Térouanne B, Carrié M L, Nicolas J C, Crastes de Paulet A
Anal Biochem. 1986 Apr;154(1):118-25. doi: 10.1016/0003-2697(86)90504-x.
We describe a bioluminescent immunoassay procedure which does not require a separation step to remove excess free label. A luminescent immunosorbent constituted of bacterial luciferase, FMN oxidoreductase, and an antibody coimmobilized on Sepharose is used to determine specifically the label enzyme (glucose-6-phosphate dehydrogenase, coupled to an antigen) bound by a specific antibody. The immunosorbent confines the bioluminescent reaction in a small volume, and the bound label produces NADH, which is directly used by the nearby luciferase FMN oxidoreductase enzyme system. On the contrary NADH produced by dehydrogenases in solution is directly oxidized without emitting light. Dehydrogenases contained in the biological sample do not interfere with the assay, which can be performed directly on 25 microliter of serum. In this paper we describe the general procedure and we analyze the different parameters that must be optimized.
我们描述了一种生物发光免疫分析方法，该方法无需进行分离步骤以去除过量的游离标记物。一种由细菌荧光素酶、FMN氧化还原酶和共固定在琼脂糖上的抗体构成的发光免疫吸附剂，用于特异性测定与特异性抗体结合的标记酶（与抗原偶联的葡萄糖-6-磷酸脱氢酶）。免疫吸附剂将生物发光反应限制在小体积内，结合的标记物产生NADH，其被附近的荧光素酶FMN氧化还原酶系统直接利用。相反，溶液中脱氢酶产生的NADH被直接氧化而不发光。生物样品中含有的脱氢酶不会干扰该分析，该分析可直接在25微升血清上进行。在本文中，我们描述了一般方法，并分析了必须优化的不同参数。
