Enzyme-linked immunosorbent assays using recombinant envelope protein expressed in COS-1 and Drosophila S2 cells for detection of West Nile virus immunoglobulin M in serum or cerebrospinal fluid.

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.

Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay.

BACKGROUND: Currently, the detection of HCV infection in blood donors relies on the ability of immunoassays to detect circulating HCV antibodies. However, a significant delay exists between the time of infection and the development of antibodies. This delay (window period) can last up to 70 days. The introduction of NAT for the detection of HCV RNA has reduced this window period dramatically. However, NAT is labor intensive, prone to contamination, and expensive as compared with standard serologic tests. STUDY DESIGN AND METHODS: An automated, microparticle-based chemiluminescent assay for the detection of HCV core antigen in human serum and plasma was developed. The specificity and sensitivity of this prototype assay were evaluated by testing a population of normal blood donors and commercially available seroconversion panels. RESULTS: The HCV core antigen assay exhibited a 99.9-percent specificity by detecting a single repeatably reactive sample out of 1004 normal donors tested. Assay sensitivity was determined by comparing the HCV core antigen detection rate with the antibody seroconversion profile and the rate of HCV RNA detection. Among 15 seroconversion panels examined, core antigen was detected in 69 of 70 antibody-negative and/or RNA-positive samples for a sensitivity relative to NAT of 98.6 percent. CONCLUSION: These data indicate that the automated, microparticle-based chemiluminescent HCV core antigen assay can reduce the window period for detection of potentially infected blood donors by 32.7 days, and it represents a viable alternative to HCV RNA testing.