猪戊型肝炎病毒血清抗体间接ELISA检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2018.10.020

Abstract

Abstract:

Swine hepatitis E virus (HEV) can infect human by cross-species transmission. Therefore, it is important for surveillance by detection of swine HEV infection in pig herds. In this study, an indirect ELISA for detecting anti-swine HEV IgG antibodies was developed using a truncated swine HEV ORF2 protein expressed in a prokaryotic system as the coating antigen. The procedures of indirect ELISA were also optimized. The sensitivity, specificity, reproducibility, and comparisons with the commercial kit were analyzed. In addition, the application of indirect ELISA for detecting anti-swine HEV IgG antibodies in the field serum samples was also evaluated. SDS-PAGE and Western blot analysis showed that the truncated ORF2 protein was successfully expressed and reacted with the positive pig sera for swine HEV antibodies. The optimal amount of the coating antigen was determined at 200 ng per well and the dilution of sera was 1:200. The optimal coating and blocking buffers were carbonate buffer (pH=9.6) and 2.5% dry milk buffer, respectively. The cut-off value of the indirect ELISA was determined as OD_{450 nm} ≥ 0.296. The sensitivity and specificity were 100% and 1:6 400, respectively. Both intra-and inter-assay variabilities were less than 10%. The confidence between the indirect ELISA and the Wantai commercial kit was 91.2%. The developed indirect ELISA can also be used to detect anti-swine HEV IgG antibodies in the sequential sera from the pigs challenged with swine HEV and the clinical pig sera. Collectively, the developed indirect ELISA have good sensitivity, specificity and accuracy, and can be used for detecting anti-swine HEV IgG antibodies in pig sera from the field and ser-epidemiology investigation for swine HEV infection in pig herds.

CLC Number:

S852.659.6

DING Jun, ZHAO Xiao-jing, YU Kai-rui, ZHOU Tian-ming, LI Hui-xia, SHENG Ya-min, CHEN Yi-yang, LIU Bao-yuan, SUN Ya-ni, ZHOU En-min, ZHAO Qin. Development and Application of an Indirect ELISA for Detecting Antibodies against Swine Hepatitis E Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2240-2248.

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Development and Application of an Indirect ELISA for Detecting Antibodies against Swine Hepatitis E Virus

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