Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay: application in determining epitope accessibility on ribosomes

Abstract

A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined. We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay. Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes. This method of detecting antibody migration is more sensitive than the conventional means of using A260nm to monitor the antibody-mediated dimerization of ribosomes. Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies. Thus, the relationship of different accessible epitopes in situ can be readily examined. Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems.