THE USE OF TWEEN-20 AS A BLOCKING-AGENT IN THE IMMUNOLOGICAL DETECTION OF PROTEINS TRANSFERRED TO NITROCELLULOSE MEMBRANES

Determination of the immunoreactivity of protein antigens in complex mixtures is greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents with each other or with others of potential merit, using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae were compared. A method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins, is described. This system was equivalent or superior to other methods evaluated in the detection of immunoreactive proteins in hyperimmune rabbit and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. Individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay.