Removal of Species Constraints in Antibody Detection

Serum antibodies from myriad species, particularly birds, can provide key information regarding the transmission and the expansion of the territory of emerging pathogens. Expedient antibody analysis is constrained by a lack of species-specific reagents, a deficiency potentially highlighted by the recent swine-origin influenza A virus (H1N1) outbreak. Available methodologies present difficulties that discourage thorough serologic monitoring of potential disease vectors or hosts. Rapid high-throughput procedures that combined serum amine labeling via biotinylation, contaminant removal, and microsphere-based immunoassays for antibodies to three arboviruses were developed. Agent-specific adaptations of this simple format should facilitate expanded surveillance and diagnostic capabilities regarding pathogens of human and veterinary importance.Serologic analysis of samples obtained from nondomestic animals, birds in particular, can provide key information regarding the transmission and the expansion of the territory of emerging pathogens (1). Unfortunately, species-specific reagents such as antispecies capture antibodies and detection conjugates, necessary for most rapid diagnostic methods, are frequently unavailable due to a lack of commercial interest. Traditional methods for circumventing species constraints often use live pathogens, are technically challenging, or are so time-consuming that large-scale testing becomes impractical (2). Methods that utilize protein G (4) are able to capture and detect mammalian IgG but cannot be used when the antibodies of interest are IgM and IgY, the former being the first of the Igs to be generated after infection and the latter being the avian and reptilian equivalent of IgG. The results of blocking enzyme-linked immunosorbent assay (ELISAs) (3) are sometimes inconsistent, and test sensitivities are dependent on the relative affinities of the competing monoclonal and serum antibodies to the antigens. Disease surveillance that involves the analysis of antibodies in nondomestic species is thus limited.The ideal method for the detection of antibodies in a range of species would be rapid, be capable of high throughput, provide a positive signal (as opposed to a signal reduction via competition), and require a small sample volume. Total antibody measurement would be advantageous, because surveillance is usually conducted without knowledge of the timing of infection. Biotin (vitamin H) is well-known to react with free amine groups on proteins (18). We determined that virus-specific serum antibodies independent of the species of origin could be biotinylated and directly detected in microsphere immunoassays (biotin-MIAs) that were modified from established protocols (11). Here we describe the development of two species-independent antibody detection methods for use with arboviruses, which involve animals as vectors or hosts. The first is a duplex procedure for the detection of antibodies to West Nile (WN) and St. Louis encephalitis (SLE) viruses, and the second is a procedure for the identification of anti-eastern equine encephalitis (anti-EEE) virus antibodies.