Purification of antibodies to bacterial antigens by an immunoadsorbent and a method to quantify their reaction with insoluble bacterial targets.
A combination of procedures was employed to develop a radioimmunoassay which quantified the binding of antibodies to antigens of either intact Propionibacterium acnes or to antigens of insoluble extracts derived from the bacteria. Reactive antibody populations were purified by use of bacterial immunoadsorbents which were prepared by coupling P. acnes to diethylaminoethyl cellulose. Binding of antibodies was detected with [125I]staphylococcal protein A ([125I]SpA) and optimal conditions for the assay defined by varying the amounts of antibodies, bacterial antigenic targets and [125I]SpA. In antibody excess, 100% of available [125I]SpA was bound by the target-antibody complexes. However, when antibody concentration was limiting, a linear relationship was demonstrated between per cent specific binding of [125I]SpA and antibodies bound to bacterial targets. These results were achieved only with immunoadsorbent-purified antibody populations and not with hyperimmune sera or IgG. The radioimmunoassay detected subtle antigenic differences and similarities between P. acnes, P. acnes extracts and a variety of unrelated microorganisms.
D000818:Animals; D000907:Antibodies, Bacterial / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex / Q000494:pharmacology*; D000937:Antigen-Antibody Reactions; D000942:Antigens, Bacterial; D002417:Cattle; D007164:Immunosorbents / Q000494:pharmacology*; D008089:Listeria monocytogenes / Q000276:immunology; D009163:Mycobacterium bovis / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000276:immunology; D011425:Propionibacterium acnes / Q000276:immunology; D011817:Rabbits; D012995:Solubility; D013211:Staphylococcus aureus / Q000276:immunology
D000907:Antibodies, Bacterial; D000936:Antigen-Antibody Complex; D000942:Antigens, Bacterial; D007164:Immunosorbents