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Development of enzyme-linked immunosorbent assays using 2 truncated ORF2 proteins for detection of IgG antibodies against hepatitis E virus.

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 Ni(2+)-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.

SUBMITTER: Taherkhani R

PROVIDER: S-EPMC3948824 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Development of enzyme-linked immunosorbent assays using 2 truncated ORF2 proteins for detection of IgG antibodies against hepatitis E virus.

Taherkhani Reza R Makvandi Manoochehr M Farshadpour Fatemeh F

Annals of laboratory medicine 20140213 2

<h4>Background</h4>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.<h4>Methods</h4>Two truncated forms of the ORF2 protein were expressed ...[more]

PMID: 24624347

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Project description:BACKGROUND:Since 2010, outbreaks of genotype 2 (G2) porcine epidemic diarrhea virus (PEDV) have caused high mortality in neonatal piglets and have had devastating impacts on the swine industry in many countries. A reliable serological assay for evaluating the PEDV-specific humoral and mucosal immune response is important for disease survey, monitoring the efficacy of immunization, and designing strategies for the prevention and control of PED. Two PEDV spike (S) glycoprotein-based indirect enzyme-linked immunosorbent assays (ELISAs) were developed using G2b PEDV-Pintung 52 (PEDV-PT) trimeric full-length S and truncated S1-501 proteins derived from the human embryonic kidney (HEK)-293 cell expression system. The truncated S1-501 protein was selected from a superior expressed stable cell line. The sensitivity and specificity of these two ELISAs were compared to immunostaining of G2b PEDV-PT infected cells and to a commercial nucleocapsid (N)-based indirect ELISA kit using a panel of PEDV negative and hyperimmune sera. RESULTS:The commercial N-based ELISA exhibited a sensitivity of 37%, a specificity of 100%, and a fair agreement (kappa?=?0.37) with the immunostaining result. In comparison, the full-length S-based ELISA showed a sensitivity of 97.8%, a specificity of 94%, and an almost perfect agreement (kappa?=?0.90) with the immunostaining result. Interestingly, the S1-501-based ELISA had even higher sensitivity of 98.9% and specificity of 99.1%, and an almost perfect agreement (kappa?=?0.97) with the immunostaining result. A fair agreement (kappa<?0.4) was seen between the commercial N-based ELISA and either of our S-based ELISAs. However, the results of the full-length S-based ELISA shared an almost perfect agreement (kappa?=?0.92) with that of S1-501-based ELISA. CONCLUSIONS:Both full-length S-based and S1-501-based ELISAs exhibit high sensitivity and high specificity for detecting antibodies against PEDVs. Considering the high protein yield and cost-effectiveness, the S1-501-based ELISA could be used as a reliable, sensitive, specific, and economic serological test for PEDV.

Project description:Porcine epidemic diarrhea virus (PEDV) can infect pigs of all ages, especially piglets. PEDV has spread across Asia since the 1980s. The highly virulent variant PEDV broke out on a large scale and caused huge economic losses to the pig industry in late 2010 in China. Rapid detection methods with high specificity and sensitivity are urgently needed for the diagnosis and control of the disease. In this study, we divided the PEDV S1 gene into three segments and constructed the recombinant plasmids pFastBac1-S1T1 (aa 21-279), pFastBac1-S1T2 (aa 280-539) and pFastBac1-S1T3 (aa 540-788), which carry the different antigenic regions of the S1 gene. Truncated S1 proteins PEDV-S1T1/S1T2/S1T3 were obtained by a Bac-to-Bac expression system, with protein sizes of 36 kDa, 38 kDa and 38 kDa, respectively. Recombinant proteins presented high reactivity with the monoclonal antibody against PEDV and positive pig serum. Based on full-length S1 protein and these truncated proteins, we established indirect ELISA methods for the detection of PEDV IgA antibody. A total of 213 clinical serum samples were tested by the above indirect ELISA methods, and IFA was used as the gold standard. ROC curves revealed a significant correlation between S1-ELISA and S1T2-ELISA with a 0.9134 correlation coefficient and favourable sensitivity and specificity of S1-ELISA (93.24%, 95.68%) and S1T2-ELISA (89.33%, 94.16%). Our results also indicated that serum with higher neutralizing activity (SNT ≥ 40) had a higher IgA antibody level based on S1-ELISA, S1T1-ELISA and S1T2-ELISA. In conclusion, both S1-ELISA and S1T2-ELISA can be used as candidate systems for detecting anti-PEDV IgA antibody titers in serum, which can reflect the level of neutralizing activity in pigs after natural infection or vaccination. The above research results provide a basis for the prevention and control of PEDV and can be used in the detection of host anti-infective immunity and evaluation of vaccine immune effects.

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Development of enzyme-linked immunosorbent assays using 2 truncated ORF2 proteins for detection of IgG antibodies against hepatitis E virus.

Publications

Development of enzyme-linked immunosorbent assays using 2 truncated ORF2 proteins for detection of IgG antibodies against hepatitis E virus.

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