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 α-peptide of β-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 α-peptide. The α-peptide is allowed to react (complementation reaction) with the remaining part of the β-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.