ENZYME-LINKED IMMUNOASSAY - CONJUGATION OF FAB' FRAGMENT OF RABBIT IGG WITH BETA-D-GALACTOSIDASE FROM ESCHERICHIA-COLI AND ITS USE FOR IMMUNOASSAY

Abstract

The conjugation method consists of 2 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 .beta.-D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab''-.beta.-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''-.beta.-D-galactosidase complex for immunoassay of macromolecular antigens is shown by measuring human IgG [immunoglobulin G] 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 ficomol of human IgG.