A simple quantitative dot-immunobinding assay for glial and neuronal marker proteins in SDS-solubilized brain tissue extracts

Abstract

Immunoassays for quantitative determinations of the S-100 protein, the glial fibrillary acidic protein, the neuron specific enolase and the neurofilament proteins with molecular weight of 68 and 200 kDa in hot SDS sonicated rat brain extracts have been developed and characterized. The assays utilize a dot immunobinding technique, poly- or monoclonal antibodies and 125I-protein A. The SDS-sonication procedure was not found to affect the radioactivity recovery in the assay of the soluble S-100 protein or the neuron specific enolase. All 5 antigens can be measured with a within-assay variance below 10%. Even at a coefficient of variation less than or equal to 5%, the working ranges are approximately 30–100-fold with regard to the different antigens. It was found that gelatin-coated nitrocellulose membranes considerably increase the recovered radioactivity in the assay of the purified bovine S-100 protein, possibly by protein-protein interaction. This effect was not observed when SDS-sonicated rat brain extracts were assayed. The assay appears to be reproducible, convenient and rapid, and provides a high degree of precision in the determination of large number of samples.