Heterobifunctional linker between antibodies and reporter genes for immunoassay development Original Research Article

The amplification inherent in transcription and translation of DNA has already been exploited for the development of highly sensitive immunoassays by using a reporter gene as a label that, upon in vitro expression, generates multiple enzyme molecules in solution (expression immunoassay). The most challenging task in the development of an expression immunoassay is to link the antibody to a reporter gene that also contains control elements for transcription/translation. In this work, we prepare heterobifunctional linkers that consist of a modified avidin or streptavidin covalently attached to an oligonucleotide (dA)40. (Strept)avidin interacts with a biotinylated detection antibody whereas the oligonucleotide hybridizes with a complementary poly(dT) tail added enzymically to the 3′ end of the reporter gene. The linker is evaluated in a model two-site (sandwich-type) immunoassay performed in microtiter wells. A 4.3 kb plasmid containing the firefly luciferase cDNA is used as a reporter.