[Enzyme immunoassay for rotavirus using nylon as the solid phase].

Abstract

An enzyme-linked immunoassay (EIA) to detect Rotavirus in stools is described. Antibodies prepared in rabbits were immobilized on small nylon cubes as capture phase and enzyme conjugated antibodies were used to reveal the reaction. The conjugate was prepared with horseradish peroxidase by the Nakane periodate oxidation method. The solid phase consisted of 3 mm nylon cubes (66 CNL Du-cilo) previously submitted to partial acid hydrolysis to liberate amino-reactive groups. Glutaraldehyde was employed to couple the capturing antibody to the solid phase resulting in a covalent linkage between the gamma-globulin and the nylon. Phenylenediamine in citrate buffer pH 5.0 with 0.5% hydrogen peroxide was used as revealing substrate. EIA was performed as follows: stools watery extracts were incubated 1 h at 37 degrees C with antibody-treated nylon cubes, and then with enzyme conjugate, rinsed with distilled water and substrate-added. Samples developing colour, with optical density of at least 0.350 at 492 nm, were considered positive. The method showed good correlation with a commercial kit.