New Enzyme Immunoassay with Visual Detection Based on Membrane Photoimmobilized Antibodies

Abstract

New method for visual enzyme immunoassay of some model antigens in solution by using covalently photoimmobilized antibodies has been developed. This approach is based on the quantitative photoimmobilization of antibodies on the surface of porous matrixes. It is easy to control the dimensions and shape of the activated zones and the quantity of the active groups on it by this technique. A strip of the membrane impregnated with p-azidobenzaldehyde was illuminated by the light. A s a result, .the quantity of aldehyde groups developed on the surface of membrane is proportional to the time of illumination. After the covalent immobilization of antibodies, the membrane has separate zones with an exact surface concentration of antibodies. The antigens of different types were assayed: human IgG, human chorionic gonadotropin, Shigella Sonnei. The lowest detection limit was 1 μg/ml, 20 U/L 1×10₄ cells/ml. The method allows measure of thyroxire concentrations in the range 50 to 200 nM, the precision of replicate measurements has the coefficient of variation 7%. The reasons for the background signal appearance were accurately analyzed. The choice of support was substantiated, the optimal conditions of it.s pretreatment being defined. This method makes possible the visualization of the results based on comparison the color intensity of zones with the control.