Rapid polyelectrolyte-based immunofiltration technique for testosterone detection in serum samples.
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.
Zherdev
Anatoly V
AV
Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, 119071 Moscow, Russia. zherdev@inbi.ras.ru
Byzova
Nadezhda A
NA
Izumrudov
Vladimir A
VA
Dzantiev
Boris B
BB
D005374:Filtration; D006801:Humans; D007120:Immunochemistry / Q000379:methods; D008297:Male; D011109:Polymethacrylic Acids; D013205:Staphylococcal Protein A; D013739:Testosterone / Q000097:blood*
D011109:Polymethacrylic Acids; D013205:Staphylococcal Protein A; C030613:polymethacrylic acid; D013739:Testosterone