Method and kit for detecting GnRH antibody in dog serum

Abstract

The invention relates to the technical field of canine serum GnRH antibody detection, in particular to a method and a kit for detecting a GnRH antibody in canine serum. The KLH-GnRH dimer polypeptide is obtained by coupling a KLH dimer polypeptide with a GnRH dimer polypeptide; the amino acid sequence of the GnRH dimer polypeptide is as shown in SEQ ID NO. 1. The KLH-GnRH dimer polypeptide is used as an antigen, and an indirect ELISA method is adopted to detect the GnRH antibody titer, so that the hormone state and reproductive capacity of dogs at different stages are evaluated. The method has the advantages of low cost, simplicity and convenience in operation, high sensitivity, strong specificity, good accuracy and the like, stable, rapid, accurate and convenient dog serum GnRH antibody level detection can be realized, and meanwhile, the repeatability is good.

Description

Technical Field

[0001] This invention relates to the field of canine serum GnRH antibody detection technology, and more specifically, to a method and kit for detecting GnRH antibodies in canine serum. Background Technology

[0002] Gonadotropin-releasing hormone (GnRH) is a decapeptide hormone secreted by the hypothalamus. As a core regulator of the reproductive axis, it regulates gonadal development and sex hormone production through pulsatile release via the pituitary portal system. GnRH immunocastration technology involves immunizing animals with KLH (keyhole hemocyanin)-conjugated GnRH polypeptide antigens, stimulating the body to produce high-titer specific neutralizing antibodies. KLH, as a high-molecular-weight carrier protein, has advantages such as strong immunogenicity, breaking immune tolerance to small-molecule polypeptides through T-cell-dependent antigenic epitopes; its high-density lysine residues on its surface enable multivalent GnRH polypeptide conjugation, significantly improving antigen presentation efficiency; it exhibits no cross-reactive antibodies in canines, avoiding detection interference; and the steric hindrance it creates reduces GnRH polypeptide degradation, maintaining conformational stability. The induced antibodies bind to endogenous GnRH, blocking its interaction with pituitary receptors, thereby inhibiting the function of the hypothalamus-pituitary-gonadal axis (HPGA) and achieving reversible chemical castration. KLH-GnRH dimer conjugates can enhance antibody affinity and establish a sensitive and specific method for detecting canine serum GnRH antibodies. This method is invaluable for evaluating the efficacy of castration vaccines, monitoring GnRH antibody titers, optimizing booster immunization dosages and frequency, and determining reproductive physiological status. Serum GnRH antibody titers are an important standard for detecting immune neutralization capacity, but previously there was no canine antibody detection technology based on the KLH-GnRH dimer immunogen. Summary of the Invention

[0003] The purpose of this invention is to provide a method and kit for detecting GnRH antibodies in canine serum, achieving highly sensitive and specific detection of GnRH antibodies in canine serum, and providing reliable technical support for evaluating the immunization effect of castration vaccines in dogs.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a KLH-GnRH dimer polypeptide, which is obtained by coupling KLH and GnRH dimer polypeptide; the amino acid sequence of the GnRH dimer polypeptide is as shown in SEQ ID NO.1.

[0005] In a second aspect, the present invention provides an ELISA kit for detecting GnRH antibodies in canine serum, comprising the above-mentioned KLH-GnRH dimer polypeptide, wherein the KLH-GnRH dimer polypeptide serves as an antigen.

[0006] The preferred formulation includes KLH-GnRH dimer peptide, coating solution, blocking solution, enzyme-labeled secondary antibody, standard serum sample, washing solution, antibody dilution solution, substrate chromogenic solution, and stop solution; the blocking agent is 5% skim milk powder.

[0007] In the preferred embodiment, the coating buffer is 50mM Na2CO3-NaHCO3 buffer with a pH of 9.1±0.2; the enzyme-labeled secondary antibody is HRP-labeled goat anti-canine IgG; the washing buffer and antibody dilution buffer are PBST buffers; the PBST buffer is obtained by diluting 10×PBS buffer 10 times to 1×PBS buffer and then adding 0.05% Tween 20; the substrate chromogenic solution is TMB chromogenic solution, which includes solution A and solution B; and the stop solution is 0.55mol / L H2SO4 solution.

[0008] In the preferred scheme, solution A and solution B are prepared and used immediately in a 1:1 ratio.

[0009] In the preferred embodiment, the KLH-GnRH dimer peptide is coated with a coating solution and then blocked with a blocking solution to prepare the encapsulated antigen; the coating concentration of the KLH-GnRH dimer peptide is 2 μL / mL.

[0010] A third aspect of the present invention provides a method for detecting GnRH antibodies in canine serum based on the above-described ELISA kit, comprising the following steps: S1. Coating antigen: Dilute the KLH-GnRH dimer peptide with coating buffer and add it to a 96-well microplate, 100 μL / well. Coat at 4℃ for 12-16 h. Discard the liquid and wash the microplate 3 times with washing buffer, 5 min / time. Discard the liquid and pat dry to obtain the coated antigen microplate. S2. Blocking: Add blocking solution to the coated antigen ELISA plate, 200 μL / well, block at 37℃ for 3 h, wash the ELISA plate 3 times with washing solution, 5 min / time, discard the liquid and pat dry to obtain the coated antigen ELISA plate; S3. Serum / primary antibody incubation: Dilute the standard or test sample at a ratio of 1:100 and add it to the encapsulated antigen ELISA plate at 100 μL / well. Incubate at 37°C for 1 h. Wash the ELISA plate 3 times with washing buffer for 5 min each time. Discard the liquid and pat dry to obtain the ELISA plate after serum / primary antibody incubation. S4. Secondary antibody incubation: Dilute the enzyme-labeled secondary antibody with antibody dilution buffer at a ratio of 1:5000, add 100 μL / well to the enzyme-labeled plate after serum / primary antibody incubation, incubate at 37°C for 30 min, wash the enzyme-labeled plate 3 times with washing buffer for 5 min each time, discard the liquid and pat dry. The enzyme-labeled plate after secondary antibody incubation. S5. Color development: Add the substrate color development solution to the microplate after incubation with the secondary antibody, 100 μL / well, and develop the color at 37°C in the dark for 30 min to obtain the color developed microplate. S6. Termination: Add the stop solution to the developed microplate at 100 μL / well and read the values ​​using a microplate reader. D 450nm Value, if D 450nm A value ≥0.1773 is considered positive. D 450nm A value <0.1773 is considered negative.

[0011] Beneficial effects of the present invention This invention provides an indirect ELISA method for detecting GnRH antibody titers in canine serum. The method uses a keyhole hemocyanin (KLH)-conjugated GnRH dimer polypeptide antigen as the coating antigen, and detects GnRH antibody titers via indirect ELISA to assess the hormonal status and reproductive capacity of dogs at different stages. This method offers advantages such as low cost, simple operation, high sensitivity, strong specificity, and good accuracy. It enables stable, rapid, accurate, and convenient detection of GnRH antibody levels in canine serum, while exhibiting good reproducibility.

[0012] Specifically, this invention uses KLH-conjugated GnRH dimer peptides as the specific coating antigen, fully utilizing the characteristic of KLH carriers in canines that they have no cross-reactive antibodies, effectively avoiding non-specific interference during the detection process and significantly improving detection specificity. The optimized combination of 50mM Na2CO3-NaHCO3 coating buffer (pH 9.1±0.2) and 5% skim milk blocking solution reduces background signal, further improving detection sensitivity and accuracy. All components in the kit, such as washing solution, diluent, chromogenic solution, and stop solution, are commercially available raw materials and reagents. The entire detection process can be completed within 4 hours, with simple operation steps and no special equipment required, providing a powerful tool for rapid assessment of canine immunization and castration effects. Furthermore, the reagents used in this method are inexpensive, facilitating large-scale application and possessing significant practical value for canine breeding management, pet health protection, and reproductive regulation in livestock production. Attached Figure Description

[0013] Figure 1 The graph shows the P / N ratio results of the optimized canine serum ELISA antigen coating concentration and serum dilution in Example 3.

[0014] Figure 2 The graph shows the P / N ratio results for optimizing the serum incubation time in canine serum ELISA in Example 3.

[0015] Figure 3The graph shows the P / N ratio results of the screening of canine serum ELISA blocking solution in Example 3.

[0016] Figure 4 The graph shows the P / N ratio results of the optimization of canine serum ELISA enzyme-labeled secondary antibody concentration and incubation time in Example 3.

[0017] Figure 5 The graph shows the P / N ratio results for optimizing the substrate color development time of canine serum ELISA in Example 3.

[0018] Figure 6 The graph shows the P / N ratio results for the sensitivity detection of canine serum ELISA in Example 4. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0020] Unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods used in the examples are conventional methods. Where specific conditions are not specified in the examples, they should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] Example 1: GnRH dimer peptide and KLH-GnRH dimer peptide In this embodiment, the KLH-GnRH dimer polypeptide was obtained by coupling KLH and GnRH dimer polypeptide. The amino acid sequence of the GnRH dimer polypeptide used to synthesize the KLH-GnRH dimer polypeptide is shown in SEQ ID NO.1. Both the GnRH dimer polypeptide and the KLH-GnRH dimer polypeptide were obtained by chemical synthesis commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0022] SEQ ID NO.1: QHWSYGLRPGGGGGSGGGGSGGGGSQHWSYGLRPGC.

[0023] Example 2: ELISA kit An ELISA kit for detecting GnRH antibodies in canine serum includes the following components: 1) Antigen: KLH-conjugated GnRH dimer polypeptide (prepared in Example 1); 2) Coating solution: 50mM Na2CO3-NaHCO3 buffer (pH 9.1±0.2); 3) Sealing solution: 5% skim milk powder; 4) Enzyme-labeled secondary antibody: HRP-labeled goat anti-canine IgG; 5) Washing solution: PBST solution; PBST solution: Dilute 10×PBS buffer to 1×PBS + 0.05% Tween 20; 10×PBS buffer: 80g NaCl, 2g KCl, 36.3g Na2HPO4·12H2O, 2.4g KH2PO4, distilled water to a final volume of 1L; 6) Antibody dilution solution: PBST solution (same as the washing solution mentioned above); 7) Substrate solution: TMB (A solution: B solution = 1:1, prepare fresh before use); A solution is a peroxide donor, and B solution is a hydrogen donor (colorimetric substrate). 8) Termination solution: 0.55 mol / L H2SO4 solution; 9) Standard serum sample: Standard negative serum.

[0024] Example 3: A method for detecting GnRH antibodies in canine serum based on an ELISA kit This embodiment will optimize the detection conditions of the ELISA kit provided in Example 2 and determine the final detection method.

[0025] 1. Optimization of detection method conditions Unless otherwise specified, the detection process and conditions in the following optimization experiments shall be carried out in accordance with the methods provided in Section 2 of this embodiment.

[0026] 1.1 Determination of the optimal coating concentration and optimal serum dilution of indirect ELISA antigen The optimal reaction conditions were determined using a checkerboard titration method, utilizing different dilutions of KLH-GnRH peptides with varying coating concentrations and GnRH-positive and negative sera. Results are shown below. Figure 1 ,by D 450nm,p / D 450nm,n The optimal antigen coating concentration and optimal serum dilution of the well with the larger value are taken as the optimal antigen coating concentration and optimal serum dilution.

[0027] The results showed that the optimal KLH-GnRH dimer polypeptide antigen coating concentration in this embodiment was 2 μg / mL, and the optimal dilution of GnRH positive and negative sera was 1:50.

[0028] 1.2 Optimization of serum incubation time Following the optimal antigen coating concentration described above, positive and negative sera were incubated at 37°C for 10, 15, 20, 30, 45, and 60 min, respectively. Serum samples were diluted 1:50, 1:100, 1:200, and 1:400, and then subjected to ELISA according to the above procedure. D 450nm Value and calculate D 450nm,p / D 450nm,n Value, with the maximum D 450nm,p / D 450nm,n The value determines the optimal incubation time for the serum.

[0029] See results Figure 2 When incubated with the primary antibody at 37°C for 60 minutes, D 450nm,p / D 450nm,n The value was the highest, indicating that the optimal serum incubation time in this embodiment was 60 min.

[0030] 1.3 Selection of the optimal sealing solution ELISA plates were coated with the optimized antigen coating concentration and divided into four groups. The plates were blocked at 37°C for 3 hours with 3% skim milk powder, 5% skim milk powder, 10% FBS, and 3% BSA, respectively. Serum samples were diluted 1:50, 1:100, 1:200, and 1:400. ELISA assays were performed using the optimized antigen coating concentration. The OD values ​​of negative and positive sera in each group were compared. D 450nm,p / D 450nm,n To determine the appropriate blocking solution, different blocking solutions were used to block the coated antigen, and the blocking effect was tested.

[0031] See results Figure 3 This indicates that the concentration of 5% skim milk powder measured in this embodiment... D 450nm,p / D 450nm,n The value is the highest, so 5% skim milk powder is chosen as the best sealing liquid.

[0032] 1.4 Determination of the optimal concentration and time of enzyme-labeled secondary antibody Based on the optimal antigen coating concentration and conditions determined above, the antigen was diluted and coated, and the optimal blocking conditions were determined for blocking. The primary antibody was incubated for the determined optimal time. HRP-labeled goat anti-canine IgG was diluted at dilutions of 1:5000, 1:7000, 1:9000, and 1:10000, and used as enzyme-labeled secondary antibodies for ELISA detection of GnRH-positive and negative sera. Incubation times were set at 37℃ for 15 min, 30 min, 45 min, and 60 min, respectively. The results were compared... D 450nm,p / D 450nm,n The optimal concentration of enzyme-labeled secondary antibody and incubation time are determined by the value.

[0033] See results Figure 4 The enzyme-labeled secondary antibody was diluted 1:5000 and incubated for 30 minutes to detect the results. D 450nm,p / D 450nm,n The value was the highest, indicating that the optimal conditions for this example were a 1:5000 dilution of the enzyme-labeled secondary antibody and an incubation period of 30 minutes.

[0034] 1.5 Determination of substrate color development time The antigen was diluted and coated according to the optimal antigen coating concentration and conditions determined above. Blocking was performed under the optimal blocking conditions determined above. Incubation was carried out using the optimal antigen concentration and serum incubation conditions determined above. Serum samples were diluted 1:50, 1:100, and 1:200. After adding the chromogenic solution, the optimal chromogenic conditions were determined at 37℃ for 10 min, 37℃ for 15 min, 37℃ for 20 min, 37℃ for 25 min, and 37℃ for 30 min. The readings were taken after adding the stop solution. D 450nm Value, selection D 450nm,p / D 450nm,n The hole with the largest value is used to determine the optimal termination condition.

[0035] The results are as follows Figure 5 As shown, when the colorimetric solution is added and incubated at 37°C for 30 minutes... D 450nm,p / D 450nm,n The value was the highest, indicating that incubation at 37°C for 30 min was the optimal condition for this example.

[0036] 1.6 Critical Value for Yin-Yang Identification Eighty negative canine serum samples were selected, diluted 1:50, and analyzed using the optimized ELISA method. The mean (X) and standard deviation (SD) were calculated based on the OD values. The results showed that for 50 negative canine serum samples diluted 1:50, the highest value was 0.1374, the lowest was 0.1014, the mean was 0.1185, and the standard deviation was 0.0196. Therefore, the indirect ELISA cutoff value was determined to be 0.1185 + 3 × 0.0196 = 0.1773, i.e., when... D 450nm A value ≥0.1773 is considered positive. D 450nm A value <0.1773 is considered negative.

[0037] 2. Determination of Detection Method The method for detecting GnRH antibodies in canine serum based on the ELISA kit provided in Example 2 includes the following steps: 1) Coating antigen: Dilute the KLH-GnRH dimer polypeptide antigen to 2 μg / mL with coating buffer, repeating each concentration 8 times, and add it to a 96-well microplate (100 μL / well). Coat overnight at 4°C. After coating overnight (14 h), remove the coating buffer from the 96-well microplate, wash the microplate 3 times with PBST buffer (300 μL / well), incubating at room temperature for 5 min each time, and then blot dry the liquid in the microplate to obtain the coated antigen microplate.

[0038] 2) Blocking: Prepare 5% skim milk powder with PBST buffer and add it to the 96-well ELISA plate containing the coated antigen (200 μL / well). Block at 37°C for 3 h. Wash the ELISA plate 3 times with PBST buffer (300 μL / well) for 5 min each time. Pat the liquid in the ELISA plate dry to obtain the antigen-coated ELISA plate.

[0039] 3) Serum / primary antibody incubation: Standard positive and negative serum samples or canine serum samples to be tested are diluted in a 1:100 gradient and added to the encapsulated antigen ELISA plate at 100 μL / well. The plate is incubated at 37°C for 1 h. The plate is washed three times with PBST buffer (300 μL / well) for 5 min each time. The liquid in the plate is then patted dry to obtain the serum / primary antibody incubated ELISA plate.

[0040] 4) Secondary antibody incubation: Dilute the enzyme-labeled secondary antibody (HRP-labeled goat anti-canine IgG) 1:5000 with PBST buffer, add it to the ELISA plate after serum / primary antibody incubation (100 μL / well), incubate at 37℃ for 30 min, wash the deenzyme plate 3 times with PBST buffer (300 μL / well), 5 min each time, pat dry the liquid in the ELISA plate, and obtain the ELISA plate after secondary antibody incubation.

[0041] 5) Color development: Add TMB color development solution to the microplate after incubation with secondary antibody, 100 μL / well, and develop at 37℃ in the dark for 30 min to obtain the color developed microplate.

[0042] 6) Termination: Add the stop solution to the developed microplate at 100 μL / well and read the values ​​using a microplate reader. D 450nm Value, that is, when D 450nm A value ≥ 0.1773 is considered positive. D 450nm A value < 0.1773 is considered negative.

[0043] Example 4: Sensitivity Test Enzyme-labeled plates were coated with optimized antigen concentrations. Positive serum samples were diluted at ratios of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200 according to the procedure provided in Section 2 of Example 3. ELISA assays were performed on the positive serum samples under selected experimental conditions, and OD values ​​were measured to determine the sensitivity of the method. Standard strongly positive serum samples were also diluted proportionally, and ELISA assays were performed on the positive serum samples under selected experimental conditions, and OD values ​​were measured to determine the sensitivity of the method.

[0044] See results Figure 4 The positive serum was diluted 12,800 times and the test result was positive, while it was diluted 25,600 times and the test result was negative. Therefore, the sensitivity was determined to be 1:12,800.

[0045] Example 5: Comparison of GnRH dimer peptide and KLH-GnRH dimer peptide Using GnRH dimer peptide and KLH-GnRH dimer peptide as antigens, respectively, and following the detection method provided in Section 2 of Example 3, positive serum samples were tested at antigen coating concentrations of 1 μL / mL, 1.5 μL / mL, 2 μL / mL, and 2.5 μL / mL, and serum dilution concentrations of 1:50, 1:100, 1:200, and 1:400. The results are shown in Table 1.

[0046] Table 1. Detection results of GnRH dimer peptide and KLH-GnRH dimer peptide antigens.

[0047] As shown in Table 1, under the same serum dilution factor and antigen coating concentration, the detection of KLH-GnRH dimer peptides... D 450nmThe sensitivity of KLH-GnRH dimer peptide was significantly higher than that of GnRH dimer peptide. When the serum dilution was 1:400, GnRH dimer peptide could not be detected. However, according to the sensitivity test results of Example 4, the sensitivity of KLH-GnRH dimer peptide was 1:12800, which was much higher than that of GnRH dimer peptide. This indicates that conjugation of GnRH dimer peptide with KLH can significantly improve the detection sensitivity.

[0048] Example 6: Specificity Detection The method provided in Section 2 of Example 3 was used to test clinical serum samples of dogs immunized with different vaccines and post-immunization serum samples of dogs in pet hospitals in Jilin City, Jilin Province.

[0049] Among them, the serum sample of recombinant GnRH-0806 protein from immunized dogs was collected from serum prepared after immunization; the serum samples of canine hexavalent vaccine (containing canine distemper virus, canine parainfluenza virus, canine infectious hepatitis virus, canine parvovirus and canine coronavirus) + rabies virus, and the serum samples of canine quadrivalent vaccine (canine distemper virus, canine parvovirus, canine adenovirus, canine parainfluenza virus) + rabies virus vaccine were all collected from pet hospitals in Jilin Province.

[0050] Table 2 shows the test results. This method was used to test 75 canine serum samples from groups 2 and 3. The results showed that in group 2 (40 serum samples), 39 were negative and 1 was weakly positive; all 35 serum samples from group 3 were negative. However, all serum samples from immunized dogs testing for recombinant GnRH-0806 protein were positive. The experimental results indicate that this method can effectively detect GnRH antibodies in canine serum. In groups B and C (75 samples in total), 74 showed no cross-reactivity, and only 1 was weakly positive.

[0051] Table 2 Results of specificity assay for canine serum ELISA

[0052] In summary, this detection method shows no significant cross-reactivity with the seven antibodies produced by canine distemper virus, canine parainfluenza virus, canine infectious hepatitis virus, canine parvovirus, canine coronavirus, canine adenovirus, and rabies virus immunization. It has good specificity and high sensitivity, and can stably, rapidly, accurately, and conveniently quantify the level of GnRH antibodies in canine serum, thus helping to more accurately understand the release of gonadotropin-releasing hormone in canines.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A KLH-GnRH dimer polypeptide, characterized in that, The KLH-GnRH dimer polypeptide is obtained by coupling KLH and GnRH dimer polypeptide; the amino acid sequence of the GnRH dimer polypeptide is shown in SEQ ID NO.

1.

2. An ELISA kit for detecting GnRH antibodies in canine serum, characterized in that, Includes the KLH-GnRH dimer polypeptide of claim 1, wherein the KLH-GnRH dimer polypeptide is used as an antigen.

3. The ELISA kit for detecting GnRH antibodies in canine serum according to claim 2, characterized in that, The package includes KLH-conjugated GnRH dimer peptide, coating buffer, blocking buffer, enzyme-labeled secondary antibody, standard serum sample, washing buffer, antibody dilution buffer, substrate chromogenic solution, and stop solution; the blocking agent is 5% skim milk powder.

4. The ELISA kit for detecting GnRH antibodies in canine serum according to claim 2, characterized in that, The coating buffer was 50 mM Na2CO3-NaHCO3 buffer with a pH of 9.1 ± 0.2; the enzyme-labeled secondary antibody was HRP-labeled goat anti-canine IgG; the washing buffer and antibody dilution buffer were PBST buffer; the PBST buffer was obtained by diluting 10×PBS buffer 10 times to 1×PBS buffer and then adding 0.05% Tween 20; the substrate chromogenic solution was TMB chromogenic solution, which included solution A and solution B; the stop solution was 0.55 mol / L H2SO4 solution.

5. The ELISA kit for detecting GnRH antibodies in canine serum according to claim 4, characterized in that, Solution A and solution B should be prepared fresh in a 1:1 ratio and used immediately.

6. The ELISA kit for detecting GnRH antibodies in canine serum according to claim 2, characterized in that, The KLH-GnRH dimer peptide was coated with a coating solution and then blocked with a blocking solution to prepare the encapsulated antigen; the coating concentration of the KLH-GnRH dimer peptide was 2 μL / mL.

7. A method for detecting GnRH antibodies in canine serum based on the ELISA kit according to any one of claims 2 to 6, characterized in that, Includes the following steps: S1. Coating antigen: Dilute the KLH-GnRH dimer peptide with coating buffer and add it to a 96-well microplate, 100 μL / well. Coat at 4℃ for 12-16 h. Discard the liquid and wash the microplate 3 times with washing buffer, 5 min / time. Discard the liquid and pat dry to obtain the coated antigen microplate. S2. Blocking: Add blocking solution to the coated antigen ELISA plate, 200 μL / well, block at 37℃ for 3 h, wash the ELISA plate 3 times with washing solution, 5 min / time, discard the liquid and pat dry to obtain the coated antigen ELISA plate; S3. Serum / primary antibody incubation: Dilute the standard serum sample or the sample to be tested at a ratio of 1:100 and add it to the encapsulated antigen ELISA plate at 100 μL / well. Incubate at 37°C for 1 h. Wash the ELISA plate 3 times with washing buffer for 5 min each time. Discard the liquid and pat dry to obtain the ELISA plate after serum / primary antibody incubation. S4. Secondary antibody incubation: Dilute the enzyme-labeled secondary antibody with antibody dilution buffer at a ratio of 1:5000, add 100 μL / well to the enzyme-labeled plate after serum / primary antibody incubation, incubate at 37°C for 30 min, wash the enzyme-labeled plate 3 times with washing buffer for 5 min each time, discard the liquid and pat dry. The enzyme-labeled plate after secondary antibody incubation. S5. Color development: Add the substrate color development solution to the microplate after incubation with the secondary antibody, 100 μL / well, and develop the color at 37°C in the dark for 30 min to obtain the color developed microplate. S6. Termination: Add the stop solution to the developed microplate at 100 μL / well and read the values ​​using a microplate reader. D 450nm Value, if D 450nm A value ≥0.1773 is considered positive. D 450nm A value <0.1773 is considered negative.

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