1. Insulin, rabbit IgG and its Fab' fragments were coupled to β-D-galactosidase (EC 3. 2. 1. 23) from Escherichia coli by a two-step procedure with aid of the reaction between sulfhydryl groups and maleimide residues. Maleimide residues were introduced into insulin molecules with N, N'-o-phenylenedimaleimide after mercaptosuccinylation or directly with the active ester of 4-(maleinimidomethyl) cyclohexane-1-carboxylic acid or 4-(maleinimidomethyl) benzoic acid. Maleimide residues were also introduced into molecules of reduced IgG or Fab' fragments using N, N'-o-phenylenedimaleimide. Maleimide residues attached to molecules of insulin, IgG or Fab' fragments were allowed to react with sulfhydryl groups of β-D-galactosidase.
2. Sepharose 4B, glass rods and silicone pieces were used as solid phase. Rabbit IgG was coupled to CNBr-activated Sepharose 4B.
Small rods of aminoalkylsilyl glass were loaded covalently (using glutaraldehyde) or noncovalently with insulin, rabbit IgG or its (Fab')2 fragments. Small rods of plainglass or small silicone pieces were also loaded with rabbit IgG or its (Fab')2 fragments by simpleadsorption.
3. The enzymoimmunoassay was performed by using the insulin- or antibody-β-D-galactosidase complex and the insulin- or antibody-loaded solid phases. Antigens and antibody subjected to the assay were insulin, anti-insulin antibody, ornithine δ-aminotransferase (EC 2. 6. 1. 13) from rat liver, human IgG, 2, 4-dinitrophenyl human IgG and α-fetoprotein.
4. Studies performed thus far indicated that attomole (10-18 mole) quantities of antigens could be measured with presently available techniques if antibodies of adequate quality were provided.
