Monoclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying West Nile Virus-Neutralizing Antibodies in Horse Sera

Abstract

A rapid immunoassay for detecting and quantifying West Nile virus (WNV)-neutralizing antibodies in sera was developed as an alternative to the plaque reduction neutralization test (PRNT), the gold standard test for WNV. The assay is a competitive, enzyme-linked immunosorbent assay using neutralizing monoclonal antibody 5E8 (NT-ELISA). A cutoff percent inhibition (PI) value of 35% (mean PI plus 3 standard deviations), with a specificity of 99%, was established based on analysis of 246 serum samples from horses free of WNV. The NT-ELISA detected neutralizing antibodies in all sera collected 7 or 14 days postinoculation from mice (n ‫؍‬ 11) infected with lineage I (strain NY385-99) or II (strain B956) WNV. When sera from WNV-vaccinated horses (n ‫؍‬ 212) were tested by NT-ELISA and PRNT, the NT-ELISA gave a positive result for 96.1% (173/180) of the PRNT-positive sera and 3.1% (1/32) of the PRNT-negative sera. Discrepancies between the two tests were observed mainly with sera with low PRNT 90 titers (expressed as the reciprocal of the highest dilution yielding >90% reduction in the number of plaques) for WNV or low PIs by NT-ELISA. The overall agreement (k value) between the two tests was 0.86. A good correlation (r 2 ‫؍‬ 0.77) was also observed between the tests for endpoint titration of sera (n ‫؍‬ 116). In conclusion, the newly developed NT-ELISA may be a good alternative serologic assay for detecting WNV that can be used for large-scale testing of WNV-neutralizing antibodies in multiple species.

West Nile Virus Infection among Animals and Humans in India

“…But HI cannot differentiate between closely related flaviviruses and cannot be used as a confirmatory diagnostic method. Various enzyme-linked immunosorbent assays (ELISA) has been developed for WNV (Blitvich et al, 2003;Choi et al, 2007;Johnson et al, 2003;Kitai et al, 2007;Long et al, 2006). These tests have the advantage of being rapid, reproducible and less expensive.…”

A Diagnostic Algorithm to Serologically Differentiate West Nile Virus from Japanese Encephalitis Virus Infections and Its Validation in Field Surveillance of Poultry and Horses

“…Poultry serum samples were screened for WNV using an IgG ELISA assay developed in-house, according to the method described by Choi and associates (Choi et al 2007). All poultry serum samples testing positive for WNV IgG were further investigated using a previously validated immunocapture WNV IgM ELISA assay to evaluate the time of infection ( Johnson et al 2003), which was modified by using WNV-reactive monoclonal antibody 5E8 (NVRQS) instead of SLE 6B6C-1.…”

Differentiation of West Nile Virus-Infected Animals from Vaccinated Animals by Competitive ELISA Using Monoclonal Antibodies Against Non-Structural Protein 1

“…The OD was measured at a wavelength of 492 nm, and was converted to PI of monoclonal antibody binding by competition with serum antibodies using the following formula: PI = [1 -(OD of serum-monoclonal antibody mixture/ OD of monoclonal antibody alone)] · 100. To compare antibody responses to the structural envelope protein (E), serum samples were simultaneously tested using an in-house IgG ELISA (NT-ELISA, an enzyme-linked immunosorbent assay using 5E8 neutralizing monoclonal antibody [NVRQS, Anyang, the Republic of Korea]), according to the method described by Choi and colleagues (Choi et al 2007).…”

“…The use of vaccines interferes with serological screening because conventional serological diagnosis of West Nile virus (WNV) uses either a virus neutralization assay or an enzyme-linked immunosorbent assay (ELISA; , Lindsey et al 1976Morens et al 1985;Wang et al 2002;Blitvich et al 2003;Choi et al 2007) to detect antibodies against the structural proteins of the virus, and they cannot distinguish between vaccinated and infected animals. A diagnostic method that distinguishes WNV-infected animals from vaccinated animals has not been established, although significant progress has been made in the development of diagnostic methods for the detection of antibodies against WNV nonstructural protein 1 (NS1; Jozan et al 2003;Hukkanen et al 2006;Lieberman et al 2007;Chung and Diamond 2008), which can indicate a present or past infection by WNV and/or other flaviviruses (Mason 1989;Winkler et al 1989;Young et al 2000;Alcon et al 2002;Libraty et al 2002;Macdonald et al 2005;Avirutnan et al 2006).…”