A simple colorimetric detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification assay using hydroxynaphthol blue metal indicator.

A simple reverse transcription loop-mediated isothermal amplification combined with visual detection method (vRT-LAMP) assay was developed for rapid and specific detection of porcine epidemic diarrhea virus (PEDV) in this study, which overcomes the shortcomings of previously described RT-LAMP assays that require additional detection steps or pose a risk of cross-contamination. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubating for 40 min at 62 °C. The assay specifically amplified PEDV RNA and no other viral nucleic acids. The limit of detection of the assay was less than 50 RNA copies per reaction, which was 100 times more sensitive than conventional reverse transcription polymerase chain reaction (RT-PCR) and comparable to real-time RT-PCR (RRT-PCR). In the clinical evaluation, the PEDV detection rate of vRT-LAMP was higher than that of RRT-PCR, showing 99 % concordance, with a kappa value (95 % confidence interval) of 0.97 (0.93-1.01). Considering the advantages of high sensitivity and specificity, simple and direct visual monitoring of the results, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vRT-LAMP assay will be a valuable tool for detecting PEDV from clinical samples, even in resource-limited laboratories.

Development of an epitope-blocking-enzyme-linked immunosorbent assay to differentiate between animals infected with and vaccinated against foot-and-mouth disease virus.

An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes of FMDV. Sera from uninfected and regularly vaccinated cattle, pigs, goats, and sheep (raised in FMDV free areas) were screened to evaluate the specificity of the EB-ELISA. The specificity scores of the assays were 99.8-100% and 100%, respectively. Reference sera from cattle, pigs, goats, and sheep experimentally infected with FMDV tested positive, with only a single exception. Antibodies formed in response to FMDV 3B appeared 1 week after infection and persisted at high levels for more than 8 weeks within the sera collected from serial bleeding of animals infected with FMDV O/SKR/2000. The EB-ELISA was used to differentiate between farms vaccinated against and those infected with FMDV (FMDV Asia serotype) during the 2005 epidemic in Mongolia by detecting antibodies against the FMDV Asia serotype in outbreak farms. This EB-ELISA method shows promise as an effective tool for FMDV control and eradication.

Development of a monoclonal antibody-based ELISA system for glyceraldehyde-derived advanced glycation end products.

We have previously found that glyceraldehyde-derived advanced glycation end products (glycer-AGEs) elicit oxidative stress generation and evoke inflammatory and thrombotic reactions through their higher binding affinity to RAGE (receptor for AGEs), thereby playing a role in vascular complications in diabetes. Furthermore, circulating levels of glycer-AGEs are elevated in diabetes. We characterized a monoclonal antibody (mAb) raised against glycer-AGEs and prepared its specific ELISA system in human serum. We developed here mAb reacted specifically with glycer-AGEs or glyceraldehyde-derived pyridinium, but not other structurally identified AGEs or AGE precursors. The mAb not only completely neutralized the deleterious effects of glycer-AGEs on endothelial cells, but also detected glycer-AGEs in the aorta of type 2 diabetic rats. Intra and inter-assay coefficient variations of the ELISA were 6 and 2.6%, respectively. ELISA linearity was shown intact within 5-fold dilution, and recovery ratio of added glycer-AGEs was 88-117%. Results of serum and plasma were comparable, and repeated freeze-thawing of samples did not affect the results (90.1-112.4%). Serum glycer-AGEs levels in 30 healthy subjects evaluated by the ELISA were strongly correlated with those by polyclonal Ab-based one (r=0.82). Our present study suggests the clinical utility of mAb for evaluating glycer-AGE levels in both tissue and serum.

Field application of a recombinant protein-based ELISA during the 2010 outbreak of foot-and-mouth disease type A in South Korea.

A recombinant protein-based enzyme-linked immunosorbent assay (RP ELISA) exists for the detection of antibodies to foot-and-mouth disease virus (FMDV) type A. In this study, the efficacy of the RP ELISA was compared to that of other current tests by examining sera collected in the field during an FMD type A outbreak in South Korea in 2010. The RP ELISA detected early antibodies to FMDV with the same sensitivity as the liquid-phase blocking ELISA (LPB ELISA), identifying FMD farm outbreaks correctly on a herd basis. In addition, the two assays exhibited a high correlation coefficient (γ(2)=0.83) when testing thirty seven sera from one outbreak farm exhibiting various antibody titers. The sensitivity and specificity of the RP ELISA relative to the LPB ELISA were 84% and 97%, respectively, and excellent agreement (kappa=0.82) was observed between the two tests. Taken together, the RP ELISA should be a useful alternative to the LPB ELISA for the detection of early antibodies to FMDV type A during an outbreak.

Use of a baculovirus-expressed structural protein for the detection of antibodies to foot-and-mouth disease virus type A by a blocking enzyme-linked immunosorbent assay.

A blocking enzyme-linked immunosorbent assay (ELISA) with a baculovirus-expressed structural protein was developed for the detection of antibodies to foot-and-mouth disease virus type A. It exhibited 99% specificity with a cutoff of 53% inhibition. Its sensitivity was comparable to the sensitivities of the virus neutralization test and the liquid-phase blocking ELISA, indicating its potential as an alternative assay.

A recombinant protein-based ELISA for detecting antibodies to foot-and-mouth disease virus serotype Asia 1.

A recombinant protein-based ELISA was evaluated for detecting antibodies to foot-and-mouth disease virus (FMDV) serotype Asia 1. The recombinant protein (rP13C) was derived from the P1 precursor and 3C protease genes that were cloned into a single expression vector and expressed in insect cells. This protein elicited a low titer of FMDV neutralizing antibodies in pigs. Its utility as a diagnostic antigen was explored in a blocking ELISA using monoclonal antibodies. The rP13C ELISA yielded higher endpoint titers than the liquid phase blocking (LPB) ELISA and virus neutralization test performed on sera from goats challenged with FMDV post-vaccination. The rP13C ELISA correctly scored the FMD international reference weak positive serum. The relative sensitivity between the rP13C ELISA and LPB ELISA was equivalent for vaccinated sera. With this comparable sensitivity, the rP13C ELISA exhibited a specificity of 99.7% for domestic naive swine, bovine and caprine sera. This report demonstrates that an ELISA using recombinant proteins has the potential to replace the LPB ELISA using an inactivated FMDV antigen as a simple and robust serological tool for screening antibodies to FMDV serotype Asia 1.