AbstractA novel enzyme immunoassay of anti‐insulin IgG in human serum is described. A serum sample containing anti‐insulin IgG was treated with dextran‐charcoal at pH 6.0 to remove endogenous insulin and subsequently incubated with dinitrophenyl biotinyl nonspecific rabbit IgG‐insulin conjugate. The reaction mixture was further incubated with a rabbit (antidinitrophenyl bovine serum albumin) IgG‐coated polystyrene ball to trap the complex formed between anti‐insulin IgG and the conjugate. After washing to eliminate nonspecific IgG in the test serum, the polystyrene ball was incubated with dinitrophenyl‐L‐lysine to elute the complex. The eluate was incubated with an avidin‐coated polystyrene ball. Finally, the amount of human anti‐insulin IgG in the complex trapped onto the avidin‐coated polystyrene ball was measured by incubation with rabbit (antihuman IgG (γ‐chain)) Fab'‐peroxidase conjugate. This enzyme immunoassay was 1,000‐fold more sensitive than the conventional enzyme immunoassay, in which an insulin‐bovine serum albumin‐coated polystyrene ball was incubated with a serum sample containing anti‐insulin IgG and subsequently with rabbit (antihuman IgG (γ‐chain)) Fab'‐peroxidase conjugate. The principle of the novel enzyme immunoassay can be used to more sensitively measure antibodies for most kinds of haptens and antigens t