Basic Study on Development of Ultrasensitive Enzyme Immunoassay for Antibodies

抗体超灵敏酶联免疫分析技术发展的基础研究

• 批准号: 62570119
• 负责人: ISHIKAWA Eiji
• 金额: $ 1.34万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570119/
• 关键词: Insulin; Anti-insulin IgG; Enzyme immunoassay; Antibody; Radioimmunoassay; 糖尿病

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

建立了一种超灵敏的血清抗胰岛素IgG酶免疫分析方法。将试验血清与胰岛素一起孵育，葡聚糖-活性炭处理以除去游离胰岛素，用聚乙二醇沉淀胰岛素-抗胰岛素IgG复合物，对沉淀物进行酸处理以除去抗胰岛素IgG，并通过超灵敏夹心酶免疫测定法测量胰岛素。血清中抗胰岛素IgG的检测限比使用13 I标记胰岛素的放射免疫测定法低10- 30倍<125>，比常规酶免疫测定法低1，000 - 3，000倍，在常规酶免疫测定法中，胰岛素包被的聚苯乙烯球与测试血清和（抗IgG）Fab '-过氧化物酶缀合物一起孵育。用此方法检测了用胰岛素治疗0.6 ~ 10个月的糖尿病患者血清中抗胰岛素IgG抗体的阳性率为89%，而常规酶法仅占3%。 ...更多信息 免疫测定法。抗胰岛素IgG在治疗前的一些I型糖尿病患者中也被证明，尽管在非常低的水平。为了以高灵敏度测量其他抗原的血清抗体，还开发了使用抗胰岛素IgG作为抗体模型的替代技术。将试验血清与胰岛素孵育，并上样至（抗IgG）IgG-琼脂糖4 B柱，以将胰岛素-抗胰岛素IgG复合物与游离胰岛素分离。用酸从柱中洗脱胰岛素，并在抗胰岛素IgG灭活后，通过超灵敏夹心酶免疫测定法测定。敏感性是令人满意的，这种技术的适用性支持的发现，如生长激素和促甲状腺激素的抗原的抗原性，保留酸处理后。对于抗半抗原抗体的测量，从各种考虑中得出结论，与先前开发的技术不同的技术仍有待开发。少