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

Abstract

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.