THE JOURNAL OF I NVESTIGAT IV E D ERMATOLOGY. 67:526-53 1. 1976 Vol. 67. :-':0.4 Copyri ght © 1976 by T he Wi ll iams & Wi lkins Co. Prin ted in U.S.A. MEASUREMENT OF THE STAPHYLOCOCCAL EPIDERMOL YTIC TOXIN: A COMPARISON OF BIOASSAY, RADIAL IMMUNODIFFUSION, AND RADIOIMMUNOASSAY KIRK D. WUEPPER, M.D., DIA NE HAAS BAKER, M.D., AND ROBERT L. DIMO ND , t:A .D. Department of Dermatology, School of M edicine, University of Oregon Health Sciences Center, Portland, Oregon, U.S.A. T hree methods of m easuring t he epidermolytic toxin of Staphy lococcus aureus-bioassay in newborn m ice, radial immunodiffusion , a nd ra dioimmunoassay-were compa red for reproducibility, specificity, and sensit ivity. The bioassay is highly specifi c a nd remains the only functional assay. It is reproducible only if newb orn mice of t he same age a re used . The time required fo r epidermolysis fo llows a dose- response relationship only if concent rations of toxin large enough to cause peeling in 90 min or less are used . This limits the sensit ivity of t he bioassay to a bout 5 J.L g per m\. S ingle radial immunodiffusion in agar is a specific and reproducible assay method , but its sensit ivity is also about 5 J.L g per m\. A radioimmunoassay was established by t he F arr technique using purified epidermolysin radiolabeled wi t h '25 iodine. This assay was highly reproducible a nd specific. The staphylo­ coccal products, a-toxin and enterotoxins A and B , did not cross-react with ant i-epider­ m olysin ant ibodies. The sensitivity of t he radioimmunoassay is 20 n g per m\. In 1970 M elish and G lasgow proposed t ha t a n oped other more sensit ive a nd reproducible ways to epidermolytic toxin was responsible for t he staphy­ m easure t he epidermolytic toxin, radial immuno­ lococcal scalded-skin syndrome [1]. Since t hen t his diffusion, and radioimmunoassay. toxin, (called by various a ut hors epidermolysin, epidermolytic toxin, exfoliatin, exfoliating toxin), MATERIALS AND METHODS has been purified and part ia lly characterized by Epidermolytic toxin was obtain ed and purified as several investigators [2-6]. Accurate measurem ent previously described l6 ] from Strain EV , a phage group of toxin is necessary to furt her ch aracteri ze it a nd II, type 55/71, coagulase-positive S . aureus, isolated from to delineate its role in Staphy lococcus aureus­ a case of staphylococcal scalded- skin sy ndrome. An ti­ serum to epidermolysin was produced in rabbits as associated scalded -skin syndrome. previously desc ri bed [6 j. M easurement of epidermolysin has been limited to a functional bioassay in newborn mice. In our Bioassay laboratories, bioassay resul ts were found to be Newborn Webster/Swiss mice (Simo nso n Laborato­ q ui te vari able when t he same amount of toxin was ri es, Gi lroy , Cali f.) of known age (1 to 5 days) we re injected into several m ice from different li tters. injected subcutaneo usly, in duplicate, in to the nape of Because of t his observation, t he fo llowing study the neck. We used 25-gauge needles, and 0.02- ml volumes was underta ken to examine the factors producing of materi al were assayed for epidermolytic tox in activity. variability in b ioassay resul ts. We have a lso d evel- The time required for production of a locali zed Nikolsky sign in the skin ove rlyi ng the site of injection was recorded as the end poin t. Manuscript received March 25, 1976 ' accepted fo r publication May 24, 1976. ' Radial Immunodiffusion Dr. Wuepper is the rec ipient of Research Caree r Development Awa rd 5 K04 AM 40086. Radial immunodiffusion in agar was carried out ac­ Dr. Baker is the rec ipient of an Individual Research cording to the method of Mancini et al [7] . In brief, a Fellowship 5 F22 AM 00900 and the Joseph Gardner 1.5% agar ge l plate (lonagar No.2, Difco , Detroit, Mich.) Hopkins Prize presented at the Society fo r Investigative was prepared with 7.5 % rabbi t ant iserum to epider­ Dermatology meeting, Atlantic City, N. J., May 3, 1975. molys in in corporated into the agar. Circul ar well s were A preliminary report of these studies has bee n pub­ cut into the agar to all ow introduction of 7- /l1 amounts of li shed (C lin Res 23: 167 A, 1975). the material to be assayed fo r epidermolys in . Radi al Reprint requests to : Dr. K. D. Wu epper, Department diffusion took place over a 7- to 10-day peri od at roo m of Derm atology, School of Medi cin e, Uni ve rsity of Ore­ gon Health Sciences Ce nter, Portland, Oregon 97201. temperature in humidity chambers. The diameters of the Abbrev iations: circ ul ar prec ipi tin bands that formed were measured ABC-33: antigen-binding capacity ca lculated from using a mi croscope oc ul ar eq uipped with a micrometer, the reciprocal of the serum dilu tion which prec ipi­ and the areas of the circles were calcul ated. Known tates 33 % of the antigen added amoun ts of epiderm olysi n we re introduced in to wells in BS A: bov in e se ru m albumin the same agar ge l pl ate in which unknown s were mea- 526 Oct. 1976 MEASUREMENT OF S. aureus EPlDERMOLYTIC TOXIN 527 M ou~e !\eJ! lOa ys) s ured , allowing a standard cu rve to be prepared for each '00 0----<> 0 ~ , o ~ assay run. ~25 ! 05 ~45 ! 05 Radiolabeling of Epidermolysin .. !5'" 100 Purifi ed epid ermolysin was radioiodinated wi t h , I" iodine (1 mCi/50 Ill , New England Nuclear, Boston, ~ o ~ o Mass.) and 50 III of chloramine T (1 mg/ ml) were added " ~ to purified epidermolysin (1200 Il g in 0. 5 mI). Fifty III of ~ sodium metabisulfi te (1 mg/ml) we re added to terminate the reaction. Bovine se rum albumin (BSA) (Schwa rz/ Mann, Orange burg, N .Y.), 0.2% in saline, was added as ® carrie r. Free "'I was separated from radiolabeled epider­ rnolys in by extensive dia lysis and passage over a Sepha ­ d ex G-50 column [8 1. ~ Saturated ammonium sulfate precipitation was inves­ 240 210 t igated as a means of separating free from ant ibody­ bound epidermolysin [9 J. Radiolabeled epidermolys in FI G. 1. Dose- response curve to highly purified epider­ was in cubated overnight at 4°C. with phosphate-buffered molysin injected intracutaneously in mice of varying ages. saline, ra bbit serum, human serum, or rabbit antiserum The time required for production of a locali zed Nikolsky to epidermolysin . Cold sat urated ammonium sulfate was sign in skin overlying t he injection site was recorded as then added in amounts sufficient to obtain final concen­ the end point. t rations of ammonium sulfate from 20 to 90%. The precipitate which formed was centrifuged, separated from the supernatant, and counted in a we ll- type ga mma overl yi ng the injection site in response to rubbing counter. with a finge r (Nikolsky sign), was clear-cut and could be measured accurately wi t hin a 2- to 5- min Characterization of the Rabbit Antiserum Used in the in terval. There was close agreement between du­ Radioim munoassay plicate animals. Rabbit antiserum to epidermolysin was tested for its The relationship between t he amount of epider­ ability to bi nd radiolabeled epidermolysi n as fo ll ows: 0.5 molysin injected (dose) and the time required for m! of doubling di lutions of anti-epidermolysin was incu­ epidermolysis to develop (response) was linear only bated overni ght at 4°C. with constant amounts of if the response occurred in less t han 60 to 90 min. (I2>IJepidermolysin (100 ng in 0.5 mI) . Following incuba­ The results became erratic and nonlinear there­ tion, 1.0 ml of saturated ammonium sulfate was added. Precipitates were cent rifuged at 4°C at 3000 g for 20 min, after. As shown in Figure 1, the dose- response washed with 50% saturated ammonium sulfate, recen ­ varied with t he age of t he mice injected ; t he assay t rifuged, and radioactivity in precipitates was co unted in was more sensitive in mice less t han 24 hr old . a well-type ga mma counter. To exclude the possibility that deterioration of the toxin was responsible for the delayed responses Assay System observed in older animals, experiments were re­ To assay unknown samples for epidermolysin the peated in 1-day-old mice. The results coincided fo llowing system was used: 0.5 ml of' rabbit anti-epider­ with the first determination, confirming that the molysin (1 :750) was added to 0.5 ml of [1251] epidermolysin toxin was stable. The minimum time required for and 0.5 ml of varying dilutions of sample to be assayed . epidermolysis was about 15 min. This was pre­ Following incubation overnight at 4°C, 1.5 ml of satu­ sumably due to the time required for passive dif­ rated a mmonium sulfate was added to effect precipitation fusion from the intradermal injection site to the of antibody-bound epidermolysin. Precipitates were cen­ trifuged, washed with 50% saturated a mmonium sulfate, overlying epidermis. Apparently, systemic adsorp­ recentrifuged, and radioactivity was determined . A stan­ tion also takes place, since animals which received dard curve was co nstructed with known q uantit ies of higher doses would develop a positive Nikolsky sign epidermolysin, measured by the method of Lowry et al at any skin site several hours after the initial injec­ (1 0 I with BSA as standard .