Correlation of Indirect ELISA Based on the Neutralizing Epitopes on the S Protein of Porcine epidemic diarrhea virus Genotype Ⅱ Strain with Virus Neutralization Assay
ZHOU Yi-Mei1,2,*, DONG Wan-Yu1,*, WANG Xin-Yu1, ZHENG Mou-Feng1, WANG Zhi-Peng1, ZHU Xu-Hang1, ZHOU Ying-Shan1, YANG Yong-Chun1, SONG Hou-Hui1,**, WANG Xiao-Du1,**
1 College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology/Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/China-Australia Joint Laboratory for Animal Health Big Data Analytics, Hangzhou 311300, China; 2 Bureau of Ruian Agricultural and Affairs, Ruian 325200, China
Abstract：Porcine epidemic diarrhea (PED) is a highly contagious, highly lethal disease caused by Porcine epidemic diarrhea virus (PEDV). It causes severe diarrhea in 3~10 day-old piglets (Sus scrofa). The immune protection of PEDV depends on the neutralizing activity of colostrum and serum neutralizing antibodies against PEDV S1 protein. The establishment of rapid and high-throughput detection method of neutralizing antibodies is conducive to the prevention and control of PED. In order to evaluate PEDV neutralizing antibodies, an indirect ELISA method based on the S neutralizing peptide of PEDV genotype Ⅱ strain were established. In this study, the optimal conditions were determined that coating concentration of antigen was 0.2 μg/well by the equation titration. The dilution of serum was 1∶100, the reaction time of serum was 30 min, the optimal concentration of secondary antibody was 1∶6 000, the reaction time of secondary antibody was 60 min, and the reaction time of substrate was 10 min. The sensitivity, specificity of indirect ELISA were determined to compare with the neutralization test. The sensitivity and specificity of the indirect ELISA employed in the clinical sera samples were 94.74%, 91.67%, respectively and the Kappa was 0.93. The coefficient of variation of each serum sample after 3 repetitions was less than 10% in which 3 PEDV negative sera and 3 positive sera were tested repeatedly in inter group and intra group by the indirect ELISA. The sera against other pathogens (Swine fever virus, Porcine pseudorabies virus, Porcine reproductive and respiratory syndrome virus) showed no cross-reactivity with neutralizing peptide of PEDV S1 by the indirect ELISA. The correlation between the indirect ELISA and the virus neutralization test were performed by statistical analysis from 29 positive sera and 18 negative sera, thus the formula Y=0.370 8X+1.377 2 (X: The S/P value; Y: lg (1/neutralizing titer-32), R2=0.963) was obtained by correlation analysis. Since the neutralizing antibodies were indication about the evaluation of PEDV vaccine efficacy, this indirect ELISA provides a better tool for the large scale detection of anti-PEDV neutralizing antibodies, compared with the neutralization test.
周一媚, 董婉玉, 王馨雨, 郑谋凤, 王志鹏, 祝徐航, 周莹珊, 杨永春, 宋厚辉, 王晓杜. 基于猪流行性腹泻病毒基因Ⅱ型毒株S蛋白中和表位的间接ELISA和病毒中和试验的相关性分析[J]. 农业生物技术学报, 2022, 30(12): 2464-2472.
ZHOU Yi-Mei, DONG Wan-Yu, WANG Xin-Yu, ZHENG Mou-Feng, WANG Zhi-Peng, ZHU Xu-Hang, ZHOU Ying-Shan, YANG Yong-Chun, SONG Hou-Hui, WANG Xiao-Du. Correlation of Indirect ELISA Based on the Neutralizing Epitopes on the S Protein of Porcine epidemic diarrhea virus Genotype Ⅱ Strain with Virus Neutralization Assay. 农业生物技术学报, 2022, 30(12): 2464-2472.
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