Prevention of nonspecific binding of immunoglobulin to Staphylococcus aureus protein A in ELISA assays.

The Fc region of IgG of most mammals binds protein A on S. aureus resulting in high backgrounds when measuring specific antibodies to S. aureus in the ELISA. Removal of protein A from S. aureus or modification of the Ig Fc to prevent binding to protein A could affect specific antibody binding. We compared effects of blockage of Fc binding to protein A with purified protein A to trypsin removal of protein A from S. aureus, on specific antibody binding. When NMS was incubated without and with protein A (0 microgram, 50 micrograms, 200 micrograms and 400 micrograms) and high protein A Cowan I was the bound S. aureus antigen in the ELISA, absorbance OD405 was 0.769, 0.240, 0.224 and 0.210 +/- SE 0.026. When mouse Mab (IgG1, kappa) to bovine IgA was incubated without and with protein A (400 micrograms) prior to reaction with bovine IgA in the ELISA, absorbance was 0.645 and 0.639, indicating protein A had no effect on specific antibody binding. To determine the effect of trypsin on specific binding, Becker S. aureus was trypsin treated before linking it to microtiter wells. When Mab (IgM) to Becker (Nelles et al., Infect. Immun. (1985) 49, 14) was incubated with protein A (400 micrograms) before use in the ELISA, trypsin treatment of Becker resulted in reduced specific antibody activity (untreated Becker = 1.306, trypsin treated Becker = 0.331). These results suggest that purified protein A can be used to block nonspecific binding via Fc of Ig to S. aureus, thus avoiding trypsin denaturation of surface antigens.

Effect of anticapsular antibodies on neutrophil phagocytosis of Staphylococcus aureus.

One of the major virulence factors of Staphylococcus aureus is development of an exopolysaccharide capsule in vivo, which inhibits recognition of antibodies to highly antigenic cell wall by neutrophils. To circumvent this inhibition, an attempt was made to produce anticapsular antibodies. Three cows per group were immunized in midlactation by injections in the area of the supramammary lymph node and intramuscularly and were boosted on d 14, 42, and 70 with three variants of Smith S. aureus: compact, unencapsulated; diffuse, rigid capsule; and diffuse large clearing, exceptionally large flaccid capsule using dextran sulfate as adjuvant. Serum agglutination and ELISA titers of cows immunized with diffuse and diffuse large clearing increased after immunization and after each boost and remained elevated to the end of the experiment at 112 d. Phagocytosis of diffuse and diffuse large clearing, measured by flow cytometry, was enhanced by immunization with either organism. No antibody response to capsule or enhanced phagocytosis of diffuse developed in cows immunized with compact. However, anticompact antibodies were opsonic for diffuse large clearing. These data show that bovine antibodies to S. aureus capsule are opsonic for bovine neutrophils and that capsule plays a role in inhibition of cell-wall opsonization of S. aureus.