Toxic-shock-syndrome toxin-1 (TSST-1), a 22-kilodalton (kDa) polypeptide, was proteolyzed by papain, generating three distinct fragments, identified as 16, 12, and 10 kDa (based on molecular masses estimated from the predicted amino acid sequence). The NH2-terminal sequence analysis of the fragments indicated that the peptide bonds between Tyr-52 and Ser-53 and between Gly-87 and Val-88 were cleaved. Functional activity, evaluated through enzyme-linked immunosorbent and inhibition assays, was demonstrated only with the 16- and 12-kDa fragments. The presence of homologous and heterologous antigenic determinants on the fragments was demonstrated by immunoblotting. In in vitro stimulation of human peripheral blood mononuclear cells, the 12-kDa fragment was significantly (P = .003) more active than the 16-kDa fragment. The former composed 75% of the latter and occupied the COOH-terminal portion of the holotoxin. The functional domains were located on two-thirds of the TSST-1 molecule, toward the COOH-terminal end, and mitogenicity apparently was separable from serological activity.
When toxic shock syndrome toxin 1 was subjected to papain hydrolysis, two serologically active fragments of 16.3 kilodaltons (16K fragment) and 12.4 kilodaltons (12K fragment) were generated, whereas a third fragment of 9.7 kilodaltons (1OK fragment) was inactive. The biologic activities of the fragments were evaluated in vitro by determining their ability to promote nonspecific proliferation of human peripheral blood mononuclear cells. The 12K fragment was significantly (P c 0.013) more stimulatory than the 16K fragment. When human peripheral blood mononuclear cells were preincubated for a period of 24 h with various concentrations of the 16K fragment, followed by incubation with a constant amount (2 x 10-2 ng/ml) of whole toxin, the level of DNA synthesis induced by the holotoxin was reduced by approximately 60% when compared with that of controls exposed to whole toxin alone. The 12K fragment did not demonstrate a similar blocking effect. Immunoblots of the toxic shock syndrome toxin 1 digest, which were exposed to monoclonal antibodies (MAbs) developed against native toxin, depicted the presence of two different antigenic regions (epitopes). One MAb, 8-5-7, which has been shown previously to inhibit the biologic activity of the holotoxin in vitro and in vivo, reacted primarily with the 12K fragment. A second MAb, 10-6-1, that did not neutralize interleukin-1 production reacted primarily with the 16K fragment. On the basis of the differential mitogenic responses and the identification of heterologous epitopes, it was concluded that the functional region of the holotoxin can be partitioned into at least two functional segments encompassed between amino acid residues 53 and 87 and between amino acid residues 88 and 194 on the polypeptide chain.
Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of >106 to 107 when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.
A 21-mer synthetic peptide (KGEKVDLNTKRTKKSQHTSEG), designated TSST-1(58-78), was constructed from the primary structure of the toxic shock syndrome toxin 1 (TSST-1). The peptide reacted with a panel of neutralizing monoclonal antibodies (MAbs) to whole TSST-1 in solid-phase immunoassays. TSST-1(58-78) promoted the in vitro proliferation of human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Minimum dose required for stimulation (P less than or equal to .05 microM) was 0.75 microM peptide. This mitogenic effect was abrogated by incubation of the peptide with MAbs to whole TSST-1 before addition to PBMC. The ability of TSST-1(58-78) to stimulate the proliferation of highly purified resting human T cells was analyzed. Significant proliferation (P less than or equal to .01) was observed only in the presence of increasing populations of monocytes added to the cultures. Adherent human monocytes exposed to TSST-1(58-78) released tumor necrosis factor. Thus, some of the immunoregulatory properties attributed to TSST-1 are demonstrated by the region of the toxin represented by the peptide TSST-1(58-78).
A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin A (SEA) was developed by using monoclonal antibodies (MAb) to SEA as primary capture antibodies. The antigen was detected with purified rabbit anti-SEA antibody as the secondary antibody. The secondary antibody was identified by direct conjugation with biotin or via biotinylated sheep F(ab')2 fragments to rabbit antibody. The biotin was then reacted with avidin-alkaline phosphatase (AP) conjugate, avidin-biotin-AP conjugated complex, or streptavidin-AP conjugate. The enzyme was identified by using p-nitrophenylphosphate. The incorporation of the avidin-biotin-AP conjugated complex or streptavidin-AP conjugate augmented the sensitivity 32-fold over that of the enzyme-linked immunosorbent assay without these reagents. Controls were run by substitution of the anti-SEA MAb with unrelated MAb of the same isotype. Sample values were considered positive when the A405 exceeded those of the negative controls by 3 standard deviations (>99% confidence interval). The toxin could be quantitated with purified SEA standards through linear regression analysis with lower detection limits of 4 ng/ml (r = 0.99) and 0.25 ng/ml (r-0.98). Concentrations of protein A up to 10 ,ug/ml did not cause interference. Analyses of crude growth extracts of SEA-secreting strains of Staphylococcus aureus were reproducible and were expressed in terms of 95% confidence intervals. Lack of cross-reactivity was seen with extracts of other toxigenic and nontoxigenic strains of S. aureus. The assay can be completed in one working day, provided that MAb-coated plates are available.
The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C,, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfatepolyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, Cl, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.
Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.
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