Journal of Immunological Methods, 105 (1987) 133-137 133 Elsevier JIM 04564 Mitogenic activity of staphylococcal protein A is due to contaminating staphylococcal enterotoxins Hubert Schrezenmeier and Bernhard Fleischer Department of Medical Microbiology and Immunology, University of Ulm, Ulm, F.R.G. (Received 27 April 1987, revised received 10 July 1987, accepted 21 July 1987) Soluble protein A from S. aureus is widely used as a polyclonal activator of human T cells. However, recombinant protein A produced in E. coli does not show any mitogenic properties, although its IgG-binding activity is identical to protein A purified from S. aureus. Antisera against the staphylococcal enterotoxins A and B are able to specifically inhibit the response of human T lymphocytes to protein A from S. aureus. Therefore, the mitogenic principle of this extensively used T cells activator is due to minute contaminations by enterotoxins that are active in picomolar concentrations. Key words: Staphylococcalprotein A; Mitogen; T lymphocyte, human; Enterotoxin Introduction Since it was first reported in 1972 that prepara- tions of protein A of Staphylococcus aureus (SPA) activate human T lymphocytes (Rodey et al., 1972), SpA has been widely used as a polyclonal T cell activator to induce proliferation or production of interleukin-2, gamma-interferon or other lympho- kines in human T lymphocytes. A literature search for the use of SpA as a T cell mitogen revealed approximately 300 publications and the actual number is likely to be much higher. Preparations of SpA can be contaminated with small amounts of other staphylococcal proteins. Since the staphylococcal enterotoxin A (SEA) is a potent mitogen (Langford et al., 1978), it has been Correspondence to: B. Fleischer, Department of Medical Microbiology and Immunology, University of Ulm, P.O. Box 4066, D-7900 Ulm, F.R.G. Abbreviations: IL-2, interleukin-2; PHA, phytohemagg- lutinin; PBMC, peripheral blood mononuclear cells; SEA, staphylococcal enterotoxin A; SEB, staphylococcalenterotoxin B; SpA, staphylococcal protein A; recSpA, SpA produced in E. coli. suspected that SEA may be one of the T cell- activating principles of SpA (Smith et al., 1983). However, a complete separation of the T cell- stimulating activity from the protein A itself has never been achieved. In this report we demon- strate that protein A is not mitogenic and that most of the mitogenic activity of SpA is probably due to contamination with staphylococcal enter- otoxins. Materials and methods Reagents SpA was purchased from Pharmacia (Freiburg, F.R.G., lot no. 11126), recombinant staphylococ- cal protein A (recSpA) produced in E. coli from Calbiochem (Frankfurt, F.R.G.), phytohemag- glutinin-P (PHA) from Difco (Hamburg, F.R.G.) and staphylococcal enterotoxins A and B as well as antisera to these enterotoxins from Serva (Heidelberg, F.R.G.). SDS-PAGE of iodinated en- terotoxin revealed only a single band in the 30 kDa region. The antisera neutralized the mito- 0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)