A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments

Abstract

A simple and sensitive radioimmunoassay using E. coli β-galactosidase as a model protein has been developed for the detection of specific translation products of foreign gene fragments cloned into plasmid or phage vectors. This immunoassay is based upon the coupling to an insoluble matrix of F(ab)′ 2 fragments derived from the specific antiserum by pepsin digestion. The in situ analysis of phage plaques or of bacterial colonies is performed by overlaying the phage plaques or lysed bacterial colonies with a cellulose filter to which F(ab)′ 2 fragments have been chemically coupled. The antigen bound to the filter is detected by subsequent incubations with undigested antiserum and with 125I-labeled Staphylococcus aureus protein A followed by autoradiography. By coupling the F(ab)′ 2 fragments to the wells of a plastic microtiter plate, liquid cultures can be analyzed quantitatively for the presence of antigen, making possible the analysis of heterogeneous cultures by sib selection. The detection threshold of the microtiter plate assay for liquid culture is shown to be <2 × 10 8 molecules, or about 1 molecule of β-galactosidase per cell. The in situ immunoassay for bacterial colonies, which permits examination of about 1000 clones per plate, can easily detect microcolonies producing about 10 molecules of β-galactosidase per cell, while the in situ phage plaque assay, also capable of screening about 1000 plaques per plate, is even more sensitive, detecting <1 × 10 7 molecules per bacteriophage plaque.