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Enzyme-Linked Immunoassay: Conjugation of the Fab' Fragment of Rabbit IgG with -D-Galactosidase from E. Coli and its Use for Immunoassay

From the Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan

Abstract

1. A method for the conjugation of the Fab' fragment of rabbit IgG with -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 -D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab'--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.

2. The application of the rabbit Fab'--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.

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