Date Published: September 25, 2012
Publisher: Public Library of Science
Author(s): Madhuri Namekar, Mukesh Kumar, Maile O’Connell, Vivek R. Nerurkar, Robyn Klein. http://doi.org/10.1371/journal.pone.0045851
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.
West Nile virus (WNV), a mosquito-borne flavivirus that causes lethal encephalitis, has emerged as a significant cause of viral encephalitis in the United States . Although, WNV infection in humans is mainly acquired after mosquito bite, human-to-human transmission can occur through blood transfusion, organ transplantation and breastfeeding , . Currently, no antiviral or vaccine is available to counteract or protect against WNV infection in humans . WNV immunopathogenesis studies in animal models such as mice provide important information for the development of antivirals and vaccines against WNV infection in humans. In WNV-infected mice, IgM and IgG antibodies are produced early after the infection and persist for a long time. These WNV-specific antibodies limit viremia and dissemination of virus into the CNS and provide protection against lethal infection . Induction of these antibodies is also a critical determinant for the efficacy of WNV vaccines . Therefore, it is important to detect low levels of both anti-WNV IgM and IgG antibodies during early and late phase of the infection. WNV E-protein enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT) have been used for detection of both anti-WNV IgM and IgG antibodies, and WNV-neutralizing antibodies, respectively, in mice . Though these assays are sensitive and specific, they are expensive and time consuming. Luminex-based microsphere immunoassays (MIA) have been developed and used for detection of anti-WNV antibodies in humans and in mice model using purified recombinant proteins (E, NS3 and NS5) of WNV –. WNV E-MIA is sensitive, cost-effective and requires less time than traditional ELISA and PRNT assays for detection of anti-WNV antibodies , . MIA has also been used for improved serological detection of several other viruses such as respiratory syncytial virus , HIV , WNV , , , human papillomaviruses , equine arteritis virus , and avian influenza virus .