Expression immunoassay.

Abstract

Expression immunoassay is a sensitive analytical method that takes advantage of coupled in vitro transcription and translation as a signal amplification technique. Essentially, the immunoassay is performed using a detection antibody that is labeled with an expressible fragment of DNA. The product of expression is a protein that can be used to generate a signal. Here we describe two distinct expression immunoassays; both are based on expression of DNA labels to produce active enzyme molecules which are subsequently detected through their enzymatic activities. The luciferase expression immunoassay uses a 2.1-kb DNA template as a reporter molecule. The DNA is attached to a biotinylated detection antibody via biotin-streptavidin linkage. After the detection antibody is immunoreacted with immobilized antigen and excess antibody is removed, the DNA label is expressed in vitro. A linear relationship exists between the bioluminescent signal, from luciferase activity, and the immobilized antigen. This expression immunoassay allows the detection of 5 x 10(4) antigen molecules. The second expression immunoassay makes use of the well-characterized alpha-complementation of beta-galactosidase by employing an alpha-peptide encoding DNA as a reporter molecule. By monitoring the resulting beta-galactosidase activity with a fluorogenic substrate it was possible to detect as little as 3 fmol of immobilized antigen. Both expression immunoassays are amenable to automation and demonstrate the potential sensitivity that can be achieved using in vitro expression as a signal amplification method.