Development of Novel, Ultrasensitive and Practical Enzyme Immunoassay for Anti-Thyroglobulin Autoantibodies

新型、超灵敏且实用的抗甲状腺球蛋白自身抗体酶免疫测定法的开发

• 批准号: 02557019
• 负责人: ISHIKAWA Eiji
• 金额: $ 4.22万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557019/
• 关键词: Enzymme immunoassay; Antibody; Thyroglobulin; Anti-thyroglobulin IgG; Autoantibody; Graves' disease; Hashimoto's disease; Chronic thyroditis; 自己抗体; 酵素免疫測定法; 免疫複合体転移測定法; 自己免疫疾患; 甲状腺

A number of experiments were carried out to develop a practical and ultrasensitive enzyme immunoassay for anti - thyroglobulin IgG in serum. 1) By testing some variations of immune complex transfer enzyme immunoassay, the following version was found to be the most sensitive and practical. Anti - thyroglobulin IgG in serum was reacted simultaneously with 2, 4 - dinitrophenyl - thyroglobulin and thyroglobulin - beta - D - galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with antiN-2, 4 - dinitrophenyl group) IgG. After washing, the complex was eluted from the polystyrene balls with epsilonN-2, 4-dinitrophenyl - L - lysine and transferred to polystyrene balls coated with (anti - human IgG gamma - chain) IgG. B - D - Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. 2)NSulfosuccinimidyl - 6 - [6 - ((2, 4 - dinitrophenyl) amino) hexanoylamino] hexanoate was synthesized and used to in … More troduce 2, 4 - dinitrophenyl groups into thyroglobulin molecules more easily than before. 3) Anti - thyroglobulin IgG in serum was reacted with thyroglobulin labeled with europium ion, which can be detected more quickly than enzymes. The complex formed was trapped onto polystyrene balls coated with (anti - 2, 4 - dinitrophenyl group) IgG. After washing, the complex was eluted from the polystyrene balls with epsilon N-2, 4 - dinitrophenyl - L - lysine and transferred to polystyrene balls coated with (anti - human IgG gamma - chain) IgG. Europium ion bound to the last polystyrene balls was measured by time - resolved fluorometry. This assay was simpler than the above assay, although less sensitive with larger assay variations. 4) In the above assay with beta-D - galactosidase, the background in the presence of excess of thyroglobulin was unexpectedly high with some serum samples. This interference was suspected to be due to the presence of anti - beta - D - galactosidase antibodies in test serum. and eliminated by using inactive beta-D - D - galactosidase, making it possible to detect anti - thyroglobulin IgG in almost all healthy subjects as well as in all patients with Graves' disease and chronic thyroiditis. Less

本实验旨在建立一种实用的、超灵敏的血清抗甲状腺球蛋白IgG酶免疫分析法。1)通过测试免疫复合物转移酶免疫分析的一些变化，发现以下版本是最敏感和实用的。血清中抗甲状腺球蛋白IgG同时与2，4 -二硝基苯-甲状腺球蛋白和甲状腺球蛋白-β- D -半乳糖苷酶结合物反应.由三种组分组成的复合物被捕获到用抗N-2，4 -二硝基苯基）IgG包被的聚苯乙烯球上。洗涤后，用ε-N-2，4-二硝基苯基- L -赖氨酸从聚苯乙烯球上洗脱复合物，并转移到涂有（抗人IgG γ链）IgG的聚苯乙烯球上.通过荧光测定法测定与最后的聚苯乙烯球结合的B - D -半乳糖苷酶活性。2)N-磺基琥珀酰亚胺-6- [6 -（（2，4 -二硝基苯基）氨基）己酰氨基]己酸酯的合成 ...更多信息 在甲状腺球蛋白分子中引入2，4 -二硝基苯基比以前更容易。3)血清中的抗甲状腺球蛋白IgG与铕离子标记的甲状腺球蛋白反应，比酶检测更快。将形成的复合物捕获到用（抗2，4 -二硝基苯基）IgG包被的聚苯乙烯球上。洗涤后，用N，N-2，4 -二硝基苯基- L -赖氨酸从聚苯乙烯球上洗脱复合物，并转移到涂有（抗人IgG γ链）IgG的聚苯乙烯球上.结合到最后的聚苯乙烯球的铕离子通过时间分辨荧光法测量。该测定法比上述测定法简单，但灵敏度较低，测定法变异较大。4)在上述β-D-半乳糖苷酶测定中，在存在过量甲状腺球蛋白的情况下，某些血清样本的背景出乎意料地高。怀疑这种干扰是由于试验血清中存在抗β- D -半乳糖苷酶抗体所致。并通过使用无活性的β-D-D-半乳糖苷酶消除，使得可以在几乎所有健康受试者以及所有Graves病和慢性甲状腺炎患者中检测抗甲状腺球蛋白IgG。少

项目成果

Takeyuki Kohno,et al: "Sensitive time-resolved fluorimetric immune-complex-transfer immunoassay for antithyroglobulin IgG in serum." Journal of Clinical Laboratory Analysis. 4. 224-230 (1990)

Takeyuki Kohno 等人：“血清中抗甲状腺球蛋白 IgG 的灵敏时间分辨荧光免疫复合物转移免疫测定。”

Takeyuki Kohno Hiroyuki Katsumaru, Hidetaka Nakamoto, Taeko Yasuda, Tomohiro Mitsukawa, Shigeru Matsukura, Yoshizo Tsunetoshi and Eiji Ishikawa.: "Use of inactive beta-D-galactosidase for elimination of interference by anti-beta-D-galactosidase antibodies

Takeyuki Kohno Hiroyuki Katsumaru、Hidetaka Nakamoto、Taeko Yasuda、Tomohiro Mitsukawa、Shigeru Matsukura、Yoshizo Tsunetoshi 和 Eiji Ishikawa。：“使用非活性 β-D-半乳糖苷酶消除抗 β-D-半乳糖苷酶抗体的干扰

Takeyuki Kohno,et al.: "Sensitive timeーresolved fluorimetric immuneーcomplexーtransfer immunoassay for antithyroglobulin IgG in serum." Journal of Clinical Laboratory Analysis. 4. 224-230 (1990)

Takeyuki Kohno 等人：“血清中抗甲状腺球蛋白 IgG 的灵敏时间分辨荧光免疫复合物转移免疫测定”，《临床实验室分析杂志》4. 224-230 (1990)。

Takeyuki Kohno,et al.: "Use of inactive β-D-galactosidase for elimination of interference by anti-β-D-galactosidase antibodies in immune complex transfer enzyme immunoassay for anti-thyroglobulin IgG in serum using β-D-galactosidase from Escherichia coli

Takeyuki Kohno 等人：“使用来自埃希氏菌的 β-D-半乳糖苷酶来消除血清中抗甲状腺球蛋白 IgG 的免疫复合物转移酶免疫测定中抗 β-D-半乳糖苷酶抗体的干扰大肠杆菌

Takeyuki Kohno, Tomohiro Mitsukawa, Shigeru Matsukura and Eiji Ishikawa.: "Sensitive time-resoloved fluorimetric immune-complex-transfer immunoassay for antithyroglobulin IgG in serum." Journal of Clinical Laboratory Analysis. 4. 224-230 (1990)

Takeyuki Kohno、Tomohiro Mitsukawa、Shigeru Matsukura 和 Eiji Ishikawa.：“针对血清中抗甲状腺球蛋白 IgG 的灵敏时间分辨荧光免疫复合物转移免疫测定。”