EXPRESSION IMMUNOASSAY BASED ON ANTIBODIES LABELED WITH A DEOXYRIBONUCLEIC-ACID FRAGMENT ENCODING THE ALPHA-PEPTIDE OF BETA-GALACTOSIDASE

An immunoassay is reported which uses, as a label, an expressible DNA fragment encoding the alpha-peptide of beta-galactosidase, This inacti ve peptide consists of 97 amino acid residues containing an amino-term inal portion of the enzyme. Antigen (an anti-thyrotropin immunoglobuli n) immobilized in microtiter wells is allowed to react with specific a ntibodies which are then linked to the DNA label via biotin-streptavid in interaction. After completion of the immunoreaction, the solid phas e bound DNA is subjected to a cell-free, one-step transcription/transl ation reaction to produce the alpha-peptide. The alpha-peptide is allo wed to react (complementation reaction) with the remaining part of the beta-galactosidase (M15 protein, also inactive) to give fully active enzyme molecules, 4-Methylumbelliferyl galactoside is used as a substr ate. The fluorescence is linearly related to the amount of antigen in the well. As little as 3 fmol of antigen can be detected. The RSDs (wi thin-run) obtained for 8 and 20 fmol of antigen were 10.7 and 9.3 %, r espectively (n = 4). The present work illustrates the utility of expre ssing a non-detectable peptide capable of triggering a signal generati ng system.