Staphylococcal scalded-skin syndrome: development of a primary binding assay for human antibody to the exfoliative toxin

Abstract

Exfoliative toxin (ET) from a phage group II Staphylococcus aureus strain causing staphylococcal scalded-skin syndrome was purified by electrofocusing. Ampholytes and salts were removed from the final product by column chromatography on G-50 Sephadex. Sodium dodecyl sulfate-polyacrylamide gels of the final product yielded a single band upon gel electrophoresis, even when 60 ,ig of protein was placed in the gels. Radiolabeling of the purified toxin with 125I yielded a product that still caused exfoliation of suckling mice, indicating that the toxin was still biologically active. A radioimmunobinding assay was developed and used to test rabbit and human sera for antibodies to exfoliative toxin. Although the maximum percentage of binding was not as high as expected (approximately 40%), it was postulated that either iodination had not been sufficiently vigorous or the toxin had sustained immunological damage. The assay was reproducible and more sensitive than the existing neutralization method and readily applicable to the testing of human sera for exfoliative toxin antibodies. 120 rpm at 35 C and flushed thrice daily with 100% CO2 for 10 min.Sera. Specimens of human serum from cases of scalded-skin syndrome were kindly supplied by physicians. The sera from "normal" humans were col-513

Production and properties of a staphylococcin genetically controlled by the staphylococcal plasmid for exfoliative toxin synthesis

“…Isoelectric density gradient electrophoresis. Isoelectric fractionation was performed as described previously (25). One milliliter of a concentrated Bac R. solution obtained after lyophilizing the (NH4)2SO4precipitated preparation was electrofocused for approximately 48 h at 4°C in a glycerol density gradient with 10% carrier ampholytes (Brinkmann) in the pH range of 2 to 10.…”

“…The covalently closed circular structure of this large plasmid spontaneously converts to the open circular form, and this conversion is enhanced in the presence of either sodium dodecyl sulfate, Pronase, or ethidium bromide (19,24). The 56S plasmid is believed to carry at least three different types of genetic markers that include (i) a temperature-sensitive genetic site for replication, (ii) genetic loci for the production of a specific type of exfoliative toxin (ET) that is responsible for the dermatological manifestations of the staphylococcal scalded-skin syndrome (17,19,25), and (iii) a genetic site for the production of a specific bacteriocin (19,23,24) that has been designated as Bac R. in this laboratory. Genes that code for Bac R. appear to be strictly extrachromosomal, whereas genes that code for ET appear to be located both chromosomally and extrachromosomally (19).…”

Staphylococcal scalded skin syndrome: detection of antibody to epidermolytic toxin by a primary binding assay

“…Of interest, Wiley et al (1976) did not observe higher titres of antibody to epidermolysin in adults as compared with children. He did recognize a smaller proportion of children who had prior exposure to this toxin as reflected by the presence of measurable antibody in their serum.…”

“…The number of healthy adults studied was small, but all but 2 of those studied had antibody present. In addition Wiley et al (1976) reported finding anti-epidermolysin antibodies by a primary binding assay in sera from 64 randomly chosen subjects of all ages.…”

“…In humans, antibodies to epidermolysin are thought to be neutralizing and protective also. Recently, in another study anti-epidermolysin antibodies were measured by a primary binding assay in sera from patients with the scalded skin syndrome (Wiley et al, 1976). However, paired acute and convalescent sera were not studied so it is difficult to draw any conclusions from that data.…”