T_4—酶联免疫测定法的的研究—Ⅰ、半抗原T4(甲状腺素)的酶标记

Methodological study on T_4-Enzyme-immunoassay Ⅰ.Enzyme-Linking Immunology assay of hapten T_4(Thyroxine)

采用戊二醛二步法使辣根过氧化物酶(HRP)与半抗原 T_4相偶联。研究了酶醛化时戊二醛的浓度,T_4和酶的克分子比与 T_4-HRP 结合物活性的关系。戊二醛的最佳浓度为 HRP 的光1600倍,T_4/酶克分子比以1∶3为宜。经纯化及分离后的 T_4-HRP 用于 EIA 考察其活性,所测光密度值维持在1.0以上,具有一定的灵敏度。用于测定血清标本所得的结果与放射免疫法测定值接近。T_4-HRP 用 SDS-PAGE 法确定的分子量约为4×10~4dal。标记效果取决于酶与戊二醛反应程度和反应物的克分子比。

Thyroxine (T_4) was coupled with horseradish Peroxidase(HRP) by a two-step linking method of glutaraldehyde.The optimal concentration of glutaraldehy-de used in the cross-linking reaction between T_4 and HRP was studied.The mo-lar ratio of T_4/HRP in relation to its activity has been investigated.Experime-ntal results showed that concentration ratio of HRP to glutaraldehyde was bestat 1∶1600 and the optimal molar ratio of T_4/HRP was 1∶3.The activity ofpurified T_4-HRP was examined.The optical density measured could be maintai-ned above 1.0,sensitivity of present method was tested。The value of T_4 measu-red for sera according to the present method met fairly good with those by RIA.The molecular weight of T_4-HRP determined by SDS-PAGE was about 4×10~4dal.