Expression immunoassay based on antibodies labeled with a deoxyribonucleic acid fragment encoding the α-peptide of β-galactosidase

Abstract

An immunoassay is reported which uses, as a label, an expressible DNA fragment encoding the alpha-peptide of beta-galactosidase. This inactive peptide consists of 97 amino acid residues containing an amino-terminal portion of the enzyme. Antigen (an anti-thyrotropin immunoglobulin) immobilized in microtiter wells is allowed to react with specific antibodies which are then linked to the DNA label via biotin-streptavidin interaction. After completion of the immunoreaction, the solid phase bound DNA is subjected to a cell-free, one-step transcription/translation reaction to produce the alpha-peptide. The alpha-peptide is allowed 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 substrate. 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 (within-run) obtained for 8 and 20 fmol of antigen were 10.7 and 9.3%, respectively (n = 4). The present work illustrates the utility of expressing a non-detectable peptide capable of triggering a signal generating system.