Removal of species constraints in antibody detection

Abstract

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.

FIG. 1.

Receiver operator characteristic curves for WN, SLE, and EEE viral antigens in the biotin-MIAs. The sensitivities and the specificities for each antigen were calculated by using the results of PRNT as the gold standard. Areas under the curve and 95% confidence intervals are shown.

FIG. 2.

V/N ratios for samples used to develop the tests are shown. •, PRNT negative; +, PRNT positive; dashed vertical lines, positive V/N cutoff for that antigen. Species are listed in the following order: birds (the normal type above the boldface type). mammals (boldface type), and reptile (the normal type below the boldface type). (A) WN/SLE virus biotin-MIA. Results are shown only for known negative results and positive results for the homologous viral antigens. (B) EEE virus biotin-MIA.

FIG. 3.

Cross-reactivity of the WN/SLE virus biotin-MIA (a) and the EEE virus biotin-MIA (b) with antisera to other confirmed arboviral infections. No dual infections were included in the analyses. The cutoffs for a positive reaction were V/N ratio values of 10.0 for WN virus, 10.23 for SLE virus, and 8.97 for EEE virus.