Studies have been extended on neoantigens of immune complexed IgG by development of mouse monoclonal antibodies that react with the neoantigens. The monoclonals were developed in mice tolerized with monomeric or nonspecifically aggregated IgG. Several different monoclonal antibodies were characterized by their ability to recognize neoantigen or antigen bound IgG compared to monomeric plate bound IgG. We have continued work on quantitative analysis of pneumococcal adherence and phagocytosis by human monocytes. The assay employs two fluorescent labels and dual laser flow cytometry. The pneumococci are labeled with Lucifer Yellow, which fluoresces independent of pH. Antibodies to pneumococcal capsular polysaccharides cleaved to F(ab')2 fragments and biotinylated, then stained with Streptavidin-Texas Red were used to stain adherent but not ingested pneumococci. The assay has been used to study opsonization requirements for adherence and rate analysis of ingestion via the C3b receptor. Antibodies to Lucifer Yellow have been developed in rabbits which recognize the Lucifer labeled pneumococci without changing the Lucifer fluorescence.
