Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus.
10.3343/alm.2014.34.2.118
- Author:
Reza TAHERKHANI
1
;
Manoochehr MAKVANDI
;
Fatemeh FARSHADPOUR
Author Information
1. Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Scien, Ahvaz, Iran. f.farshadpour@yahoo.com
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Hepatitis E virus;
ORF2;
Optimization;
Expression;
Escherichia coli;
ELISA
- MeSH:
Amino Acid Sequence;
Antibodies/*blood;
*Enzyme-Linked Immunosorbent Assay;
Escherichia coli/metabolism;
Hepatitis E virus/*metabolism;
Humans;
Immunoglobulin G/*blood;
Molecular Sequence Data;
Recombinant Proteins/biosynthesis/immunology/isolation & purification;
Sequence Alignment;
Viral Proteins/chemistry/*immunology/metabolism
- From:Annals of Laboratory Medicine
2014;34(2):118-126
- CountryRepublic of Korea
- Language:English
-
Abstract:
BACKGROUND: Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. METHODS: Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by Ni2+-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. RESULTS: High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P=0.625). CONCLUSIONS: The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.
