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  • Title: Enzyme-linked immunoassay: conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from E. coli and its use for immunoassay.
    Author: Kato K, Fukui H, Hamaguchi Y, Ishikawa E.
    Journal: J Immunol; 1976 Jun; 116(6):1554-60. PubMed ID: 774986.
    Abstract:
    1. A method for the conjugation of the Fab' fragment of rabbit IgG with beta-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 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. 2. The application of the rabbit Fab'-beta-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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