Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids

The monoclonal antibody solution hybridization assay is a novel enzyme immunoassay for detection of RNA with a biotinylated DNA probe. To increase the sensitivity of this test, a fluorescent substrate and an enzymatic amplification cycling system were compared with a conventional colorigenic substrate for alkaline phosphatase. The fluorescent, cycling, and colorigenic substrates detected, respectively, 10, 10, and 100 amol of unbound alkaline phosphatase in 2 h. With a prolonged incubation period of 16.6 h, the conventional substrate measured 10 amol of the enzyme. In the immunoassay for RNA detection, the fluorescence and cycling assays were faster than that using the colorigenic substrate and reached an endpoint sensitivity of 3.2 pg/ml (0.16 pg per assay) of cRNA. However, longer incubation periods (16.6 h) for optimal generation of the colorigenic product led to a comparable level of sensitivity for the conventional substrate.