COMPARISON OF 3-ENZYME-LINKED PROCEDURES FOR QUANTITATIVE-DETERMINATION OF GUINEA-PIG ANTI-PORCINE INSULIN ANTIBODY

Guinea pig anti-porcine insulin was determined quantitatively by three enzyme-linked procedures. (Anti-insulin immunoglobulins in the antiserum used were mostly IgG.) Procedure a: Anti-insulin to be assayed was bound to insulin immobilized on a small piece of silicone rubber and anti-insulin IgG bound onto the solid phase was allowed to react with Fab' of rabbit anti-guinea pig IgG conjugated with β-D-galactosidase from Escherichia coli. Procedure b: Anti-insulin was trapped by insulin immobilized on silicone rubber and allowed to react with insulin conjugated with β-D-galactosidase. Procedure c: IgG in anti-insulin serum was trapped by rabbit anti-guinea pig IgG antibody (IgG) immobilized on silicone rubber and anti-insulin trapped was allowed to react with insulin conjugated with β-D-galactosidase. A linear dose-response relation of β-D-galactosidase activity bound onto the solid phase to the dilution of anti-insulin serum with a buffer was observed between 3×107-fold and 9×104-fold dilutions by Procedures a and c and between 3×105-fold and 3×103-fold dilutions by Procedure b. Non-specific adsorption of normal Guinea pig IgG onto silicone rubber resulted in an excessively high blank in the assay by Procedure a when the dilution of the antiserum with buffer was less than 9×104-fold. The binding of insulin-β-D-galactosidase conjugate to the solid phase in the assay by Procedure b was significantly inhibited by unknown factor(s) in normal serum when the dilution of the serum in the assay mixture was less than 100-fold. The amounts of serum that could be used in Procedure c was limited (to 150 μ of 9×104-fold diluted serum) by the amount of anti-guinea pig IgG antibody immobilized on the solid phase. Because of these limitations, the maximal dilution of antiserum with normal serum that still gave a linear dose-response relation was 300-, 10,000-, and 1,000-fold in Procedures a, b, and c, respectively. © 1978 By The Journal of Biochemistry.