Rapid polyelectrolyte-based immunofiltration technique for testosterone detection in serum samples

被引:19
|
作者
Zherdev, AV
Byzova, NA
Izumrudov, VA
Dzantiev, BB
机构
[1] Russian Acad Sci, Inst Biochem, Moscow 119071, Russia
[2] Moscow MV Lomonosov State Univ, Dept Chem, Moscow 119899, Russia
关键词
D O I
10.1039/b303288d
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
A new immunofiltration assay for testosterone is proposed. During the first step of the assay, testosterone molecules in serum samples compete in solution with the testosterone-peroxidase conjugate for interaction with anti-testosterone antibodies pre-bound to the conjugate between staphylococcal protein A and polymethacrylate polyanion. The reaction mixture is then filtered through a membrane charged with immobilized poly(N-ethyl-4-vinylpyridinium) polycation. The filtration is accompanied by a rapid separation of the polyanion containing complexes due to high-affinity electrostatic interactions. Following removal of unbound compounds the immobilized peroxidase is detected using a substrate that produces an insoluble coloured product. The proposed assay has been shown to combine high speed (20 min) and sensitivity (0.1 ng ml(-1)), and to be applicable for out-of-laboratory conditions. Based on densitometric measurements, the RSD of the assay is calculated to be 3.2-5.1% (n = 4). The proposed assay is 4 times faster than the microplate enzyme immunoassay (ELISA) based on the same immunoreagents. Pre-incubation of the antibody and the polyanion-protein A conjugate at a certain ratio excludes the influence of immunoglobulins from the tested serum samples on the assay results. The polyanion-protein A conjugate can be used as a universal reagent, eliminating the necessity to modify specific antibodies for each immunoassay.
引用
收藏
页码:1275 / 1280
页数:6
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