Development of a high throughput, semi-automated, infectious center cell-based ELISA for equine infectious anemia virus

Abstract

A faster semi-automated 96-well microtiter plate assay to determine viral infectivity titers, or viral focal units (vfu), of equine infectious anemia virus (EIAV) stocks is described. Optimization of the existing method modernizes a classic virological technique for viral titer determination by quantitating EIAV in experimentally infected cells via a cell-based ELISA. To allow for automation, multiple parameters of the current assay procedures were modified resulting in an assay that required only one quarter the original amount of virus and/or serum for infectivity or neutralization assays, respectively. Equivalent reductions in the required volumes of tissue culture, cell processing, and protein detection reagents were also achieved. Additionally, the new assay decreased the time required from start to finish from 10 days to 6 days (viral titer) or 7 days (viral neutralization), while increasing the number of samples that can be processed concurrently by transition to a 96-well microtiter plate format and by automated counting.