Staphylococcal Scalded-Skin Syndrome: Development of ?· causing staphylococcal scalded-skin syndrome…

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INFZCTION AND IMMUNITY, Feb. 1976, p. 513-520Copyright C 1976 American Society for Microbiology

Vol. 13, No. 2Printed in USA.

Staphylococcal Scalded-Skin Syndrome: Development of aPrimary Binding Assay for Human Antibody to the


Departments of Microbiology and Pediatrics, College of Medicine, University of Utah, Salt Lake City, Utah84132

Received for publication 21 October 1975

Exfoliative toxin (ET) from a phage group II Staphylococcus aureus straincausing staphylococcal scalded-skin syndrome was purified by electrofocusing.Ampholytes and salts were removed from the final product by column chroma-tography on G-50 Sephadex. Sodium dodecyl sulfate-polyacrylamide gels of thefinal product yielded a single band upon gel electrophoresis, even when 60 ,ig ofprotein was placed in the gels. Radiolabeling of the purified toxin with 125Iyielded a product that still caused exfoliation of suckling mice, indicating thatthe toxin was still biologically active. A radioimmunobinding assay was devel-oped 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 beensufficiently vigorous or the toxin had sustained immunological damage. Theassay was reproducible and more sensitive than the existing neutralizationmethod and readily applicable to the testing of human sera for exfoliative toxinantibodies.

The staphylococcal scalded-skin syndrome isrepresented by a spectrum of clinical entitiesthat include: a generalized exfoliative dermati-tis (Ritter's disease, toxic epidermal necrolysis),bullous impetigo, and a scarlatiniform rash(15). The majority of clinical reports of the dis-ease have emphasized the occurrence of diseaseprimarily in infants and children (15). Initiallythis age-related susceptibility also appeared toapply to the mouse model described by Melishand Glasgow (14). Subsequently, a small num-ber of cases has been reported in human adults(3-5, 10, 11, 16, 19). In the majority of thesecases in adults, with the exception of thosereported by Epstein and co-workers (5), somepredisposing factor or underlying disease, suchas alcoholism, immunosuppression, or compro-mised cell-mediated immunity was present andmay have played a role in the development ofthe disease. The susceptibility of adult skin tothe action of the exfoliative toxin (ET) in bothhumans (3, 24) and experimental animals (4)has now been demonstrated convincingly.These data suggest that the relatively low inci-dence of dermatological manifestations ofphage group II staphylococcal infection may berelated to the presence of antibody (antitoxin)that is capable of neutralizing the toxin respon-sible for the exfoliation of the superficial layer

of the epidermis. The purpose of this report is todescribe the development of a radioimmuno-binding assay for the ET and to present initialdata concerning: (i) the frequency of occurrenceof antibody to ET in the general population, (ii)the incidence of antibody correlated with age,(iii) the presence of antibody in patients withscalded-skin syndrome, and (iv) the sensitivityof the radioimmunobinding assay comparedwith the in vitro neutralization assay.

MATERIALS AND METHODSCultures. The ET used in this study was produced

in vitro from the UT 0002 strain of Staphylococcusaureus, a previously (17) described phage group IIisolate that was grown in heart infusion broth (HIB,Difco) on a gyratory water bath shaker with a gashood (New Brunswick Scientific Co.). Cultures weregrown in 100-ml amounts in 250-ml screw-cappedErlenmeyer flasks. Organisms for seeding weregrown on 5% sheep blood agar plates and suspendedin HIB to yield an optical density of 0.15 when readat 650 nm against an HIB blank in a Spectronic 20spectrophotometer. The inoculum per flask was 0.3ml. The flasks with loosened caps were shaken at120 rpm at 35 C and flushed thrice daily with 100%CO2 for 10 min.

Sera. Specimens of human serum from cases ofscalded-skin syndrome were kindly supplied by phy-sicians. The sera from "normal" humans were col-


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lected at the Primary Children's Hospital and Uni-versity of Utah Medical Center, Salt Lake City,from patients who had no known history of scalded-skin syndrome.

Isolation and purification of toxin. Crude toxinwas precipitated from the HIB cultures by additionof aqueous saturated (NH4)2SO4 to a final concentra-tion of 80%. The crude toxin was collected by centrif-ugation after standing at 4 C for 18 h, dissolved indistilled water, and dialyzed against 0.0015 M phos-phate buffer, pH 7.3, to free it of (NH4)2S04. Afterlyophilization, the crude toxin was electrofocused ina glycerol density gradient with 1% carrier ampho-lytes (LKB Instruments Inc.), pH 6 to 8, and thefractions between pH 6 and 7 were collected andpooled. Glycerol and carrier ampholytes were re-moved by column chromatography on Sephadex G-50 (coarse) equilibrated with the 0.0015 M phosphatebuffer. The final product from a single run wasrelyophilized and made up in distilled water to yielda solution with 500 to 1,000 ,ug of protein per ml.

Estimation of protein. The method ofLowry et al.(12) employing Folin reagent was employed to esti-mate the protein content of ET prior to titration insuckling mice or radioiodination.

Polyacrylamide gel electrophoresis. The electro-focused toxin was subjected to electrophoresis in7.5% polyacrylamide gels with sodium dodecyl sul-fate at pH 7.2 by the methods of Seehafer and Weil(20) and Weber and Osborn (21). Gel tubes measured0.5 by 80 mm. The protein was dissociated withmercaptoethanol and sodium dodecyl sulfate prior toelectrophoresis, and the following proteins were runas standards: bovine serum albumin, ovalbumin,alpha chymotrypsinogen, and beta lactoglobulin.Gels 60 cm in length were used, and the electropho-resis was carried out in a Buchler gel electrophoresistank using a Buchler power supply. A constant cur-rent of 5 mA/gel was employed.

Staining of gels. A special procedure developed byFairbanks et al. (6) using Coomassie blue in twoconcentrations was used for staining gels. Gels weredecolorized by diffusion destaining in a Hoefer de-stainer (Hoefer Instruments Co.).

Molecular weight estimation. The method of We-ber and Osborn (21) was used to estimate the molec-ular weight of ET. A bromophenol blue dye markerwas employed, and the ratio of the distance of mi-gration of the dye front to that of the protein bandwas computed (Rf value). Rf as a function of molecu-lar weight was plotted on single-cycle semilog pa-per, and the best straight line was extrapolated.From the Rf of the ET band, the molecular weight ofthe toxin could be estimated using the standardcalibration curve.

Radioiodination. 1251 was obtained from Amer-sham/Searle and diluted to 1 ACi/1l. in 5.5 ,uM KI bythe method of Marchalonis (13). Lactoperoxidasewas obtained from Sigma Chemical Co. and made upfresh each time. A sample protocol was as follows:purified ET, 600 .Lg in 200 Al of 0.0015 M phosphatebuffer; lactoperoxidase 8 ,ug in 80 Al of phosphate-buffered saline (PBS), pH 7.4; 125I (1 ,uCi/ul), 50Ml in 5.5 MuM KI and H202 (freshly diluted), 0.88 mMin PBS, 80 ,l. The above mixture was allowed to

stand at room temperature for 10 min; then an addi-tional 100 ,ul of the 0.88 mM H2O2 in PBS was addedwith mixing. After an additional 10 min, the addi-tion of H202 was repeated and the mixture wasallowed to stand again for 10 min. The reaction wasfinally stopped by the addition of 4 ml of 5 mMcysteine hydrochloride in PBS. Unbound iodine wasremoved by column chromatography on Bio-Gel P-2(Bio-Rad Laboratories) equilibrated with 0.0015 Mphosphate buffer, pH 7.3."Normal" human serum. It is always difficult to

define what is normal with respect to an organismas ubiquitous as S. aureus. However, a human se-rum (obtained from B.W.) that, when diluted 1:40 inTS-BSA buffer [0.15 M NaCl, 0.1 M tris(hydroxy-methyl)aminomethane), 1 mg of bovine serum albu-min per ml, pH 7.41, bound less than 10% of theradioiodinated ET was used. Although the donorhad no history of scalded-skin syndrome, contactwith toxin-producing strains of staphylococci atsome time in the past is highly probable.

Counting of radioactivity. Gamma disintegra-tions were counted on a Beckman Biogammacounter with an 125I isoset. The radioimmunobindingtests were carried out in glass disposable tubes (10by 60 mm) with parafilm caps. This permitted theiraccommodation into the Beckman Biogamma plasticcounting vials. Since the vials were not in contactwith the actual radioactivity, they could be reused.Tests were set up in duplicate, and 50 Al of superna-tant fluid was counted for 20 min in the antigen-binding assays. Controls consisted of antigen plusnormal human serum diluted 1:40 in TS-BSA. Theerror of the count was 3%, and the counting effi-ciency for '25I in the instrument was 86%.

Titration of biological activity of ET. SwissWebster mice less than 5 days old were randomizedand injected intracutaneously in the scapular regionwith 0.05 ml of a dilution of the ET preparationbeing titered. Ten mice per dilution were used, andmice were examined 18 h after injection for evidenceof a positive Nikolsky sign as an indication of exfol-iative activity. Biological activity was expressed asexfoliative units per milliliter or units per milli-gram of protein defined as the reciprocal of thehighest dilution of ET that produced a positive Ni-kolsky sign in 50% of the mice after 18 h.

Immunization. Young, 5- to 7-lb (ca. 2.27 to 3.18kg) New Zeala


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