Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin.
Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin.
复制标题
测量溶液中铕螯合物的时间分辨免疫荧光测定：纤连蛋白灵敏测定的应用。
DOI：
10.1016/0003-2697(91)90386-8
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发表时间：
1991
影响因子：
2.9
通讯作者：
A. Gressner
中科院分区：
文献类型：
--
作者：
J. Kropf;E. Quitte;A. Gressner
本文描述了一种适用于时间分辨荧光分光光度法对生物感兴趣的分子进行灵敏测量的新型检测原理。我们的方法是基于定量的Eu3+螯合剂4,7-二（氯磺苯基）-1,10-菲罗啉-2,9-二羧酸（BCPDA）在溶液中过量的Eu3+离子的存在。用尿素/SDS/Eu3+作为解离和荧光镧离子试剂，将bcpda标记的固相配合物转移到溶液中。基于上述方法实现了两种“三明治型”检测变体，用于测定生物体液中少量的纤维连接蛋白（FN）。FN分别通过固相包被明胶或单克隆抗体从溶液中捕获。兔抗fn抗血清作为第二抗体，用生物素化兔抗IgG抗体检测。用链霉亲和素- bcpda孵育和解离固相配合物后测量荧光。两种检测方法的检出限（空白+ 3 × SD）均小于0.5 ng/ml FN，动态范围高达300 ng/ml，序列内变异系数分别为4.4和6.3%。健康人唾液中FN的中位浓度分别为104 ng/ml（明胶）和36 ng/ml（双抗体）。
A novel detection principle applicable for sensitive measurement of molecules of biological interest by time-resolved fluorescence spectrophotometry is described. Our method is based on the quantification of the Eu3+chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in solution in presence of an excess of Eu3+ions. BCPDA-labeled solid phase complexes obtained by conventional immunoassay procedures are transferred into solution using urea/SDS/Eu3+as dissociating and fluorescent lanthanide ion reagent. Two ‘sandwich-type’ assay variants based on the above methodology were realized for the determination of small amounts of fibronectin (FN) in biological fluids. FN is captured from solution by solid phase coated gelatin or a monoclonal antibody, respectively. Rabbit anti-FN antiserum used as second antibody is detected with a biotinylated anti-rabbit IgG antibody. Fluorescence is measured after incubation with streptavidin-BCPDA and dissociation of solid phase complexes as described. Both assays have a detection limit (blank + 3 × SD) of less than 0.5 ng/ml FN, a dynamic range of up to 300 ng/ml, and intraserial coefficients of variation of 4.4 and 6.3%, respectively. Median FN concentrations in saliva of healthy individuals were 104 (gelatin) and 36 ng/ml (double antibody), respectively.