使用葡萄糖-6-磷酸脱氢酶的生物发光测定法：在生物素和链霉亲和素检测中的应用。

Bioluminescent assays using glucose-6-phosphate dehydrogenase: application to biotin and streptavidin detection.

作者信息

Térouanne B, Bencheick M, Balaguer P, Boussioux A M, Nicolas J C

机构信息

INSERM Unité 58, Montpellier, France.

出版信息

Anal Biochem. 1989 Jul;180(1):43-9. doi: 10.1016/0003-2697(89)90084-5.

DOI:10.1016/0003-2697(89)90084-5

PMID:2479285

Abstract

A streptavidin-glucose-6-phosphate dehydrogenase (G6PDH) conjugate was synthesized and its properties were studied, along with those of biotin-G6PDH conjugates. Two bioluminescent assays were used. Streptavidin was assayed in two steps: streptavidin samples were first incubated with a small amount of biotin-G6PDH and then with biotinylated rabbit gamma-globulins. The complex was immobilized on a bioluminescent immunoadsorbent. In the single-step biotin assay, free biotin was allowed to compete with biotin linked to rabbit gamma-globulins for binding to streptavidin-G6PDH in the presence of the bioluminescent immunoadsorbent. Neither assay required washing or separation steps and the sensitivity was 0.2 ng for streptavidin and 100 fg for biotin. Different applications are described: studies of biotin reactivity when linked to probes in solution or immobilized, and quantitation of biotin in biotinylated DNA probes and oligonucleotides.

摘要

合成了链霉亲和素 - 葡萄糖 -6- 磷酸脱氢酶（G6PDH）结合物，并研究了其性质，同时也研究了生物素 -G6PDH 结合物的性质。使用了两种生物发光测定法。链霉亲和素分两步测定：首先将链霉亲和素样品与少量生物素 -G6PDH 孵育，然后与生物素化的兔γ - 球蛋白孵育。该复合物固定在生物发光免疫吸附剂上。在单步生物素测定中，在生物发光免疫吸附剂存在的情况下，使游离生物素与连接到兔γ - 球蛋白上的生物素竞争结合链霉亲和素 -G6PDH。两种测定均不需要洗涤或分离步骤，链霉亲和素的灵敏度为 0.2 ng，生物素的灵敏度为 100 fg。描述了不同的应用：研究生物素与溶液中或固定化探针连接时的反应性，以及生物素化 DNA 探针和寡核苷酸中生物素的定量。