Role of Protein A in Nonspecific Immunofluorescence of Staphylococcus aureus

Arne Forsgren, Urban Forsum
1970 Infection and Immunity  
yG-globulin from nonimmunized rabbits and from rabbits immunized with various bacteria reacted in the immunofluorescence technique with protein A-containing Staphylococcus aureus. Pepsin digestion of most immunoglobulin preparations eliminated the reaction, thus showing that the Fc fragment is involved and that the reaction is not a true antigen-antibody reaction. As the specific immunological activity of the immunoglobulin molecules was intact after digestion, it is suggested that the method
more » ... d that the method be used to eliminate reactions with S. aureus in the fluorescent-antibody technique. So-called "normally occurring antibodies" against Staphylococcus aureus have been demonstrated with the fluorescent-antibody (FA) technique in sera from different species (1, 3, 4, 17, 22) . The well-known fact that many strains of S. aureus react with antisera to other bacteria has been a major problem in the FA technique (2, 5, 6, 18). Protein A has been shown to be associated with the cell surface of most S. aureus strains (13, 27) . A direct reaction between protein A and rabbit -yG-globulin through interaction with structures on the Fc portion of the -y-globulin is now well documented (11, 26) . This paper provides evidence that the FA reaction between S. aureus and -yG-globulin from nonimmunized rabbits and from rabbits immunized against various bacterial species is not due to an antigen-antibody reaction. Instead it seems to be a reaction between the Fc part of the 'yG-globulin molecules and protein A from S. aureus. MATERIALS AND METHODS Strains and culture techniques. S. aureus types Cowan I and Wood 46 were used. The other bacteria used were isolated in the routine laboratory of the Institute of Medical Microbiology. All strains were cultivated overnight on conventional solid media before use. In some experiments, S. aureus type Cowan I was grown on Mannitol Salt Agar plates (Difco). Sera. Pooled serum from nonimmunized rabbits and antisera from rabbits immunized against Neisseria gonorrhoeae, N. meningitidis, Escherichia coli type 0111, and Shigella boydi type 3 were used. The antisera were obtained by immunization according to the current methods at the Institute of Medical Microbiology (5, 9). The antisera had agglutination titers against the corresponding bacteria ranging from 1/64 to 1/512. Preparation of yG-globulin. Rabbit -yG-globulin was prepared from sera by precipitation at 37% saturation with ammonium sulfate. The precipitate was dissolved in distilled water and the procedure was repeated twice. The precipitated and redissolved material was dialyzed against 0.0175 M Na2HPO4 (pH 6.3), further purified by chromatography on a diethylaminoethyl (DEAE)-cellulose column equilibrated and eluted with the dialysis buffer (11), and finally concentrated with an ultrafiltration cell (Diaflo m. 50, Amicon Corp.) to a protein concentration of 18 to 20 mg/ml. The protein concentration was determined by measuring the optical density at 280 nm. Each sample was divided into two portions. One portion was dialyzed against 0.15 M NaCl and diluted with 0.15 M NaCl and carbonate-bicarbonate buffer (pH 9.0, 0.5 M) to a final concentration of about 10 mg of protein per ml (23). Carbonate-bicarbonate buffer made up 20% of the final solution. The other portion was dialyzed against 0.1 M sodium acetate. For two sera, the purification step on DEAEcellulose was omitted. After precipitation with ammonium sulfate, the crude -yG-globulin fraction was dissolved in distilled water, divided into two portions, and dialyzed as described above. Pepsin digestion. Pepsin digestion of -yG-globulin from normal and immunized rabbits was carried out by the method of Nisonoff (24). After the dialysis against 0.1 M sodium acetate, the pH was lowered to 4.5. Pepsin (Worthington Biochemical Corp.) dissolved in 0.1 M sodium acetate (pH 4.5) was added in the proportion 1 mg of enzyme per 50 mg of protein. The digestion was allowed to proceed at 37 C for 8 hr, after which time the pH was adjusted to 8.0. The di-387 on May 9, 2020 by guest http://iai.asm.org/ Downloaded from
doi:10.1128/iai.2.4.387-391.1970 fatcat:bmtas5nrwzhmrgqlmu354vzfhy