Detection of human anti-flavivirus antibodies with a west nile virus recombinant antigen microsphere immunoassay

Abstract

We report a new, suspended-microsphere diagnostic test to detect antibodies to West Nile (WN) virus in human serum and cerebrospinal fluid (CSF). The microsphere immunofluorescence assay can be performed in less than 3 h on specimens of <or=30 microl. A recombinant WN virus envelope (E) protein antigen is covalently coupled to fluorescent polystyrene microspheres. After incubation with diluted serum or CSF, antibodies bound to the E protein antigen are detected with fluorescently labeled anti-human immunoglobulin antibody and flow analysis in a dual-laser Luminex 100 instrument. Retrospective testing of 833 sera from New York patients with suspected viral encephalitis demonstrated concordance with results obtained with the traditional enzyme-linked immunosorbent assay for immunoglobulin G (IgG) antibodies to WN virus (kappa = 0.85). One hundred eighty-eight (22.4%) of the samples, which were collected from June to November 2002, tested positive for antibodies to WN virus in the microsphere assay. Specimens depleted of IgG with anti-IgG antibody were reassayed to measure anti-E protein IgM antibodies and to provide an indication of current or recent WN virus infection. The assay also detects antibodies to E proteins from related flaviviruses, including St. Louis encephalitis, Japanese encephalitis, and dengue viruses. The new microsphere immunoassay provides a sensitive and rapid alternative to traditional enzyme-linked immunosorbent assays that detect antibodies to flavivirus E proteins. This assay can aid physicians and public health workers in the management of outbreaks of WN virus and related flaviviruses.

FIG. 1.

rWNV-E MIA analysis of serially diluted serum specimens. Sera from patients with WN virus infection (closed symbols), and negative control human sera (open symbols) were serially diluted and evaluated in the rWNV-E MIA with a polyvalent detector antibody. Results are reported as MFI per 100 microspheres.

FIG. 2.

rWNV-E MIA and ELISA analyses of anti-WN virus antibodies in sequential serum specimens from a patient infected with WN virus. (A) Unadsorbed sera were evaluated in the rWNV-E MIA with a polyvalent detector antibody. Sera adsorbed with anti-IgG (IgG adsorbed) or anti-IgM (IgM adsorbed) were evaluated in the rWNV-E MIA with a polyvalent detector antibody. The IgM-adsorbed sera were also analyzed in the rWNV-E MIA with an anti-IgM detector antibody (M conjugate). (B) Comparison of results with the MAC-ELISA and indirect IgG ELISA with sequential sera.

FIG. 3.

Retrospective parallel rWNV-E MIA and WN virus IgG ELISA analyses of sera from patients with suspected viral encephalitis. Dashed lines indicate P/N cutoff values for a positive result (n = 702, r² = 0.60, and slope = 1.68).