ULTRASENSITIVE ENZYMATIC RADIOIMMUNOASSAY USING A FUSION PROTEIN OF PROTEIN-A AND NEOMYCIN PHOSPHOTRANSFERASE-II IN 2-CHAMBER-WELL MICROTITER PLATES

A new sensitive method for antigen detection employing a phosphorylation reaction is described using human serum albumin as a model. The antigen is initially bound to the surface of polystyrene microtiter plates and reacted with an antibody (rabbit). A microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (NPT II) serves as a second immunological reagent by virtue of its protein A component. The detection is based on the phosphorylation of an aminoglycoside antibiotic by the NPT II moiety of the fusion protein using [γ-32P]ATP as a cosubstrate. This reaction is performed in solution and the evaluation is accomplished by dotting aliquots of the reaction mixture onto phosphocellulose paper, washing with water, and autoradiography. Microtiter plates with a specially designed 10-μl-volume reaction chamber are particularly advantageous for this procedure. The sensitivity of detection is currently 10 fg (1 pg/ml) of antigen. © 1990.