A novel, spectrophotometric microneutralization assay for respiratory syncytial virus

We describe a simple and rapid microneutralization assay for respiratory syncytial virus (RSV) based on the colorimetric quantitation of the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a formazan product by the mitochondria of viable cells. Conditions for RSV infectivity were first optimized for sensitivity and reproducibility based on cell density and on RSV concentration as a function of multiplicity of infection (MOI) and time post-infection and the resulting optical densities were shown to be inversely proportional to MOI. For RSV neutralization, dilutions of heat-inactivated human plasma were preincubated with RSV and complement prior to infection of cells in microtiter plates. Following MTT dye conversion, 50% RSV neutralization titers were determined by linear regression analysis of the optical density values and endpoints were markedly influenced by MOI. The MTT-based assay was shown to be comparably sensitive to the plaque reduction assay for quantitation of neutralizing antibody, but more readily adaptable to the screening of a large number of samples. Finally, we demonstrated that the MTT microneutralization assay for RSV was useful for quantitation assay of neutralization activity in sera of mice and cotton rats.