Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

Abstract

Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.

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“…The levels of WNV-specific IgM antibodies were measured in the serum using microsphere immunoassay (MIA) for WNV envelope (E) protein (L2 Diagnostics), as described previously (34,35). Briefly, serum samples were incubated with magnetic microspheres (MagPlexTM-C) coated with a recombinant WNV E antigen for 30 min, followed by secondary goat anti-mouse IgG or Measurement of IFN-␣, cytokines, and chemokines.…”

Multiple Immune Factors Are Involved in Controlling Acute and Chronic Chikungunya Virus Infection

“…1 , MHCII Δ/Δ ). Whether this was due to IgM responses (intact in MHCII Δ/Δ mice) or IgG2c responses remains unresolved, with IgG responses detected in mice using sensitive techniques as early as day 3 post viral infection [58] .…”

Infection with Non-Lethal West Nile Virus Eg101 Strain Induces Immunity that Protects Mice against the Lethal West Nile Virus NY99 Strain

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