Development and Application of Indirect ELISA Kits for Antibody Detection of Porcine Proliferative Enteropathy
[Objective]The objective of this study was to develop an indirect ELISA kits to detect Lawsonia intracellularis(LI)antibodies in pig,so as to provide a tool for the monitoring of LI infection and vaccine evaluation.[Method]The outer membrane protein(Omp2)of LI was expressed and purified,and an indirect ELISA method based on it to detect LI antibodies was established.The reaction conditions of the indirect ELISA and assemble the test kit was further optimized.Based on this,the prototype kit was used to test the sensitivity of the kit with serially diluted LI-positive sera,and other pathogen-positive sera were tested to study the specificity of the kit.The practicality of the kit was evaluated by testing 1 000 clinical pig serum samples collected at different times from different farms.From these clinical serum samples,the positive or negative effect of 50 sampleswere tested for by imported commercial kits,and the conformity of the kit were compared and verified.[Result]The Omp2 protein was successfully expressed and purified,with a purity of 90.31％,and was identified to react with LI-positive serum via Western blot.The optimal ELISA conditions were:coating with Omp2 at 200 ng/well at 4 ℃ for 12-16 hours;serum diluted 1∶50 and incubated at 37 ℃ for 30 minutes;goat anti-pig IgG-HRP diluted 1∶20 000,and incubated at 37℃ for 30 minutes;TMB substrate developed at 37 ℃ for 15 minutes,and the reaction was stopped with 0.5 mol·L-1 sulfuric acid.The highest detection dilution of LI-positive sera was 8-fold,indicating that the established ELISA method had the good sensitivity.The test kit tested negative for E.coli,App,Mhp,SS2,PRRSV,and PRV,demonstrating the good specificity of the ELISA method.Sensitivity and specificity testing standards for the kit have been established.The kit remains stable in terms of appearance,sensitivity,and specificity for 15 months when stored at 4 ℃.The criteria for the kit were set as follows:OD450nm≥1.25 for positive standard sera and OD450nm＜0.3 for negative standard sera.The intra-batch and inter-batch variability coefficients of the prototype kit were all less than 10％.The concordance rate with foreign commercial ELISA test kits reached 86％.The developed ELISA test kit detected LI-positive samples at a rate of 59.90％among 1 000 clinical pig serum samples from the eastern part of China,indicating the widespread presence of LI in the region.[Conclusion]The LI indirect ELISA antibody test kit in pig developed in this study had high specificity and sensitivity,and a high concordance rate with commercial test kits,making it suitable for clinical detection of LI antibodies.
Lawsonia intracellularisouter membrane protein 2indirect enzyme-linked immunosorbent assayspecificitysensitivitycoincidence rate
国家科技期刊平台
NSTL
万方数据
