Enzyme-linked immunoassay: conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from E. coli and its use for immunoassay.
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.
D000818:Animals; D001666:Binding Sites, Antibody; D004926:Escherichia coli / Q000276:immunology*; D005696:Galactosidases / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G / Q000378:metabolism*; D066298:In Vitro Techniques; D008301:Maleimides / Q000378:metabolism; D011817:Rabbits
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008301:Maleimides; D005696:Galactosidases