 parts of trehalose, 0.08-0.12 parts of sodium glutamate, 0.04-0.06 parts of recombinant Escherichia coli thioredoxin, and 850-950 parts of water.

[0041] The present application utilizes the above-mentioned antigen protection liquid to protect the standard antigen, which effectively improves the stability of the detection method.

[0042] Furthermore, the antigen protection solution also includes glutamic acid; the weight ratio of the glutamic acid to the glycerol is (0.4-0.8):100.

[0043] After experimental analysis, it can be known that compared with using any one of glycine, alanine, and leucine, the present application chooses to use glutamic acid to add to the antigen protection solution, which can further improve the stability of the detection method. Therefore, the present application chooses to use glutamic acid to add to the antigen protection solution.

[0044] In a specific embodiment, the weight ratio of the glutamic acid to the glycerol may be 0.4:100, 0.6:100, or 0.8:100.

[0045] In some specific embodiments, the weight ratio of the glutamic acid to the glycerol can also be (0.4-0.6):100 or (0.6-0.8):100.

[0046] Through experimental analysis, it is known that when the weight ratio of glutamic acid to glycerol is controlled within the above range, the stability of the detection method can be further improved. Therefore, the present application controls the weight ratio of glutamic acid to glycerol within the above range.

[0047] In a second aspect, the present application provides a blocking ELISA detection method for CSFV-E2 antibodies, which is obtained based on the above-mentioned blocking ELISA detection kit for CSFV-E2 antibodies.

[0048] Preferably, the detection method comprises the following steps:

[0049] (1) Antibody coating: dilute the capture antibody with carbonate buffer, add 80-120 μL per well of the ELISA plate, incubate at 2-8° C. for 14-20 h, and discard the coating solution;

[0050] (2) Washing: Add the washing solution to each well for washing, discard the liquid in the well, and pat dry;

[0051] (3) Blocking: Add 180-220 μL of the blocking solution to each well and block at 37° C. for 1.5-2.5 h. Discard the blocking solution and wash according to the method of step (2);

[0052] (4) Antigen coating: dilute the standard antigen with PBST, add 80-120 μL to each well of the ELISA plate, incubate at 37° C. for 50-70 min, discard the liquid in the well, and wash according to the method of step (2);

[0053] (5) Protection: Add 64-144 μL of the antigen protection solution to each well, protect at 37° C. for 50-70 min, discard the antigen protection solution, and dry;

[0054] (6) Sample addition: dilute the positive control serum, the negative control serum, and the test serum sample using the sample diluent, add 80-120 μL to each well for incubation, then discard the liquid in the well and wash according to the method of step (2);

[0055] (7) Antibody incubation: dilute the enzyme-labeled antibody using the enzyme-labeled antibody diluent, add 80-120 μL to each well for incubation, then spin dry, and wash according to the method of step (2);

[0056] (8) Color development: Add 80-120 μL of the TMB substrate solution to each well and incubate at 25° C. for color development;

[0057] (9) Termination: Add 40-60 μL of the stop solution to each well to terminate the reaction;

[0058] (10) Reading: Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm.

[0059] According to the ROC curve analysis results obtained by the above detection method, the optimal critical value (Cut-off value) of the blocking rate (PI value) in the blocking ELISA detection method of CSFV-E2 antibodies is 40%, the specificity is 100%, the sensitivity is 97.0%, the Youden index is 0.970, and the area under the ROC curve is 0.993. That is, the blocking ELISA detection method of CSFV-E2 antibodies provided in the present application has strong sensitivity and specificity.

[0060] Preferably, the method for determining CSFV-E2 antibodies in the serum sample to be tested is:

[0061] When the PI value of the serum sample to be tested is ≥40%, the detection result of the CSFV-E2 antibody in the serum sample to be tested is judged to be positive;

[0062] When the PI value of the serum sample to be tested is less than 40%, the detection result of the CSFV-E2 antibody in the serum sample to be tested is judged to be negative.

[0063] In summary, the technical solution of this application has the following effects:

[0064] The present application is based on the principle of indirect capture and coating, and uses the CSFV-E2 protein 7C9 strain monoclonal antibody as the capture antibody coated in the ELISA plate to capture the standard antigen to form a solid phase antigen; then, the enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody is used as the competitive antibody for the sample to be tested, thereby obtaining a blocking ELISA detection kit for CSFV-E2 antibodies.

[0065] The present application uses the provided CSFV-E2 antibody blocking ELISA detection kit to obtain a CSFV-E2 antibody blocking ELISA detection method with high sensitivity, strong specificity and good repeatability by optimizing the parameters such as the capture antibody coating concentration, the standard antigen coating concentration, the enzyme-labeled antibody dilution, and the color development time.

[0066] The present application uses glycerol, trehalose, sodium glutamate, recombinant Escherichia coli thioredoxin, and water as antigen protection liquid to protect the standard antigen, which effectively improves the stability of the CSFV-E2 antibody detection method. In addition, the present application adds glutamic acid to the antigen protection liquid and screens the weight ratio of glutamic acid to glycerol, which further improves the stability of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The ROC curve diagram of the PI value in the blocking ELISA detection method of the CSFV-E2 antibody provided in the present application is shown. DETAILED DESCRIPTION

[0068] The present application provides a blocking ELISA detection kit and a detection method for CSFV-E2 antibodies.

[0069] In a first aspect, the present application provides a blocking ELISA detection kit for CSFV-E2 antibodies: comprising an ELISA plate coated with a capture antibody, a standard antigen, and an enzyme-labeled antibody;

[0070] Among them, the capture antibody is a monoclonal antibody of CSFV-E2 protein 7C9 strain; the standard antigen is a CSFV-E2 protein antigen expressed by baculovirus; and the enzyme-labeled antibody is an enzyme-labeled monoclonal antibody of CSFV-E2 protein 6E12 strain.

[0071] Specifically, the detection kit also includes positive control serum, negative control serum, sample diluent, washing solution, blocking solution, TMB substrate solution, and stop solution.

[0072] Furthermore, the coating concentration of the capture antibody is 1-4 μg / mL; the coating concentration of the standard antigen is 0.1-0.4 μg / mL; the dilution of the enzyme-labeled antibody is 1:(6000-10000); the dilution of the serum sample to be tested is 1:(1-3); and the reaction time of the TMB substrate solution is 10-15 min.

[0073] Furthermore, the sample diluent is 0.4-0.6 wt % PBSA.

[0074] Furthermore, the enzyme-labeled antibody diluent is PBSA containing 0.18-0.22 wt % sucrose and a concentration of 0.4-0.6 wt %.

[0075] Furthermore, the blocking solution is 2.5-3.5wt% BSA.

[0076] Furthermore, the stop solution is 1.8-2.2 mol / L sulfuric acid.

[0077] In addition, the detection kit also includes an antigen protection solution; the antigen protection solution includes the following components in parts by weight: 90-110 parts of propylene glycol, 8-12 parts of trehalose, 0.08-0.12 parts of sodium glutamate, 0.04-0.06 parts of recombinant Escherichia coli thioredoxin, and 850-950 parts of water.

[0078] Furthermore, the antigen protection solution also includes glutamic acid; the weight ratio of glutamic acid to glycerol is (0.4-0.8):100.

[0079] In a second aspect, the present application provides a blocking ELISA detection method for CSFV-E2 antibodies, which is obtained based on the above-mentioned blocking ELISA detection kit for CSFV-E2 antibodies, and comprises the following steps:

[0080] (1) Antibody coating: Dilute the capture antibody with carbonate buffer, add 80-120 μL to each well of the ELISA plate, incubate at 2-8°C for 14-20 hours, and discard the coating solution;

[0081] (2) Washing: Add washing solution to each well for washing, discard the liquid in the well, and pat dry;

[0082] (3) Blocking: Add 180-220 μL of blocking solution to each well and block at 37°C for 1.5-2.5 h. Discard the blocking solution and wash according to the method in step (2);

[0083] (4) Antigen coating: dilute the standard antigen with PBST, add 80-120 μL to each well of the ELISA plate, incubate at 37°C for 50-70 min, discard the liquid in the well, and wash according to the method of step (2);

[0084] (5) Protection: Add 64-144 μL of antigen protection solution to each well and protect at 37°C for 50-70 min. Discard the antigen protection solution and dry.

[0085] (6) Sample addition: dilute the positive control serum, negative control serum, and test serum sample with sample diluent, add 80-120 μL to each well for incubation, then discard the liquid in the well and wash according to the method of step (2);

[0086] (7) Antibody incubation: dilute the enzyme-labeled antibody with enzyme-labeled antibody diluent, add 80-120 μL to each well for incubation, then spin dry, and wash according to the method of step (2);

[0087] (8) Color development: Add 80-120 μL of TMB substrate solution to each well and incubate at 25°C for color development.

[0088] (9) Termination: Add 40-60 μL of stop solution to each well to terminate the reaction;

[0089] (10) Reading: Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm.

[0090] Among them, the method for determining CSFV-E2 antibodies in the serum sample to be tested is:

[0091] When the PI value of the serum sample to be tested is ≥40%, the detection result of the CSFV-E2 antibody in the serum sample to be tested is judged to be positive;

[0092] When the PI value of the serum sample to be tested is less than 40%, the detection result of the CSFV-E2 antibody in the serum sample to be tested is judged to be negative.

[0093] The present application is further described in detail below in combination with preparation examples, embodiments and performance testing experiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0094] Preparation Example

[0095] Preparation Example 1

[0096] This preparation example provides a CSFV-E2 protein.

[0097] The specific preparation method of CSFV-E2 protein in this preparation example is:

[0098] (1) Construction of recombinant plasmid pFastBacHTa-CSFV-E2: Total RNA of classical swine fever lapinized attenuated strain C was extracted and reverse transcribed to synthesize cDNA. Primers were designed with reference to the sequence of classical swine fever lapinized attenuated strain C (accession number AF531433) published in GenBank. The E2 gene was amplified by RT-PCR. Two restriction endonuclease sequences, EcoRI and HindIII, were added to the 5' and 3' ends of the gene sequence, respectively, and the recombinant plasmid pFastBacHTa was inserted into the pFastBacHTa vector to construct the recombinant plasmid pFastBacHTa-CSFV-E2, which was then sequenced and identified.

[0099] (2) Construction of recombinant bacmid DNA: The successfully sequenced pFastBacHTa-CSFV-E2 plasmid was transformed into DH10Bac competent cells, and the recombinant bacmid DNA was purified by blue-white screening and streaking, and sequenced and identified using PUC / M13 primers.

[0100] (3) Construction of recombinant baculovirus: Prepare cells in the logarithmic growth phase, with cell viability greater than 95% and a cell density of 2.0-4.0×10 6 Sf9 cells with a concentration of 100 cells / mL were transfected with recombinant Bacmid DNA according to the instructions of the commercial transfection reagent. After the cytopathic effect appeared, the cell supernatant was harvested by centrifugation, which was the recombinant baculovirus rBV-CSFV-E2 strain P0 virus.

[0101] (4) Expression and purification of recombinant E2 protein: The rBV-CSFV E2 strain was inoculated into Sf9 cells at an MOI of 1, cultured at 27°C and 120 rpm for 144 h, and the cell culture was harvested. The cell culture was centrifuged at 4°C and 3000 rpm for 15 min, and the precipitate was resuspended in lysis buffer and incubated at 4°C for 15 min. The cell culture was sonicated on ice and centrifuged at 4°C and 15000 rpm for 20 min to harvest the supernatant. The supernatant was filtered through a 0.22 μm filter membrane and purified by Ni column affinity chromatography. The eluate was collected to obtain a purified recombinant E2 protein, which was used as the raw material CSFV-E2 protein for the detection method in the present application.

[0102] It was identified that the size of the expressed recombinant E2 protein was 50kDa, and the purity of the purified E2 protein was not less than 90%. Western-blot identification showed that it only reacted with classical swine fever virus-positive serum, and did not react with classical swine fever virus-negative serum, pseudorabies virus-positive serum, porcine reproductive and respiratory syndrome virus-positive serum, porcine circovirus type 2-positive serum, and porcine parvovirus-positive serum, indicating good specificity.

[0103] Preparation Example 2-3

[0104] Preparation Example 2 provides a CSFV-E2 protein 7C9 strain monoclonal antibody.

[0105] Preparation Example 3 provides a CSFV-E2 protein 6E12 strain monoclonal antibody.

[0106] The specific preparation methods of the CSFV-E2 protein 7C9 strain monoclonal antibody and the CSFV-E2 protein 6E12 strain monoclonal antibody in the above preparation examples are:

[0107] BALB / c mice were immunized with recombinant E2 protein expressed by baculovirus. After immunization for 3 times, immune spleen cells of mice were fused with myeloma cells SP2 / 0. Positive fusion cells were screened by indirect ELISA method. The positive cell lines screened were subcloned by limiting dilution method. According to the ELISA titer, affinity, antigen recognition site and specificity analysis of monoclonal antibodies, 7C9 monoclonal antibodies and 6E12 monoclonal antibodies that recognize different antigen epitopes were selected as raw materials for the detection method in this application. The specific operation is as follows:

[0108] (1) Mouse immunization: The recombinant E2 protein expressed by baculovirus was diluted to 300 μg / mL with PBS (0.01 mol / L, pH 7.4), emulsified with Freund's adjuvant and used to immunize 6-week-old healthy female BALB / c mice. Blood was collected from the tail after three immunizations, and the serum antibody titer was detected by indirect ELISA. Mice with high serum antibody titer were selected and boosted once 3 days before fusion. The mice were killed and the immune spleen cells were harvested.

[0109] (2) Cell fusion, screening and subcloning: SP2 / 0 cells were fused with immune spleen cells at a ratio of 1:10. When the fused cell clones grew to cover 1 / 2 of the bottom of the cell wells, the cell culture supernatant was detected by indirect ELISA. The cell wells with strong positive results by ELISA were selected and subcloned three times by limiting dilution method. The strong positive cell wells with only a single clone were selected for expansion culture.

[0110] (3) Identification of monoclonal antibodies: Monoclonal positive hybridoma cells were cultured, and hybridoma cell chromosome analysis, monoclonal antibody subclass identification, and monoclonal antibody titer detection were performed. The antigenic epitopes recognized by different monoclonal antibodies were analyzed by indirect ELISA addition test. The chromosome numbers of 7C9 and 6E12 hybridoma cells were both 95-103; the heavy chain subclasses of secreted monoclonal antibodies were IgG1 and IgG2a, respectively; IFA identification showed that both monoclonal antibodies could react with classical swine fever virus (strain C) and had good reactivity; and the two monoclonal antibodies recognized different antigenic epitopes; finally, the CSFV-E2 protein 7C9 monoclonal antibody and the CSFV-E2 protein 6E12 monoclonal antibody were determined as the raw materials for the detection method in this application.

[0111] Preparation Example 4

[0112] Preparation Example 4 provides a CSFV-E2 protein 6E12 strain monoclonal antibody labeled with HRP enzyme.

[0113] The specific preparation method of the CSFV-E2 protein 6E12 strain monoclonal antibody labeled with HRP enzyme (horseradish peroxidase) in this preparation example is:

[0114] A commercial HRP coupling kit (such as Thermo Scientific's EZ-LinkMPlus activated peroxidase kit) was used to label the CSFV-E2 protein 6E12 strain monoclonal antibody with HRP enzyme according to the instructions to prepare an enzyme-labeled antibody stock solution. The enzyme-labeled monoclonal antibody was titered by a direct ELISA method, which was not less than 1:32000. An equal volume of 60% glycerol was added and stored below -70°C to obtain the CSFV-E2 protein 6E12 strain monoclonal antibody labeled with HRP enzyme.

[0115] Preparation Example 5-6

[0116] Preparation Example 5 provides a positive control serum.

[0117] Preparation Example 6 provides a negative control serum.

[0118] The specific preparation methods of the positive control serum and the negative control serum in the above preparation examples are as follows:

[0119] Select healthy susceptible pigs aged 2-4 months. They should be negative for porcine reproductive and respiratory syndrome virus ELISA antibody, negative for pseudorabies virus gB ELISA antibody, negative for bovine viral diarrhea mucosal virus antigen and neutralizing antibody, and negative for classical swine fever virus antigen and ELISA antibody.

[0120] Select the pigs that are negative for the above antigens or antibodies, randomly draw blood from the carotid artery, prepare the negative serum of classical swine fever virus, dilute it 20 times with diluent and add Coomassie brilliant blue solution with a final concentration of 0.002%, which is the negative control serum. The remaining pigs are immunized with commercial classical swine fever live vaccine. When the serum ELISA titer is not less than 1:1024, blood is collected from the carotid artery to separate the serum, and the positive serum of classical swine fever virus antibody is prepared. The positive serum is diluted with diluent to an ELISA titer of 1:16, and 1% phenol red solution is added to a final concentration of 0.002%, which is the positive control serum.

[0121] Example

[0122] The sample diluent used in Examples 1-7 was 0.5 wt % PBSA; the enzyme-labeled antibody diluent was PBSA containing 0.2 wt % sucrose at a concentration of 0.5 wt %; and the blocking solution was 3 wt % BSA.

[0123] The preparation method of the antigen protection solution in Example 1-7 is as follows: 100 g of glycerol, 10 g of trehalose, 0.1 g of sodium glutamate, and 0.6 g of glutamic acid are weighed, dissolved in 800 g of purified water and mixed evenly, the pH is adjusted to 7, and then 0.05 g of recombinant Escherichia coli thioredoxin is added, the volume is made up to 1000 mL with purified water, and stirred evenly.

[0124] Example 1

[0125] This example provides the optimization of reaction conditions in the blocking ELISA detection method of CSFV-E2 antibodies.

[0126] The specific optimization conditions in this embodiment are: optimization of the optimal coating concentration of the CSFV-E2 protein 7C9 strain monoclonal antibody and the optimal coating concentration of the CSFV-E2 protein antigen, which specifically includes the following steps:

[0127] (1) Antibody coating: The CSFV-E2 protein 7C9 strain monoclonal antibody prepared in Preparation Example 2 was diluted to 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL respectively using 0.05 mol / L carbonate buffer at pH 9.6, and 100 μL was added to each well of a 96-well ELISA plate. The plate was incubated at 4°C for 16 h, and the coating solution was discarded;

[0128] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0129] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0130] (4) Antigen coating: The CSFV-E2 protein antigen prepared in Preparation Example 1 was diluted to 3.2 μg / mL, 1.6 μg / mL, 0.8 μg / mL, 0.4 μg / mL, 0.2 μg / mL, and 0.1 μg / mL with PBST for capture coating. 100 μL was added to each well of a 96-well ELISA plate, and the plate was incubated at 37° C. for 60 min. The liquid in the wells was discarded, and the plate was washed according to the method of step (2);

[0131] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0132] (6) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, spin dry, and wash according to the method of step (2);

[0133] (7) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0134] (8) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0135] (9) Reading: Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm, as shown in Table 1.

[0136] Table 1 Optimization results of the optimal coating concentrations of CSFV-E2 protein 7C9 strain monoclonal antibody and CSFV-E2 protein antigen

[0137]

[0138]

[0139] The maximum dilution multiple when the adsorption of CSFV-E2 protein 7C9 strain monoclonal antibody on the ELISA plate reached saturation was selected as the optimal coating concentration of CSFV-E2 protein 7C9 strain monoclonal antibody; at the optimal coating concentration of CSFV-E2 protein 7C9 strain monoclonal antibody, OD 450nm The antigen concentration with a value of about 1.0 is regarded as the optimal coating concentration of CSFV-E2 protein antigen.

[0140] By analyzing Table 1, when the coating concentration of the CSFV-E2 protein 7C9 strain monoclonal antibody reached 1 μg / mL and above (4 μg / mL, 2 μg / mL, 1 μg / mL), the three OD values ​​of the CSFV-E2 protein antigen at the same coating concentration were 450nm The value did not change much, indicating that the monoclonal antibody adsorption on the ELISA plate reached saturation. When the coating concentration of the CSFV-E2 protein 7C9 strain monoclonal antibody was 1 μg / mL and the coating concentration of the CSFV-E2 protein antigen was 0.2 μg / mL, the OD 450nm The value is about 1.0. Therefore, the optimal coating concentration of the CSFV-E2 protein 7C9 strain monoclonal antibody is determined to be 1 μg / mL, and the optimal coating concentration of the CSFV-E2 protein antigen is determined to be 0.2 μg / mL.

[0141] Example 2

[0142] This example provides the optimization of reaction conditions in the blocking ELISA detection method of CSFV-E2 antibodies.

[0143] The specific optimization conditions in this embodiment are: optimization of the optimal dilution of the CSFV-E2 protein 6E12 strain monoclonal antibody labeled with HRP enzyme, specifically including the following steps:

[0144] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0145] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0146] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0147] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0148] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0149] (6) Sample addition: dilute the positive control serum and negative control serum with sample diluent at a ratio of 1:2, add 100 μL to each well, and incubate at 25° C. for 60 min; discard the liquid in the well and wash according to the method of step (2);

[0150] (7) Antibody incubation: The HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 was diluted with enzyme-labeled antibody diluent at 1:2000, 1:4000, 1:8000, and 1:16000, respectively, and 100 μL was added to each well. The mixture was incubated at 25° C. for 30 min, dried, and washed according to the method of step (2);

[0151] (8) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0152] (9) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0153] (10) Reading: Use an ELISA reader to read the absorbance of each well at 450 nm and calculate the N / P value (negative control serum OD 450nmValue / positive control serum OD 450nm values), as shown in Table 2.

[0154] Table 2 Optimization results of the best dilution of the monoclonal antibody of CSFV-E2 protein 6E12 strain labeled with HRP enzyme

[0155]

[0156] The enzyme-labeled antibody dilution at which the N / P value was the largest was taken as the optimal dilution.

[0157] By analyzing the optimization results in Table 2, when the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody was diluted at 1:8000, the N / P value was 14.056, that is, the N / P value at this time reached the maximum. Therefore, it was determined that the optimal dilution of the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody was 1:8000.

[0158] Example 3

[0159] This example provides the optimization of reaction conditions in the blocking ELISA detection method of CSFV-E2 antibodies.

[0160] The specific optimization conditions in this embodiment are: optimization of the best dilution of the sample to be tested, specifically including the following steps:

[0161] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0162] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry.

[0163] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0164] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0165] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0166] (6) Sample addition: dilute the positive control serum and negative control serum with sample diluent at a ratio of 1:2, 1:5, and 1:10, respectively, add 100 μL to each well, and incubate at 25° C. for 60 min; discard the liquid in the well and wash according to the method of step (2);

[0167] (7) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, spin dry, and wash according to the method of step (2);

[0168] (8) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0169] (9) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0170] (10) Reading: Use an ELISA reader to read the absorbance of each well at 450 nm and calculate the N / P value (negative control serum OD 450nm Value / positive control serum OD 450nm values), as shown in Table 3.

[0171] Table 3 Optimization results of the best dilution of the samples to be tested

[0172]

[0173] The dilution of the sample to be tested when the N / P value is the largest is taken as the optimal dilution.

[0174] By analyzing the optimization results in Table 3, when the serum to be tested is diluted at 1:2, the N / P value is 12.108, that is, the N / P value at this time reaches the maximum. Therefore, it is determined that the optimal dilution of the serum to be tested is 1:2.

[0175] Example 4

[0176] This example provides the optimization of reaction conditions in the blocking ELISA detection method of CSFV-E2 antibodies.

[0177] The specific optimization conditions in this embodiment are: the optimization of the best reaction conditions of the sample to be tested specifically includes the following steps:

[0178] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0179] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0180] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0181] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0182] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0183] (6) Sample addition: dilute the positive control serum and negative control serum with sample diluent at a ratio of 1:2, add 100 μL to each well, and incubate at 37°C for 30 min, 25°C for 60 min, and 25°C for 30 min, respectively; discard the liquid in the wells and wash according to the method of step (2);

[0184] (7) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, spin dry, and wash according to the method of step (2);

[0185] (8) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0186] (9) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0187] (10) Reading: Use an ELISA reader to read the absorbance of each well at 450 nm and calculate the N / P value (negative control serum OD 450nm Value / positive control serum OD 450nm values), as shown in Table 4.

[0188] Table 4 Optimization results of the best reaction conditions for the samples to be tested

[0189]

[0190] The reaction condition of the sample to be tested when the N / P value is the largest is taken as the optimal reaction condition.

[0191] By analyzing the optimization results in Table 4, when the reaction condition of the sample to be tested is 25°C for 60 min, the N / P value is 13.878, that is, the N / P value at this time reaches the maximum. Therefore, it is determined that the optimal reaction condition of the sample to be tested is 25°C for 60 min.

[0192] Example 5

[0193] This example provides the optimization of reaction conditions in the blocking ELISA detection method of CSFV-E2 antibodies.

[0194] The specific optimization conditions in this embodiment are: optimization of the optimal reaction conditions of the CSFV-E2 protein 6E12 strain monoclonal antibody labeled with HRP enzyme, specifically including the following steps:

[0195] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0196] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0197] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0198] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0199] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0200] (6) Sample addition: dilute the positive control serum and negative control serum with sample diluent at a ratio of 1:2, add 100 μL to each well, and incubate at 25° C. for 60 min; discard the liquid in the well and wash according to the method of step (2);

[0201] (7) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, 25° C. for 60 min, and 37° C. for 30 min, spin dry, and wash according to the method of step (2);

[0202] (8) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0203] (9) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0204] (10) Reading: Use an ELISA reader to read the absorbance of each well at 450 nm and calculate the N / P value (negative control serum OD 450nm Value / positive control serum OD 450nm values), as shown in Table 5.

[0205] Table 5 Optimization results of the best reaction conditions for the monoclonal antibody of CSFV-E2 protein 6E12 strain labeled with HRP enzyme

[0206]

[0207] The reaction conditions when the N / P value was the largest were taken as the optimal reaction conditions for the CSFV-E2 protein 6E12 strain monoclonal antibody labeled with HRP enzyme.

[0208] By analyzing the optimization results in Table 5, when the reaction conditions of the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody are 25°C incubation for 30 minutes, the N / P value is 14.193, that is, the N / P value at this time reaches the maximum. Therefore, it is determined that the optimal reaction conditions of the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody are 25°C incubation for 30 minutes.

[0209] Example 6

[0210] This example provides the optimization of reaction conditions in the blocking ELISA detection method of CSFV-E2 antibodies.

[0211] The specific optimization conditions in this embodiment are: optimization of the optimal reaction time of the substrate solution, which specifically includes the following steps:

[0212] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0213] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0214] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0215] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0216] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0217] (6) Sample addition: dilute the positive control serum and negative control serum with sample diluent at a ratio of 1:2, add 100 μL to each well, and incubate at 25° C. for 60 min; discard the liquid in the well and wash according to the method of step (2);

[0218] (7) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, spin dry, and wash according to the method of step (2);

[0219] (8) Color development: 100 μL of TMB substrate solution was added to each well and the color was developed at 25°C for 5 min, 10 min, 15 min, and 20 min respectively;

[0220] (9) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0221] (10) Reading: Use an ELISA reader to read the absorbance of each well at 450 nm and calculate the N / P value (negative control serum OD 450nm Value / positive control serum OD 450nm values), as shown in Table 6.

[0222] Table 6 Optimization results of the best reaction time of substrate solution

[0223]

[0224]

[0225] The reaction time when the N / P value is the largest is taken as the optimal reaction time of the substrate solution.

[0226] By analyzing the optimization results in Table 6, when the reaction time of the substrate solution is 15 min, the N / P value is 14.713, that is, the N / P value at this time reaches the maximum. Therefore, the optimal reaction time of the substrate solution is determined to be 15 min.

[0227] Example 7

[0228] This embodiment provides a blocking ELISA detection method for CSFV-E2 antibodies: the detection method determines a critical value (Cut-off value), test establishment conditions, and a method for determining CSFV-E2 antibodies in a serum sample to be tested.

[0229] In this embodiment, 135 positive pig sera and 121 clinically negative pig sera confirmed by neutralization test were used as serum samples to be tested, and then the test was carried out, which specifically included the following steps:

[0230] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0231] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0232] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0233] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0234] (5) Protection: Add 100 μL of antigen protection solution to each well and protect at 37°C for 60 min. Discard the antigen protection solution and dry.

[0235] (6) Sample addition: dilute the positive control serum, negative control serum and the test serum sample with sample diluent at a ratio of 1:2, add 100 μL to each well, and incubate at 25° C. for 60 min; discard the liquid in the well and wash according to the method of step (2);

[0236] (7) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, spin dry, and wash according to the method of step (2);

[0237] (8) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0238] (9) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0239] (10) Reading: Use an ELISA reader to read the absorbance of each well at 450 nm and calculate the N / P value (negative control serum OD 450nm Value / positive control serum OD 450nm value);

[0240] (11) Calculation of blocking rate (PI) value: The PI value of the serum sample to be tested is calculated as follows:

[0241]

[0242]

[0243] (12) Curve drawing: Using the neutralization test results as the gold standard, draw the ROC curve for the PI value of the serum sample to be tested, calculate the area under the curve, and determine the cut-off value, sensitivity, and specificity.

[0244] The ROC curve of PI value in the blocking ELISA detection method of CSFV-E2 antibody is shown in Figure 1 shown.

[0245] By analyzing Figure 1 ,The results of ROC curve analysis showed that the optimal cut-off value of PI value in the blocking ELISA detection method of CSFV-E2 antibody was 40%, the specificity was 100%, the sensitivity was 97.0%, the Youden index was 0.970, and the area under the ROC curve was 0.993.

[0246] Through the above analysis, it can be seen that the test establishment conditions in the blocking ELISA detection method of CSFV-E2 antibodies provided in the present application are: negative control serum OD 450nm The average value should be between 1.6-1.9; the average value of positive control serum PI should be >50%; the test result is judged to be valid.

[0247] According to the blocking ELISA detection method of CSFV-E2 antibodies provided in the present application, the method for determining CSFV-E2 antibodies in the serum sample to be tested is: when the PI value of the serum sample to be tested is ≥40%, it is judged as positive; when the PI value of the serum sample to be tested is <40%, it is judged as negative.

[0248] Test example

[0249] The commercial kits in the experimental examples were purchased from IDEXX Swiss Biotechnology Co., Ltd.

[0250] Test Example 1

[0251] This study investigated the sensitivity of the blocking ELISA assay for CSFV-E2 antibodies.

[0252] Using the detection method provided in Example 7, 61 positive vaccine immune sera, 59 weakly positive vaccine immune sera and 35 clinical infection sera determined by commercial kits were used as serum samples to be tested, and then tested to determine the sensitivity of the detection method in this application.

[0253] Test results: The results showed that the test results of the above 61 positive vaccine immune sera were all 61 positive, the test results of 59 weakly positive vaccine immune sera were all 59 positive, and the test results of 35 clinical infection sera were all 35 positive, that is, the positive detection rate of the blocking ELISA detection method of CSFV-E2 antibodies provided by the application was 100%. The above test results show that the detection method in the present application has good sensitivity.

[0254] Test Example 2

[0255] This test example investigated the sensitivity of the blocking ELISA detection method for CSFV-E2 antibodies.

[0256] Using the detection method provided in Example 7, the swine fever virus-positive serum prepared in Preparation Example 5 was used as the serum sample to be tested, and the serum was gradiently diluted by 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, and 1:4096 times, respectively, and then tested. The blocking rate PI value of the serum sample to be tested was calculated, and the CSFV-E2 antibody in the serum sample to be tested was determined to examine the sensitivity of the detection method in the present application.

[0257] At the same time, the commercial kit was used as a comparative example to determine the CSFV-E2 antibodies in the serum samples to be tested and to examine the sensitivity of the commercial kit.

[0258] Test results: as shown in Table 7.

[0259] Table 7 Sensitivity test results of gradient dilution positive serum

[0260]

[0261] According to the test results in Table 7, it can be seen that when the detection method provided by the present application detects CSFV-E2 antibodies in gradient diluted positive serum, when the positive serum is diluted 1:2048 times, the blocking rate is 42.64%, that is, the determination result of CSFV-E2 antibodies in the serum sample to be tested is positive; when the positive serum is diluted 1:4096 times, the blocking rate is 23.02%, that is, the determination result of CSFV-E2 antibodies in the serum sample to be tested is negative. In contrast, when the CSFV-E2 antibodies in gradient diluted positive serum are detected using a commercial kit, when the positive serum is diluted 1:1024 times, the determination result of CSFV-E2 antibodies in the serum sample to be tested is positive; when the positive serum is diluted 1:2048 times, the determination result of CSFV-E2 antibodies in the serum sample to be tested is negative.

[0262] The above test results show that the blocking ELISA detection method for CSFV-E2 antibodies provided in the present application has a higher detection sensitivity for serum samples to be tested than the commercial kit.

[0263] Test Example 3

[0264] This test example investigated the specificity of the blocking ELISA detection method for CSFV-E2 antibodies.

[0265] Using the detection method provided in Example 7, 125 clinical swine fever-negative sera, pseudorabies virus-positive sera, porcine reproductive and respiratory syndrome virus-positive sera, porcine circovirus type 2-positive sera, and porcine parvovirus-positive sera determined by commercial kits were tested to determine the specificity of the detection method in the present application.

[0266] Test results: The results show that the test results of the test method in this application for 125 clinical swine fever negative sera, swine pseudorabies virus positive sera, porcine reproductive and respiratory syndrome virus positive sera, porcine circovirus type 2 positive sera, and porcine parvovirus positive sera are all negative, that is, the test results of the test method in this application for swine fever negative sera and non-swine fever positive sera are all negative. The above test results show that the test method in this application has good specificity.

[0267] Test Example 4

[0268] This test example studied the repeatability of the blocking ELISA detection method for CSFV-E2 antibodies.

[0269] (1) Intra-batch coefficient of variation: Using the detection method provided in Example 7, 21 positive vaccine immune sera determined by commercial kits were tested respectively. Each sample was tested 5 times using the coated plates from the same batch, and the intra-batch coefficient of variation of the detection method in this application was calculated.

[0270] (2) Inter-batch coefficient of variation: Using the detection method provided in this application, 21 positive vaccine immune sera determined using a commercial kit were tested. Each sample was tested using three different batches of coated plates, and the inter-batch coefficient of variation of the detection method in this application was calculated.

[0271] Test results: as shown in Table 8.

[0272] Table 8 Repeatability study results of the blocking ELISA detection method for CSFV-E2 antibodies

[0273] Reproducibility of study results CV value range of 21 positive immune sera The average CV value of 21 positive immune sera The CV value range between batches 0.27％-6.06％ 2.04％ Intra-batch CV value range 0.33％-2.26％ 1.18％

[0274] By analyzing Table 8, the intra-batch coefficient of variation and the inter-batch coefficient of variation of the detection method in the present application are both less than 2.1%. This result shows that the detection method in the present application has good repeatability.

[0275] Example

[0276] Embodiment 8-11

[0277] Examples 8-11 each provide a blocking ELISA detection method for CSFV-E2 antibodies.

[0278] The difference between the above embodiment and embodiment 7 is that the type of antigen protection solution is different, as shown in Table 9.

[0279] Table 9 Types of antigen protection solutions in Examples 7-11

[0280]

[0281]

[0282] Example 12

[0283] This example provides a blocking ELISA detection method for CSFV-E2 antibodies.

[0284] The difference between this embodiment and embodiment 7 is that no antigen protection solution is added, and the specific steps are as follows:

[0285] (1) Antibody coating: Dilute the purified CSFV-E2 protein 7C9 strain monoclonal antibody to 1 μg / mL using 0.05 mol / L, pH 9.6 carbonate buffer, add 100 μL per well of a 96-well ELISA plate, incubate at 4°C for 16 h, and discard the coating solution;

[0286] (2) Washing: Add 300 μL of PBST washing solution to each well, wash three times, discard the liquid in the well, and pat dry;

[0287] (3) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. Discard the blocking solution and wash according to the method in step (2);

[0288] (4) Antigen coating: dilute the CSFV-E2 protein antigen prepared in Preparation Example 1 to 0.2 μg / mL with PBST for capture coating, add 100 μL per well of a 96-well ELISA plate, incubate at 37° C. for 60 min, discard the liquid in the wells, and wash according to the method of step (2);

[0289] (5) Sample addition: dilute the positive control serum, negative control serum and test serum sample with sample diluent at a ratio of 1:2, add 100 μL to each well, and incubate at 25° C. for 60 min; discard the liquid in the well and wash according to the method of step (2);

[0290] (6) Antibody incubation: dilute the HRP enzyme-labeled CSFV-E2 protein 6E12 strain monoclonal antibody prepared in Preparation Example 4 at a ratio of 1:8000 with enzyme-labeled antibody diluent, add 100 μL to each well, incubate at 25° C. for 30 min, spin dry, and wash according to the method of step (2);

[0291] (7) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25°C for 15 min;

[0292] (8) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the color reaction;

[0293] (9) Reading: Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm.

[0294] Embodiment 13

[0295] This example provides a blocking ELISA detection method for CSFV-E2 antibodies.

[0296] The difference between this embodiment and embodiment 7 is that 0.6 g of glycine is used instead of 0.6 g of glutamic acid.

[0297] Embodiment 14

[0298] This example provides a blocking ELISA detection method for CSFV-E2 antibodies.

[0299] The difference between this embodiment and embodiment 7 is that 0.6 g of alanine is used instead of 0.6 g of glutamic acid.

[0300] Embodiment 15

[0301] This example provides a blocking ELISA detection method for CSFV-E2 antibodies.

[0302] The difference between this embodiment and embodiment 7 is that 0.6 g of leucine is used instead of 0.6 g of glutamic acid.

[0303] Test example

[0304] Test Example 5

[0305] This test example studied the stability of the blocking ELISA detection method of CSFV-E2 antibodies in Examples 7-15.

[0306] The detection methods provided in Examples 7-15 were used as detection objects to examine the stability of the detection methods.

[0307] The detection method is:

[0308] The swine fever virus-positive serum prepared in Preparation Example 5 was used as the serum sample to be tested, and was diluted 1:8 times. The test was then repeated 3 times to calculate the blocking rate PI value of the serum sample to be tested, and the intra-batch coefficient of variation CV value was calculated to investigate the stability of the above detection method.

[0309] Test results: as shown in Table 10.

[0310] Table 10 Intra-batch coefficient of variation CV values ​​of the detection methods in Examples 7-15

[0311]

[0312] Combined with the test results of Table 10, the present application uses glycerol, trehalose, sodium glutamate, recombinant Escherichia coli thioredoxin, and water as antigen protection liquid to protect the standard antigen, and then detects the CSFV-E2 antibody in the serum sample to be tested, and the intra-batch coefficient of variation CV value is less than 5%. The above test results show that the use of the antigen protection liquid provided by the present application can significantly improve the stability of the blocking ELISA detection method of CSFV-E2 antibodies.

[0313] By comparing the test results of Example 7 with those of Examples 12-15, compared with not using an antigen protection solution, or using any one of glycine, alanine, and leucine to add to the antigen protection solution, the present application chooses to use glutamic acid to add to the antigen protection solution, which can further improve the stability of the detection method. Therefore, the present application chooses to use glutamic acid to add to the antigen protection solution.

[0314] By comparing the test results of Examples 7-11, when the weight ratio of glutamic acid to glycerol is controlled to be (0.4-0.8) : 100, the stability of the detection method can be further improved. Therefore, the present application controls the weight ratio of glutamic acid to glycerol to be within the above range.

[0315] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A blocking ELISA detection kit for CSFV-E2 antibodies, characterized in that: It includes an ELISA plate coated with a capture antibody, a standard antigen, an enzyme-labeled antibody, and an antigen protection solution; The capture antibody is a monoclonal antibody of the CSFV-E2 protein 7C9 strain; The standard antigen is a CSFV-E2 protein antigen expressed by baculovirus; The enzyme-labeled antibody is an enzyme-labeled monoclonal antibody of CSFV-E2 protein 6E12 strain; The antigen protection solution comprises the following components in parts by weight: 90-110 parts of glycerol, 8-12 parts of trehalose, 0.08-0.12 parts of sodium glutamate, 0.04-0.06 parts of recombinant Escherichia coli thioredoxin, and 850-950 parts of water; The antigen protection liquid also includes glutamic acid; the weight ratio of the glutamic acid to the glycerol is (0.4-0.8):

100.

2. The blocking ELISA detection kit for CSFV-E2 antibody according to claim 1, characterized in that: The detection kit also includes a positive control serum, a negative control serum, a sample diluent, an enzyme-labeled antibody diluent, a washing solution, a blocking solution, a TMB substrate solution, and a stop solution.

3. The blocking ELISA detection kit for CSFV-E2 antibody according to claim 2, characterized in that: The coating concentration of the capture antibody is 1-4 μg / mL; the coating concentration of the standard antigen is 0.1-0.4 μg / mL.

4. The blocking ELISA detection kit for CSFV-E2 antibody according to claim 2, characterized in that: The dilution of the enzyme-labeled antibody is 1: (6000-10000).

5. The blocking ELISA detection kit for CSFV-E2 antibody according to claim 2, characterized in that: The sample diluent is 0.4-0.6wt% PBSA.

6. The blocking ELISA detection kit for CSFV-E2 antibody according to claim 2, characterized in that: The enzyme-labeled antibody diluent is PBSA containing 0.18-0.22wt% sucrose and a concentration of 0.4-0.6wt%.

7. The blocking ELISA detection kit for CSFV-E2 antibody according to claim 2, characterized in that: The blocking solution is 2.5-3.5wt% BSA.

8. A blocking ELISA detection method for CSFV-E2 antibodies for non-diagnostic purposes, characterized in that: The detection method is obtained based on the blocking ELISA detection kit for CSFV-E2 antibodies according to any one of claims 2 to 7.

9. The blocking ELISA detection method of CSFV-E2 antibody according to claim 8, characterized in that: The following steps are involved: (1) Antibody coating: dilute the capture antibody with carbonate buffer, add 80-120 μL to each well of the ELISA plate, incubate at 2-8°C for 14-20 hours, and discard the coating solution; (2) Washing: Add the washing solution to each well for washing, discard the liquid in the well, and pat dry; (3) Blocking: Add 180-220 μL of the blocking solution to each well and block at 37°C for 1.5-2.5 h. Discard the blocking solution and wash according to the method in step (2); (4) Antigen coating: dilute the standard antigen with PBST, add 80-120 μL to each well of the ELISA plate, incubate at 37°C for 50-70 min, discard the liquid in the well, and wash according to the method of step (2); (5) Protection: Add 64-144 μL of the antigen protection solution to each well and protect at 37°C for 50-70 min. Discard the antigen protection solution and dry. (6) Sample addition: Use the sample diluent to dilute the positive control serum, the negative control serum, and the test serum sample, respectively, add 80-120 μL to each well for incubation, then discard the liquid in the well and wash according to the method of step (2); (7) Antibody incubation: dilute the enzyme-labeled antibody using the enzyme-labeled antibody diluent, add 80-120 μL to each well for incubation, then spin dry, and wash according to the method of step (2); (8) Color development: Add 80-120 μL of the TMB substrate solution to each well and incubate at 25°C for color development; (9) Termination: Add 40-60 μL of the stop solution to each well to terminate the reaction; (10) Reading: Use an ELISA reader to read the absorbance of each well at a wavelength of 450 nm.

10. The blocking ELISA detection method of CSFV-E2 antibody according to claim 9, characterized in that: The method for determining CSFV-E2 antibodies in the serum sample to be tested is: When the PI value of the serum sample to be tested is ≥40%, the detection result of the CSFV-E2 antibody in the serum sample to be tested is judged to be positive; When the PI value of the serum sample to be tested is less than 40%, the detection result of the CSFV-E2 antibody in the serum sample to be tested is judged to be negative.

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