Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay

Abstract

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from the Alphavirus, Flavivirus, and Bunyavirus genera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.

FIG. 1

Standardization of serum dilution for the arboviral IgG ELISAs. Antisera to EEE, SLE, and LAC were used to represent their respective viral genera. P/N ratios for each serum dilution were derived by dividing the arithmetic mean OD of the positive serum specimens reacted with viral antigen by the mean OD of the known negative serum specimens reacted with viral antigen.

FIG. 2

Comparison of representative IgG ELISA and PRNT results. Scatter plots are of EEE, SLE, and LAC. P/N ratios were determined at a serum dilution of 1:400. A P/N ratio of <2.0 was considered to be a negative result. The PRNT values plotted are of the log₁₀ endpoint titers. A PRNT titer of <5 (<log₁₀ 0.7) was considered to be a negative result.