Matsumoto T, Morita M, Shirai H, Kishi T
Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Osaka, Japan.
Biochem Med Metab Biol. 1993 Apr;49(2):164-72. doi: 10.1006/bmmb.1993.1019.
The development of a sandwich enzyme immunoassay for rat retinol-binding protein using molecular biological techniques was described. Rat retinol-binding protein gene cloned by the PCR method was expressed by a fusion vector pEZZ18 in Escherichia coli strain HB101. A recombinant retinol-binding protein fused with IgG-binding domain ZZ of protein A was purified with IgG-Sepharose. Antibody against the recombinant protein was found to be specific to rat retinol-binding protein in plasma by immunoblot analysis. Affinity-purified anti-recombinant protein IgG was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range is 1.9-60 ng/ml and the coefficients of variation within and between the assay series (assay range: 4-30 ng/ml) are 4.30 +/- 4.33 and 5.32 +/- 1.45%, respectively. Cross-reactivity of the immunoassay was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse serum. In an in vitro experiment, retinol-binding protein produced by rat hepatocytes could be measured by the sandwich enzyme immunoassay.
描述了一种使用分子生物学技术开发的用于大鼠视黄醇结合蛋白的夹心酶免疫测定法。通过PCR方法克隆的大鼠视黄醇结合蛋白基因由融合载体pEZZ18在大肠杆菌HB101菌株中表达。与蛋白A的IgG结合结构域ZZ融合的重组视黄醇结合蛋白用IgG-琼脂糖纯化。通过免疫印迹分析发现，针对重组蛋白的抗体对血浆中的大鼠视黄醇结合蛋白具有特异性。亲和纯化的抗重组蛋白IgG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1.9 - 60 ng/ml，测定系列内和系列间（测定范围：4 - 30 ng/ml）的变异系数分别为4.30 +/- 4.33和5.32 +/- 1.45%。使用牛、人及小鼠血清检测了免疫测定的交叉反应性。仅与小鼠血清存在交叉反应。在体外实验中，大鼠肝细胞产生的视黄醇结合蛋白可通过夹心酶免疫测定法进行测量。
