Basic Study on Development of Ultrasensitive Enzyme Immunoassay for Antibodies

建立了血清中抗胰岛素IgG的超灵敏酶免疫测定方法。试验血清经胰岛素培养、葡聚糖-木炭处理去除游离胰岛素、聚乙二醇沉淀胰岛素-抗胰岛素IgG复合物、沉淀物酸处理灭活抗胰岛素IgG、超灵敏三明治酶免疫分析法测定胰岛素。血清中抗胰岛素IgG的检出限比使用^<125> i标记胰岛素的放射免疫分析法低10- 30倍，比使用胰岛素包被的聚苯乙烯球与试验血清和（抗胰岛素IgG） Fab'-过氧化物酶偶联物孵卵的传统酶免疫分析法低1000 - 3000倍。在胰岛素治疗0.6 ~ 10个月的糖尿病患者血清中，大多数（89%）血清中检测到抗胰岛素IgG，而传统的酶联免疫分析法仅小部分（3%）血清中检测到抗体阳性。一些I型糖尿病患者在治疗前也检测到抗胰岛素IgG，尽管水平很低。为了高灵敏度地测定其他抗原的血清抗体，还开发了一种以抗胰岛素IgG为抗体模型的替代技术。试验血清与胰岛素孵育，并应用于（抗IgG） IgG- sepharose 4B柱，从游离胰岛素中分离胰岛素-抗胰岛素IgG复合物。用酸从柱中洗脱胰岛素，抗胰岛素IgG失活后，用超灵敏夹心酶免疫分析法测定。敏感性令人满意，酸处理后，生长激素和促甲状腺激素等抗原的抗原性得以保留，这一发现支持了该技术的适用性。对于抗半抗原抗体的测量，从多方面考虑得出结论，与以前开发的技术不同的技术仍有待开发。少

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