Basic Study on Development of Ultrasensitive Enzyme Immunoassay for Antibodies

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An ultrasensitive enzyme immunoassay technique for anti-insulin IgG in serum was developed. Test serum was subjected to successive processes of incubation with insulin, dextran-charcoal treatment to remove free insulin, precipitation of insulin-anti-insulin IgG complex by polyethylene glycol, acid-treatment of the precipitates to inactivate anti-insulin IgG and measurement of insulin by ultrasensitive sandwich enzyme immunoassay. The detection limit of anti-insulin IgG in serum was 10- to 30-fold lower than that by radioimmunoassay using ^<125>I-labeled insulin and 1,000- to 3,000-fold lower than that by the conventional enzyme immunoassay, in which an insulin-coated polystyrene ball was incubated with test serum and (anti-IgG) Fab'-peroxidase conju-gate. By this technique, anti-insulin IgG was demonstrated in most (89 %) of serum samples from diabetic patients who had been treated with insulin for 0.6-10 months, while only a small proportion (3 %) was positive by the conventional enzy … More me immunoassay. Anti-insulin IgG was also demonstrated in some of type I diabetic patients before treatment, although at very low levels. In order to measure serum antibodies for other antigens with high sensitivity, an alternative technique was also developed using anti-insulin IgG as an antibody model. Test serum was incubated with insulin and applied to a column of (anti-IgG) IgG-Sepharose 4B to separate insulin-anti-insulin IgG complex from free insulin. Insulin was eluted from the column with acid and, after inactivation of anti-insulin IgG, measured by ultrasensitive sandwich enzyme immunoassay. The sensitivity was satisfactory, and the applicability of this technique was supported by the finding that the antigenicity of antigens such as growth hormone and thyroid-stimulating hormone was retained after acid-treatment. For the measurement of anti-hapten antibodies, it has been concluded from various considerations that techniques distinct from previously developed ones remain to be developed. Less