Ultrastructural localization of specific gonococcal macromolecules with antibody-gold sphere immunological probes

In an effort to determine the ultrastructural location of specific macromolecules on the surface of intact microorganisms and in experimentally infected tissues, a new method of rapidly conjugating antibodies to gold spheres via a staphylococcal protein A intermediary has been developed. This new technique provides the excellent density of marking and versatility of sphere size provided by existing gold methods, but decreases preparation time, decreases the chance of bacterial contamination of antibody reagents, and increases specificity of marking. Staphylococcal protein A-coated gold spheres were conjugated with antibodies from rabbits immunized with purified gonococcal pili. The resulting gold-antibody conjugates allowed demonstration of antibody binding to pilus structures of the same gonococcal strain whose pili were used to raise the antibody and demonstration of the lack of antibody recognition of pilus structures on two other gonococcal strains. The failure of gold spheres to attach to isogenic nonpiliated clones of the homologous gonococcus indicated the absence of pilus antigens on the surface of these organisms. The use of a double label--small gold spheres conjugated to anti-pilus antibody and larger gold spheres conjugated to anti-protein I antibody--allowed the simultaneous localization of two gonococcal antigens.