Colony immunoblot assay for the detection of staphylococcal toxic shock syndrome toxin 1 (TSST-1) with anti-TSST-1 F(ab')2 fragments

Abstract

Toxic shock syndrome toxin 1 (TSST-1), an exoprotein of Staphylococcus aureus, is strongly implicated in the pathogenesis of TSS. Detection of TSST-1, however, is often hindered in immunoassays because of the cosecretion of protein A, a genetic trait which appears to be coordinately expressed with other exoproteins in S. aureus. We developed a colony immunoblot assay for rapid screening of TSST-1-producing S. aureus using TSST-1-specific rabbit F(ab')2 fragments. The sensitivity and specificity of this method were compared with those of a quantitative noncompetitive enzyme-linked immunosorbent assay (ELISA) for 34 S. aureus isolates (17 TSS-associated and 17 non-TSS-associated isolates). Cosecreted protein A in culture supernatants was evaluated by a quantitative competitive ELISA. The results clearly indicated the superiority of F(ab')2 fragments in eliminating nonspecific reactivity of protein A in the colony immunoblot assay. The sensitivity of the immunoblot with TSST-1-specific F(ab')2 was similar to that with whole immunoglobulin G (94 versus 82%, respectively; P = 0.601, Fisher's exact test), but the specificity was markedly improved (94 versus 59%, respectively; P = 0.039). Among TSST-1-negative isolates (as determined by ELISA), strains which gave false-positive results in the immunoglobulin G immunoblot assay produced higher amounts of protein A than strains which gave true-negative results (P = 0.08, Mann-Whitney rank sum test, one tailed). Among strains positive for TSST-1, the level of TSST-1 detected in culture supernatants correlated inversely with the amount of protein A secreted (rs = -0.64; P less than 0.01, Spearman rank correlation). These findings validate the utility of a rapid screening method for the detection of TSST-1-producing S. aureus and support the concept of coordinate secretion of exoproteins in S. aureus.

Clinical, Microbial, and Biochemical Aspects of the Exfoliative Toxins Causing Staphylococcal Scalded-Skin Syndrome

“…This method is known to be crude, unreliable, and time-consuming with poor sensitivity and is unsuitable for batch testing of large samples (139). Furthermore, our work (139) and work with diagnostic kits for TSST-1 (219,238) have shown that protein A in culture supernatants is produced by more than 90% of S. aureus strains (88,89), resulting in high rates of false-positive results due to nonspecific binding to the detecting antibodies (36,255).…”

Immunoblots for detection of toxic shock syndrome toxin 1 produced by Staphylococcus aureus

“…2. See, R. H., S. Adilman, K. H., Bartlett, and A. W. Chow. 1989. Colony immunoblot assay for the detection of staphylococcal toxic shock syndrome toxin 1 (TSST-1) with anti-TSST-1 F(ab')2 fragments.…”

Rapid assay for detection of toxic shock syndrome toxin 1 from human sera

“…To detect and quantify TSST-1, reverse passive latex agglutination (6) or enzyme-linked immunosorbent assays (ELISA) (4,9,11,14,16,20) have been used. However, reverse passive latex agglutination cannot be used with a high concentration of serum because of nonspecific agglutination.…”