Quantitative fluorescent immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus

Abstract

Optimal conditions for a fluorescence immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus ATCC 19246 are described. In the standard fluorescence immunoassay, 10(8) colony-forming units of A. viscosus reacted with an antibody preparation, were washed, and then were treated with an excess of fluorescein-conjugated goat anti-rabbit immunoglobulin G. After another set of washes, fluorescence was determined in a spectofluorometer; in most cases excitation was at 485 nm, with emission measured at 525 nm. These conditions minimized interference from light scatter and stray light. Under appropriate conditions, antibodies to A. viscosus could be readily determined, with the fluorescence of the specific antibody-treated cells more than five times the fluorescence of controls treated with normal rabbit serum. Organisms coated with specific antibody could be detected at levels approaching 10(5) colony-forming units per ml. The standard fluorescence immunoassay procedure was readily adapted to the measurement of either particulate or soluble surface antigens of A. viscosus by competition of the antigen with a fixed amount of antibody in the standard assay system; the competition resulted in an antigen dose-dependent inhibition of fluorescence. The fluorescent immunoassay system thus appears to be a general one that could be applied to other microbial systems as well.