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We developed a competitive, enzyme-linked immunosorbent assay for the quantitation of toxic shock syndrome toxin 1 (TSST-1). Polyvalent immunoglobulin G from immunized rabbits was used as the capture antibody, and alkaline phosphatase conjugated to purified toxin served as the indicator enzyme. A standard curve was-génerated with each experiment, from which the concentration of toxin in culture supernatahts was extrapolated. The assay was useful for determining toxin concentrations of 0.03 to 0.5 ,g/ml, which is a substantial, practical improvement over immunodiffusion methods.,Staphylococcal enterotoxins A through E were not significantly cross-reactive in the assay, and staphylococcal protein A did not interfere with quantitation of TSST-1. By testing a variety ot staphylococcal strains, we found 100% concordance between toxin determinations made with our assay and those made by the investigators from whom the strains were obtained. The competitive, enzyme-linked immunosorbent assay is a highly reproducible, inexpensive means of determining TSST-1 concentrations and may have broad applicability in the field of toxic shock research.

We examined the transfer of sterols and phospholipids from their site of synthesis to the plasma membrane of Acanthamoeba castellanii. Cells were labeled with [3H]acetate, and plasma membrane fractions were isolated under conditions that minimize the nonspecific exchange of lipids between subcellular membrane fractions. Sterols and phospholipids were purified from both whole-cell homogenates and isolated plasma membrane. In whole cells, 3H-labeled lipids were formed, with no apparent time lag, in a linear manner up to 1 hr. Labeled sterol and phospholipids appeared in the plasma membrane, after a 30-min lag, at approximately the same rate. However, the ratio of newly synthesized sterol to phospholipid was significantly enriched in the plasma membrane relative to the whole cell, even at the earlier time points. Pulse-chase experiments indicated that sterols and phospholipids are turned over in the plasma membrane with similar, rather short half-lives. The results of these studies suggest that, although sterols and phospholipids are transported to the cell surface with similar kinetics, some sorting of the lipids must occur at an early stage in membrane biogenesis. The data are consistent with a model of lipid translocation by vesicular transport.

The effect of Mg2+ on in vitro production of extracellular proteins and, specifically, of toxic shock syndrome toxin-1 (TSST-1), by Staphylococcus aureus in a chemically defined medium was examined. As previously observed, the organisms did not proliferate in the absence of divalent cations. Low levels of Mg2+ (0.02 to 0.04 mM) permitted submaximal proliferation and elevated production of exoproteins. When the Mg2+ concentration was raised to 0.4 mM, multiplication was optimal and exoprotein levels were depressed. Ca2+ and Mn2+ diminished the effect of limiting Mg2+. The increased levels of exoproteins were not due to cell lysis or leakage since intracellular TSST-1 levels were not high enough to account for the increase in extracellular TSST-1 and since the intracellular enzyme, lactate dehydrogenase, was not found in culture supernatants. Cells cultured in low levels of Mg2+ remained in logarithmic growth longer than did those cultured in high concentrations of Mg2+ and, unlike the latter, produced exoproteins throughout the logarithmic growth phase. Low Mg2+ had no effect on cultures in the stationary phase, and organisms cultured in low Mg2+ recovered fully when transferred to high Mg2+. We conclude that, when cultured in medium deficient in Mg2+, S. aureus responds early in the growth cycle by increasing production of many extracellular proteins, including TSST-1.

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