Pig epidemic encephalitis b virus antibody elisa detection method based on prM-EDIII fusion antigen

Abstract

The application discloses a porcine epidemic encephalitis B virus antibody ELISA detection method based on a prM-EDIII fusion antigen. 450 As a detection readout, the sample is determined as positive or negative according to a positive-negative determination critical value; the application determines the best operation condition of the method by optimizing the reaction condition, and sets the positive-negative determination critical value as OD 450 =0.3206 based on negative serum statistics. The specificity and sensitivity evaluation results show that the method does not have cross reaction to other pathogen positive serum, and the sensitivity reaches 1:1600. The method has the advantages of simple operation, suitable for large-scale application, accurate and reliable detection, and can provide technical support for JEV serum monitoring and immune evaluation.

Description

Technical Field

[0001] This invention relates to the field of serological detection technology for JEV, and more particularly to an ELISA method for detecting porcine Japanese encephalitis virus antibodies based on the prM-EDIII fusion antigen. Background Technology

[0002] JEV is an important zoonotic pathogen. Pigs are key amplification hosts for JEV, and monitoring their antibody levels is of great significance for assessing epidemic risk and immunization efficacy.

[0003] Currently, JEV antibody detection often relies on methods such as serum neutralization assays (e.g., PRNT). These methods are complex, time-consuming, and have high requirements for experimental conditions, making it difficult to meet the needs of grassroots institutions for large-scale screening.

[0004] ELISA has become the preferred technology for antibody screening due to its high throughput, ease of operation, and controllable cost.

[0005] However, most existing ELISA methods are based on a single antigen (e.g., EDIII as the core) and mainly cover the detection of neutralizing antibodies. However, they have limited coverage of non-neutralizing antibodies produced in early infection, which may lead to the risk of missed detection. This results in an incomplete detection spectrum and affects the accuracy and timeliness of antibody monitoring in pig herds.

[0006] Therefore, in order to solve the above-mentioned technical problems, it is an urgent technical problem for those skilled in the art to provide an indirect ELISA method for porcine JEV antibodies that can simultaneously recognize early infection antibodies and detect neutralizing antibodies, and has good specificity, sensitivity and reproducibility, so as to improve the reliability and application value of large-scale screening at the grassroots level. Summary of the Invention

[0007] In view of this, the present invention provides an ELISA detection method for porcine Japanese encephalitis virus antibodies based on the prM-EDIII fusion antigen. The fusion antigen of the present invention expresses prM with E protein domain III (EDIII) to expand the antibody detection spectrum. Furthermore, by optimizing the key reaction parameters of ELISA, a critical value for positive and negative results is established, and the methodological characteristics and consistency with clinical applications are verified.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] The ELISA detection method for porcine Japanese encephalitis virus antibodies based on the prM-EDIII fusion antigen includes the following steps:

[0010] S1: Prepare the prM-EDIII fusion antigen as a coating antigen and use it for ELISA coating;

[0011] S2: Add the pig serum to be tested into the coated wells and incubate;

[0012] S3: Add enzyme-labeled secondary antibody, incubate, and then perform color development;

[0013] S4: OD 450 As a test reading, the sample is determined to be positive or negative according to the positive / negative threshold; among them, the prM-EDIII fusion antigen is used to specifically bind to the JEV antibody in the sample to be tested.

[0014] Preferably, in step S1, the antigen coating concentration is 4 μg / mL, and the coating is performed overnight at 4°C.

[0015] Preferably, in step S2, the serum dilution is 1:100, and the incubation is at 37°C for 60 min;

[0016] Preferably, in step S3, the enzyme-labeled secondary antibody (HRP-goat anti-pig IgG) is diluted 1:2000, and the reaction is carried out at 37°C for 90 min;

[0017] Preferably, in step S4, the threshold value for determining positive or negative sex is OD. 450 =0.3206.

[0018] Preferably, the method has a detection sensitivity of 1:1600 for JEV-positive serum.

[0019] Preferably, the method has good specificity and does not cross-react with other pathogen-positive sera.

[0020] Preferably, the sample to be tested is a porcine serum sample.

[0021] Preferably, the method, when validated in parallel with serological neutralization tests or commercially available ELISA assays, yields application results consistent with other detection methods.

[0022] The present invention achieves the following technical effects compared to the prior art:

[0023] (1) Broader detection spectrum: Through the design of prM-EDIII fusion antigen, this invention can simultaneously recognize early infection-related antibodies and neutralizing antibodies, thereby effectively reducing the risk of missed detection and improving the comprehensiveness and accuracy of detection;

[0024] (2) Easy to operate and suitable for large-scale screening: This invention is based on the indirect ELISA process. This mature technology process is easy to operate and does not require complicated equipment or professional skills. It is well suited to the high-throughput testing needs of grassroots laboratories and is conducive to rapid screening in large populations.

[0025] (3) Good specificity and repeatability: Experimental results show that the invention does not cross-react with positive sera such as PRRSV, GETV, and SVA, indicating that it has high specificity and can accurately distinguish the target antibody from other irrelevant antibodies; at the same time, the intra-batch and inter-batch coefficients of variation are both less than 10%, indicating that the detection method has good repeatability in different experimental batches and within the same batch, and the detection results are stable and reliable.

[0026] (4) High sensitivity: The detection sensitivity for JEV positive serum can reach 1:1600, which means that even when the antibody concentration in the serum is low, the invention can accurately detect it, has a high detection capability, and can detect infection earlier.

[0027] (5) High clinical consistency: Parallel verification with PRNT (plaque reduction neutralization test, gold standard method) and commercially available kits showed that the invention has good positive and negative concordance rates and overall consistency, indicating that its detection results are highly consistent with authoritative methods and existing commercial products, and have high clinical application value.

[0028] This invention uses the fusion expression of prM and EDIII as the coating antigen. Compared with traditional single antigen detection methods, it retains the detection capability of EDIII for neutralizing antibodies and supplements the recognition range of prM for antibodies produced in the early stage of infection, thus solving the problem of high false negative rates in existing technologies. This method requires no virus culture, has a simple detection process, and all reagents can be standardized for configuration. It is suitable for large-scale JEV antibody monitoring in veterinary testing institutions at all levels, and can provide stable and reliable technical support for the prevention and control of porcine encephalitis B and the evaluation of immunization efficacy. Attached Figure Description

[0029] Figure 1 This is a diagram illustrating the amplification, construction, and identification of the prM-EDIII fusion antigen gene in this invention.

[0030] Figure 2 This is an identification diagram of the expression and purification results of the prM-EDIII fusion antigen of this invention;

[0031] Wherein, A: PCR product of JEV-prM and JEV-EDIII genes; B: PCR product of JEV-prM-EDIII fusion gene;

[0032] Figure 3 This is a diagram showing the optimization results of coating conditions for the prM-EDIII fusion antigen used in indirect ELISA according to the present invention.

[0033] Where A: different inducer concentrations; B: different induction times; C: different induction temperatures; (the optimal induction conditions are: temperature 37℃, IPTG concentration 0.8 mmol / L, and induction for 10 h).

[0034] Figure 4 The diagram shows the results of determining the negative / positive threshold values ​​for this invention (based on JEV-negative serum OD). 450 The statistical critical value was 0.3206.

[0035] Figure 5 This is a graph showing the specific detection results of the indirect ELISA of this invention (no cross-reaction occurs with positive sera from other pathogens);

[0036] Figure 6 This is a graph showing the sensitivity detection results of the indirect ELISA of this invention (detection sensitivity reaches 1:1600).

[0037] Figure 7 This is a graph showing the repeatability / stability test results of the indirect ELISA of this invention;

[0038] Figure 8 The graph shows the parallel consistency verification results of the indirect ELISA of this invention with PRNT and / or commercially available ELISA kits;

[0039] Figure 9 For the identification of the recombinant protein expression form of the present invention;

[0040] Among them, A: SDS-PAGE identification of recombinant protein; B: Western blot identification of recombinant protein. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0042] This invention discloses an ELISA method for detecting porcine Japanese encephalitis virus antibodies based on the prM-EDIII fusion antigen, comprising the following steps:

[0043] S1: Design, amplification, and prokaryotic expression of fusion antigens

[0044] Based on the design of JEV prM protein and the key antigen domain of EDIII, a prM-EDIII fusion gene (containing a linker sequence) was constructed and cloned into the pET-28a(+) expression vector. The recombinant plasmid was transformed into BL21(DE3) competent cells, and the induction expression conditions were optimized to obtain the recombinant fusion protein expression product.

[0045] S2: Purification, refolding, and preparation of fusion proteins

[0046] The expression product was purified by nickel column affinity chromatography, and the fusion protein was subjected to gradient dialysis refolding to obtain prM-EDIII fusion antigen that can be used for ELISA coating.

[0047] The fusion protein can be prepared at a level of 260 μg / mL, providing an antigenic basis for the subsequent establishment of ELISA.

[0048] S3: Establishment of Indirect ELISA Detection Method and Optimization of Reaction Conditions

[0049] The purified and refolded prM-EDIII fusion protein was used as the coating antigen, and the reaction conditions were optimized using a checkerboard titration method, wherein:

[0050] The antigen coating concentration was 4 μg / mL, and the coating was incubated overnight at 4°C.

[0051] The serum was diluted 1:100 and incubated at 37°C for 60 min.

[0052] The sealing solution is 2.5% skim milk;

[0053] The enzyme-labeled secondary antibody (HRP-goat anti-pig IgG) was diluted 1:2000 and reacted at 37℃ for 90 min.

[0054] TMB development time: 15 min.

[0055] S4: Determination of Critical Values ​​for Positive and Negative Sex

[0056] Forty JEV antibody-negative swine serum samples were randomly selected and tested using a commercially available ELISA kit. The mean and standard deviation were calculated, and the cutoff value for determining positive or negative was determined to be OD. 450 =0.3206;

[0057] OD of the sample to be tested 450 A value greater than or equal to the threshold is considered positive, while a value less than or equal to the threshold is considered negative.

[0058] S5: Methodological Performance Evaluation and Cross-Reactivity Validation

[0059] The specificity, sensitivity, and repeatability of JEV antibody detection were evaluated, and it was verified that the method does not cross-react with positive sera of pathogens such as PRRSV, GETV, and SVA. The detection sensitivity can reach 1:1600, and the intra-assay and inter-assay coefficients of variation are both less than 10%, showing good repeatability.

[0060] S6: Clinical Sample Testing and Consistency Verification

[0061] The method of this invention was verified by parallel detection with PRNT and commercially available ELISA kits.

[0062] The results showed that the positive / negative concordance rates with PRNT were 96.9% and 85.7% (Kappa=0.843), respectively, and the positive / negative concordance rates with commercially available kits were 98.5% and 94.3%, respectively, indicating good consistency and meeting the needs of clinical / epidemiological surveillance.

[0063] S7: Application Example (Pig Herd Monitoring)

[0064] Three JEV antibody-positive samples were detected in 1,145 swine serum samples from Shandong Province, with a positive rate of 0.26% (3 / 1,145). This provides basic data for regional JEV serological surveillance and epidemic risk assessment.

[0065] Example 1:

[0066] Includes the following steps:

[0067] (1) Construction and preparation of fusion antigen: The fusion antigen gene was constructed using the prM-EDIII fusion antigen design scheme. The prM-EDIII fusion protein was obtained by expression and then purified to obtain a fusion antigen that can be used for ELISA coating.

[0068] The results of fusion antigen expression and purification are shown below. Figure 1 and Figure 2 .

[0069] The concentration of the fusion protein after purification reached 260 μg / mL.

[0070] (2) Optimization of indirect ELISA reaction conditions: Using prM-EDIII fusion protein as the coating antigen, an indirect ELISA detection system was established, and key reaction conditions were optimized to obtain the best detection performance;

[0071] The optimization results of the indirect ELISA package conditions are shown below. Figure 3 .

[0072] (3) Determination of positive / negative threshold: Collect JEV negative serum samples and perform testing, using OD... 450Statistical analysis was used to establish criteria for determining positive and negative results, and the critical value for determining positive and negative results was determined to be OD. 450 =0.3206.

[0073] The results of determining the critical value are shown below. Figure 4 .

[0074] (4) Evaluation of specificity and sensitivity: The specificity of JEV antibody positive serum and other pathogen positive serum was evaluated. The results showed that the method of the present invention did not cross-react with other pathogen positive serum. At the same time, the sensitivity of the method was evaluated. The results showed that the detection sensitivity reached 1:1600.

[0075] The specificity and sensitivity results are shown below. Figure 5 and Figure 6 .

[0076] (5) Repeatability / stability evaluation: The repeatability of the indirect ELISA of the present invention was evaluated, demonstrating that the detection method has good detection stability and repeatability.

[0077] The repeatability / stability results are shown below. Figure 7 .

[0078] (6) Consistency verification: The indirect ELISA detection results of the present invention were compared with those of PRNT and / or commercially available ELISA kits in parallel. The results showed that the method of the present invention has high consistency with other detection methods at the application level.

[0079] The results of the parallel verification are shown below. Figure 8 .

[0080] In this embodiment, the coefficient of variation for intra-batch repeatability tests was 2.13%–6.87%, and the coefficient of variation for inter-batch repeatability tests was 3.25%–8.42%, both less than 10%, indicating that the method has good repeatability and the detection results are stable and reliable, meeting practical testing needs. Cross-reactivity validation results showed that this method is effective for OD values ​​of PRRSV, GETV, and SVA positive sera. 450 All values ​​were less than the negative / positive threshold of 0.3206 set by this method, and were therefore determined to be negative. This further confirms that this method does not have cross-reaction with the above-mentioned common swine pathogens and has good specificity.

[0081] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. An ELISA method for detecting porcine Japanese encephalitis virus antibodies based on prM-EDIII fusion antigen, characterized in that, Includes the following steps: S1: Prepare the prM-EDIII fusion antigen as a coating antigen and use it for ELISA coating; S2: Add the pig serum to be tested into the coated wells and incubate; S3: Add enzyme-labeled secondary antibody, incubate, and then perform color development; S4: OD 450 As a test reading, the sample is determined to be positive or negative according to the positive / negative threshold; among them, the prM-EDIII fusion antigen is used to specifically bind to the JEV antibody in the sample to be tested.

2. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, In step S1, the antigen coating concentration is 4 μg / mL, and the coating is performed overnight at 4°C.

3. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, In step S2, the serum dilution is 1:100, and the incubation is carried out at 37°C for 60 min.

4. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, In step S3, the enzyme-labeled secondary antibody (HRP-goat anti-pig IgG) is diluted 1:2000 and reacted at 37°C for 90 min.

5. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, In step S4, the threshold value for determining positive or negative sex is OD. 450 =0.3206.

6. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, The method has a detection sensitivity of 1:1600 for JEV-positive serum.

7. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, The method has good specificity and does not cross-react with other pathogen-positive sera.

8. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, The sample to be tested was a pig serum sample.

9. The ELISA detection method for porcine Japanese encephalitis virus antibody based on prM-EDIII fusion antigen according to claim 1, characterized in that, The method has been validated in parallel with serological neutralization tests or commercially available ELISA assays, and has consistent application results with other detection methods.

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