Developing an Antibody-Based MAV-1 Detection Assay
Quantitating infectious mouse adenovirus type 1 (MAV-1) particles requires cumbersome titration via in vitro plaque assay. Results obtained with the current protocol rely on macroscopic detection of visible plaques and can take up to 12 days. We describe here the development of an immunologically-based assay that shortens the input and endpoint time while increasing specificity. Input time is decreased using a higher throughput 24-well format versus traditional 60 mm plates and by eliminating the necessity of steps following viral infection. Endpoint time is shortened to 7 days. Immunodetection of MAV-1 using primary antibodies against one or more MAV-1 capsid proteins introduces specificity and subsequently may offer increased sensitivity. A secondary antibody linked to horseradish peroxidase (HRP) is then bound to the primary antibody and developed by chromogenic detection using a chloronapthol substrate. HRP-amplified detection resulted in simple identification of bluish-purple stained, MAV-1-specific plaques at 7 days post-infection. No background staining was observed in mock infected samples. Results are reported in log scale denoted as plaque forming units and are within one log of known viral titers quantified by traditional plaque assay. With optimization, this immunologically-based assay may serve as an effective and more efficient viral quantitation tool and may help illuminate previously undetectable trends in MAV-1 infection.