猪传染性胃肠炎病毒核衣壳蛋白阻断ELISA抗体检测方法的建立与应用OA

To establish a rapid,specific,and clinically applicable antibody detection method for porcine transmissible gastroenteritis virus(TGEV),this study used recombinant TGEV nucleocapsid(N)protein as the coating antigen.Key parameters including antigen coating mass concentration,serum dilution ratio,enzyme-labeled antibody dilution ratio,and incubation time and temperature were optimized to construct a blocking ELISA detection system,whose performance and clinical application value were systematically evaluated.Results showed that the optimal reaction conditions of this method were as follows:the optimal antigen coating mass concentration was 1.0 μg/mL;the optimal serum dilution ratio was 1∶2 with incubation at 37 ℃ for 45 min;the optimal enzyme-labeled antibody dilution ratio was 1∶12 000 with incubation at 37 ℃ for 30 min;and the optimal development time was 10 min.Specificity test results indicated no cross-reactivity with positive sera of common swine pathogens such as porcine epidemic diarrhea virus,porcine rotavirus,classical swine fever virus,porcine circovirus type 2,porcine reproductive and respiratory syndrome virus,and porcine sapelovirus,demonstrating good specificity.The method could detect TGEV-positive serum diluted at 1∶32,with sensitivity comparable to that of commercial kits.The coefficient of variations for both intra-batch and inter-batch repeatability tests was less than 10％,and the detection reagent remained stable when stored at 4 ℃ for 12 months.Clinical application results revealed that 124 clinical swine serum samples were tested using this method,the coincidence rate with a commercial TGEV antibody detection kit reached 96.77％,and the κ value between the two methods was 0.93,suggesting a high degree of consistency in detection results.In conclusion,the TGEV N protein-based blocking ELISA antibody detection method established in this study exhibited good specificity,sensitivity,repeatability,and stability.It could be effectively used for TGEV epidemiological surveys and vaccine immune effect evaluation,providing reliable technical support for the scientific prevention and control of swine transmissible gastroenteritis.