title: A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs.
abstract: Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Yiyang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Baoyuan
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Lizhen
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Yani
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Huixia
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Xinjie
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: Shahid Faraz
authors.initials: SF
authors.lastname: Syed
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan, China.
authors.forename: Gaiping
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.|Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
authors.forename: En-Min
authors.initials: EM
authors.lastname: Zhou
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000956:Antigens, Viral / Q000302:isolation & purification; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000378:metabolism; D005260:Female; D006508:Hepatitis Antibodies / Q000302:isolation & purification*; D016752:Hepatitis E virus / Q000276:immunology*; D006735:Horseradish Peroxidase / Q000378:metabolism; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D034421:Sus scrofa / Q000276:immunology* / Q000821:virology*
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D006508:Hepatitis Antibodies; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase