A More Sensitive Enzyme Immunoassay of Anti-Insulin IgG in Guinea Pig Serum with Less Non-Specific Binding of Normal Guinea Pig IgG

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Abstract
An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20°C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37°C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to β-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem.84, 93–102).

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