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Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.

ABSTRACT: The recent dramatic increase in reported cases of porcine epidemic diarrhea (PED) in pig farms is a potential threat to the global swine industry. Therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (PEDV) infection or vaccination would be essential in helping to control the spread of PED. We developed and validated an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant membrane (M) protein of PEDV. To detect PEDV antibodies in eight herds, 382 serum samples were collected from sows that had been immunized with a PED vaccine, and screened using the developed ELISA in parallel with a serum neutralization (SN) assay. Of the tested samples, 276 were positive for the presence of PEDV antibodies according to both assays, while 98 were negative. An excellent agreement between the ELISA and the SN assay was observed (kappa=0.947; 95% confidence interval=0.910-0.984; McNemar's test, P=0.727). No cross-reaction was detected for the developed ELISA with other coronaviruses or other common pig pathogens. The developed ELISA could be used for serological evaluation and indirect diagnosis of PED infection.

SUBMITTER: Fan JH

PROVIDER: S-EPMC7119585 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.

Fan Jing-Hui JH Zuo Yu-Zhu YZ Shen Xiao-Qiang XQ Gu Wen-Yuan WY Di Jing-Mei JM

Journal of virological methods 20150804

The recent dramatic increase in reported cases of porcine epidemic diarrhea (PED) in pig farms is a potential threat to the global swine industry. Therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (PEDV) infection or vaccination would be essential in helping to control the spread of PED. We developed and validated an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant m ...[more]

PMID: 26253335

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Project description:BACKGROUND:Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. RESULTS:One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80-100%, respectively. CONCLUSIONS:The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

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.

Dataset Information

Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.

Publications

Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.

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