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

为建立一种实用、高灵敏度的血清中抗甲状腺球蛋白抗体的酶免疫分析方法，进行了大量的实验。1)通过对免疫复合物转移酶免疫法的几种变异体的检测，发现以下版本最灵敏、实用。2，4-二硝基苯基-甲状腺球蛋白和甲状腺球蛋白-β-D-半乳糖苷酶结合物与血清中抗甲状腺球蛋白抗体同时反应。将这三种组分形成的络合物固定在包覆有Antin-2，4-二硝基苯基(Ig)的聚苯乙烯球上。洗涤后，用N-2，4-二硝基苯基-L-赖氨酸从聚苯乙烯小球上洗脱，转移到包被(抗人Ig G伽马链)抗体的聚苯乙烯小球上。用荧光分光光度法检测与聚苯乙烯小球结合的B-D-半乳糖苷酶活性。2)合成了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，并将其用于…更容易将2，4-二硝基苯基引入甲状腺球蛋白分子。3)用Eu离子标记的甲状腺球蛋白与血清中的抗甲状腺球蛋白反应，比酶法检测更快。形成的络合物被包裹在包覆有(抗2，4-二硝基苯基)抗体的聚苯乙烯球上。洗涤后，将N-2，4-二硝基苯基-L-赖氨酸从聚苯乙烯小球上洗脱下来，转移到包覆有抗人免疫球蛋白伽马链的聚苯乙烯小球上。用时间分辨荧光法测定了最后一个聚苯乙烯小球上结合的Eu离子。这种方法比上面的方法简单，但对较大的测定变异量的灵敏度较低。4)在上述用β-D-半乳糖苷酶检测甲状腺球蛋白过量的情况下，某些血清样本的背景值出乎意料地高。这种干扰被怀疑是由于测试血清中存在抗β-D-半乳糖苷酶抗体所致。通过使用失活的β-D-D-半乳糖苷酶消除，使其能够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。较少

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