Ann Lab Med.  2014 Mar;34(2):118-126. 10.3343/alm.2014.34.2.118.

Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

• Taherkhani R
• Makvandi M
• Farshadpour F

• KMID: 1791904
• DOI: http://doi.org/10.3343/alm.2014.34.2.118

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.

Keyword

Hepatitis E virus; ORF2; Optimization; Expression; Escherichia coli; ELISA

MeSH Terms

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
Antibodies
Immunoglobulin G
Recombinant Proteins
Viral Proteins