Enzyme-Linked Immunoassay: Conjugation of the Fab′ Fragment of Rabbit IgG with <i>β</i>-D-Galactosidase from E. Coli and its Use for Immunoassay

Abstract

Abstract A method for the conjugation of the Fab′ fragment of rabbit IgG with β-D-galactosidase from Escherichia coli is described. The method consists of two main steps: treatment of the Fab′ fragments containing sulfhydryl groups with excess N,N′-o-phenylenedimaleimide, to introduce maleimide residues into the fragments, and then incubation of the dimaleimide-treated Fab′ fragments with β-D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab′-β-d-galactosidase complex. More than 90% of the enzyme used can be converted to the Fab′-enzyme complex, and the complex is readily separated from free Fab′ fragments by chromatography on a Sepharose 6B column. The application of the rabbit Fab′-β-D-galactosidase complex for immunoassay of macromolecular antigens is shown by measuring human IgG by the sandwich method. The rabbit (anti-human IgG) IgG-coupled Sepharose 4B is incubated with human IgG and then with the rabbit (anti-human IgG) Fab′-enzyme complex, and the enzyme activity bound to the Sepharose is measured. In this way it is possible to determine as little as 0.3 fmoles of human IgG.