Measurement of the Staphylococcal Epidermolytic Toxin: a Comparison
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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 .