A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.

Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China

Objective In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis （LB） and screened out the appropriate antigens to support the production of a Chinese clinical ELISA （enzyme-linked immunosorbent assay） kit for LB. Methods Six recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic （ROC） curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. Results Two IgG （VISE and OspC B.g） and two IgM （OspC B.g and OspC B.a） antigens were left by the logistic regression model screened. VIsE had the highest specificity for syphilis samples in the IgG test （87.7%, P〈0.05）. OspC B.g had the highest diagnostic value in the IgM test （AUC=0.871）. Interactive effects between OspC B.a and Fla B.g could reduce the specificity of the ELISA. Conclusion Three recombinant antigens, OspC B.g, OspC B.a, and VisE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

Serological diagnosis of nasopharyngeal carcinoma by enzyme linked immunosorbant assay:optimization,standardization and diagnostic criteria

Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC).Methods We used a combination of highly purified glutathione transferase fusion proteins of the 40kD carboxy domain of EBNA1 and the 18kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/-20% of this value.Results All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%.Conclusions After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.