An immunoblot assay for the simultaneous quantification of several antigens

A radioimmunologic assay method that allows for the simultaneous quantification of several antigens in one sample is described. Polypeptide antigens are resolved electrophoretically and electroblotted to nitrocellulose. The nitrocellulose is then reacted with a mixture of several antisera simultaneously, and antibody-binding proteins are visualized by incubation with 125I-protein A and by autoradiography. Antigens are identified according to their molecular weights and quantified by counting the bound radioactivity. The sensitivity of the assay is in the low nanogram range and can be adjusted individually for each antigen by appropriately diluting the first antiserum. The procedure is presently applied to the detection of three neural antigens, neural cell adhesion molecule, neuron-specific enolase, and synaptophysin, in adult brain tissue and to the assessment of expression of the latter two during development of brain cells in primary culture. The method is fast, comparatively cheap, and associated with a low radiation exposure. It should prove especially useful when only scarce amounts of sample are available.