Matsumoto T, Morita M, Shirai H, Kishi T
Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Osaka, Japan.
Biosci Biotechnol Biochem. 1993 Mar;57(3):414-8. doi: 10.1271/bbb.57.414.
A sandwich enzyme immunoassay for rat transthyretin using a recombinant antigen approach is described. Rat transthyretin cDNA was cloned by PCR. Rat transthyretin fused with the IgG-binding ZZ domain of protein A was expressed in E. coli using the fusion vector pEZZ18. Antibody against the fusion protein was specific to rat transthyretin in plasma as shown by immunoblotting. Affinity-purified anti-rat transthyretin-ZZ was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range was 1.2-50 ng/ml and the coefficients of variation within and between the assay series (assay range: 2.5-20 ng/ml) were 1.31 +/- 0.08% and 3.50 +/- 1.46%, respectively. Cross-reactivity was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse transthyretin. In an in vitro experiment, transthyretin secreted by rat hepatocytes could be measured by the sandwich enzyme immunoassay.
描述了一种使用重组抗原方法检测大鼠甲状腺素转运蛋白的夹心酶免疫测定法。通过聚合酶链反应（PCR）克隆大鼠甲状腺素转运蛋白cDNA。使用融合载体pEZZ18在大肠杆菌中表达与蛋白A的IgG结合ZZ结构域融合的大鼠甲状腺素转运蛋白。免疫印迹显示，针对融合蛋白的抗体对血浆中的大鼠甲状腺素转运蛋白具有特异性。亲和纯化的抗大鼠甲状腺素转运蛋白-ZZ被生物素化，并用于夹心酶免疫测定。在该测定中，可测量范围为1.2-50 ng/ml，测定系列内和系列间（测定范围：2.5-20 ng/ml）的变异系数分别为1.31±0.08%和3.50±1.46%。使用牛、人及小鼠血清检测交叉反应性。仅与小鼠甲状腺素转运蛋白存在交叉反应。在体外实验中，大鼠肝细胞分泌的甲状腺素转运蛋白可通过夹心酶免疫测定法进行检测。
