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This paper describes an improved sandwich enzyme immunoassay for insulin in human serum. The detection limit was significantly improved from 0.1 mU/L to 0.02 mU/L, by incubation with guinea-pig anti-insulin Fab'-peroxidase conjugate in the presence of normal guinea-pig F(ab')2 to reduce the non-specific binding of the conjugate. The degree of serum interference was considerably reduced by coating polystyrene balls with acid-treated anti-insulin IgG and by incubating the polystyrene balls with serum samples at a lower temperature for a shorter time. The time for incubation with anti-insulin Fab'-peroxidase conjugate was also shortened. As a result, the volume of serum that could be used increased from 10 microliters to 50 microliters, and the time for immuno-reactions was reduced from 8 h to 4 h. Applicability of the present improvement to enzyme immunoassay of other antigens in human serum is discussed.