Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Quantifying Antibodies to Japanese Encephalitis Virus Nonstructural 1 Protein To Detect Subclinical Infections in Vaccinated Horses

Abstract

Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

“…Although the mechanism behind this is currently unknown, the diagnostic performances between antiprM/E and NS1 detections were similar. Previous studies report that anti-NS1 antibodies are present in low levels in serum after flavivirus infection, hence, its poor detection (10,31). This might be due to a relative abundance of anti-prM/E antibodies, which results in lower sensitivity of NS1 isotype assays.…”

“…Antibodies to nonstructural protein 1 (NS1) have been proposed as serological markers of natural infection among inactivated Japanese encephalitis virus (JEV)-vaccinated populations, and an NS1-ELISA has been developed (9)(10)(11). However, this NS1-ELISA required that either the microplates be sensitized with purified NS1 or NS1 antigen be captured by NS1-specific murine monoclonal antibodies, before adding test human serum specimens.…”

Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans

“…The novelty in our NS1-MAC-ELISA is the preabsorption of the patient serum with VLP antigens to deplete the anti-prM/E antibodies. This is based on the rationale that the binding of a low level of anti-NS1 antibodies, which are captured on the MAC-ELISA plate coated with goat anti-human IgM antibodies, to the NS1 antigens would be dramatically hampered by the presence of relatively abundant anti-prM/E IgM antibodies; hence, we see its poor detectability, particularly in asymptomatic cases (16,33,34). Furthermore, detection of the formed prM/E antibody-VLP antigen immune complexes in the preabsorption plate demonstrated that a single preabsorption step sufficiently depleted the serum antiprM/E antibodies and consequently allowed for the optimal detection of anti-NS1 antibodies.…”

Identification of Circulating Natural Antibodies against Endogenous Mediators in the Peripheral Blood Sera of Patients with Osteoarthritis of the Knee: A New Diagnostic Frontier

“…Improved analytical methods are required to accommodate the analysis of large numbers of samples for biological and epidemiological monitoring [44,[107][108][109][110][111][112]. A sensitive assay to identify markers that can accurately determine the onset of osteoarthritis-especially if the technique is of low risk to the patient, such as blood drawing -is ideal for early detection of osteoarthritis of the knee.…”