Immunosorbent Assay. TSST-1 antisera were raised by active immunization of New

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1987, p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 25, No. 2 Detection nd Quntittion of Toxic Shock Syndrome Toxin 1 In Vitro nd In Vivo by Noncompetitive Enzyme-Linked Immunosorbent Assy PATRICIA M. ROSTEN, KAREN H. BARTLETT, AND ANTHONY W. CHOW* Division of Infectious Diseses, Deprtments of Medicine nd Microbiology, University of British Columbi nd Vncouver Generl Hospitl, Vncouver, British Columbi, Cnd V5Z IM9 Received 9 June 1986/Accepted 21 October 1986 Toxic shock syndrome toxin 1 (TSST-1), n exotoxin produced by mny Stphylococcus ureus strins, is implicted s the prime cusl gent of toxic shock syndrome (TSS). A sensitive nd specific noncompetitive enzyme-linked immunosorbent ssy (ELISA) cpble of detecting TSST-1 t concentrtions from 0.5 to 16 ng/mi ws developed. This ssy did not detect other stphylococcl enterotoxins including A, B, CI, C2, C3, D, nd E. Possible interctions with protein A were redily eliminted by pretretment of test smples with 10% norml rbbit serum. The ssy ws dpted for rpid screening of TSST-1 production by S. ureus isoltes in culture superntnts in vitro nd for detection of TSST-1 in vginl wshings of TSS ptients nd helthy controls in vivo. All 35 S. ureus isoltes confirmed to be TSST positive by Ouchterlony immunodiffusion nd 59 of 60 isoltes confirmed to be TSST-1 negtive gve concordnt results by ELISA. Interestingly, toxigenic S. ureus strins isolted from TSS ptients quntittively produced significntly more TSST-1 in vitro compred with toxigenic control strins (P < 0.05, Mnn-Whitney rnk sum test). TSST-1 could be detected by ELISA in three of four vginl wshings collected within 3 dys of hospitliztion from three women with cute menstrul TSS, compred with 0 of 17 wshings from nine TSS ptients hospitlized longer thn 3 dys (P = 0.003, Fisher's exct test) nd 1 of 15 wshings from 14 helthy control women (P = 0.016). This noncompetitive ELISA should be prticulrly useful for rpid screening of TSST-1 production by S. ureus isoltes, for the purifiction nd biochemicl chrcteriztion of TSST-1, nd for humn nd niml studies of the pthogenesis of TSS. Toxic shock syndrome toxin 1 (TSST-1) is believed to be mjor cuse of the disese mnifesttions of toxic shock syndrome (TSS), first described by Todd et l. (18). This toxin, identified s 22- to 24-kilodlton protein with n isoelectric point (pi of pproximtely 7.2, is produced by lmost ll Stphylococcus ureus strins isolted from ptients with cute menstrul TSS, compred with 16 to 23% of isoltes from helthy control women or ptients with non- TSS cses of S. ureus infection (13). In ddition, TSST-1 hs been detected in the serum, urine, nd vginl wshings (M. E. Melish, F. S. Chen, nd M. S. Murt, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, B21, p. 27), s well s brest milk (19) of individuls with typicl TSS. To further explore the role of TSST-1 in the pthogenesis of TSS, we developed simple, sensitive, nd specific enzyme-linked immunosorbent ssy (ELISA) cpble of detecting TSST-1 t concentrtions of 0.5 to 16 nglml in culture superntnts nd biologic fluids. Such n ssy should be prticulrly useful for rpid screening of TSST-1 production by S. ureus isoltes in vitro, for the purifiction nd biochemicl chrcteriztion of TSST-1, nd for humn nd niml studies of the pthogenesis of TSS. MATERIALS AND METHODS Immunorectnts. Reference rbbit ntitoxin to TSST-1 (1:4-diluted stock) s well s purified reference TSST-1 (50-,ug/ml stock) were kindly provided by Merlin Bergdoll (University of Wisconsin, Mdison). Additionl rbbit nti- * Corresponding uthor. TSST-1 ntiser were rised by ctive immuniztion of New Zelnd White rbbits fter grded intrvenous chllenge with purified TSST-1 ccording to the dose schedule of Bergdoll et l. (2). Immunoglobulin frctions of ll rbbit ser to be used were prepred by precipittion with sturted mmonium sulfte by stndrd procedures (7). Briefly, 1 ml of serum nd 0.5 ml of sturted mmonium sulfte were mixed nd stirred for 2 h t room temperture. The precipitted protein ws centrifuged t 1,400 x g for 30 min t room temperture, the superntnt ws discrded, nd the pellet ws dissolved in 1 ml of sline. This procedure ws repeted twice, nd the finl pellet ws dissolved in phosphte-buffered sline (PBS; ph 8.0) nd dilyzed (12 to 14 kilodlton exclusion) ginst PBS (ph 8.0) t 4 C for 48 h with four chnges of buffer. Protein determintions were performed by the method of Lowry et l. (9). Ammonium sulfte-precipitted nti-tsst-1 ws conjugted to lkline phosphtse with glutrldehyde by the method of Voller et l. (20). A 1-mg smple of precipitted nti-tsst-1 nd 2.5 mg of lkline phosphtse enzyme (P-0405; Sigm Chemicl Co., St. Louis, Mo.) were mixed, glutrldehyde (Sigm G-5882) ws dded to finl concentrtion of 0.2%, nd this ws stirred for 2 h t room temperture to llow conjugtion. The conjugte ws then dilyzed (12- to 14-kilodlton exclusion) ginst PBS (ph 7.0) for 24 h with two chnges of buffer. Further dilysis ws performed ginst 0.05 M Tris hydrochloride (ph 8.0) for 24 h with two chnges of buffer. The conjugte ws then diluted to 2 ml with 0.05 M Tris hydrochloride contining 1% bovine 327

2 328 ROSTEN ET AL. serum lbumin nd 0.02% sodium zide nd stored t 4 C in the drk. ELISA procedure. The noncompetitive, ntibody enzymeconjugted ELISA procedure ws used for TSST-1 detection. Ammonium sulfte-precipitted reference rbbit nti- TSST-1 s well s nonimmune control ser (100 pl) diluted t 1:10,000 in 0.05 M crbonte buffer (ph 9.6) were pssively dsorbed to the inner wells of polystyrene microtiter pltes (Immulon t; Dyntech Lbortories, Inc., Alexndri, V.) by incubtion t 20 C for 18 h. Unbound ntitoxin ws removed by three 2-min wshes with PBS contining 0.05% Tween 20, ph 7.4 (PBS-T). Purified TSST-1 reference stndrd (serilly diluted from 16.0 to 0.5 ng/ml in PBS-T), test smples pretreted with norml rbbit serum (10% [vol/vol] finl concentrtion) to eliminte possible sources of protein A, nd dditionl regent controls were pipetted in duplicte in 100-pul volumes to their respective wells. This ws followed by incubtion t 37 C for 2 h nd wshing to remove unbound toxin. After ddition of the conjugte (100 pul) nd nother 2-h incubtion t 37 C, the pltes were wshed, treted with p-nitrophenyl phosphte (1 mg/ml in 10% diethnolmine buffer [ph 9.8], 100 pul) (BDH, Poole, Englnd), nd incubted t 37 C for 90 min to llow the enzyme rection to proceed. Pltes were red t 405 nm in Titertek multiscn spectrophotometer (Flow Lbortories, Inc., Mc- Len, V.) Liner regression nlyses of the stndrd reference toxin concentrtions were clculted by plotting bsorbnce (405 nm) versus log2 TSST-1 concentrtion. TSST-1 concentrtions in test smples were predicted from the reference toxin regression equtions derived during ech ssy procedure. The specificity of the ELISA for TSST-1 ws exmined by inclusion of nonimmune rbbit serum lcking nti-tsst-1, by pretretment of TSST-1 with rbbit ntitoxin, nd by exmintion for possible cross-rectivity with protein A nd other stphylococcl enterotoxins, including enterotoxins A, B, C1, C2, C3, D, nd E (Toxin Technology Inc., Mdison, Wis.) in concentrtions of 1,000, 100, nd 10 ng/ml. TSST-1 detection in S. ureus culture superntnts. The S. ureus isoltes tested for in vitro TSST-1 production were vginl isoltes from TSS ptients or helthy control women. Culture superntnts were prepred from subcultures of S. ureus grown in brin hert infusion (BHI) broth (Difco Lbortories, Detroit, Mich.) with shking t 37 C for 18 to 20 h in mbient tmosphere. Cells were removed by centrifugtion (30,000 x g for 15 min), nd culture superntnt ws filter sterilized nd frozen t -70 C before testing. Ech culture superntnt ws tested for TSST-1 both by Ouchterlony immunodiffusion nd by ELISA with nd without overnight preincubtion t 4 C in norml rbbit serum (10%, vol/vol) to eliminte ny interfering effect of protein A. Pretreted test smples were centrifuged in Beckmn Microfuge to remove prticulte mtter before testing in microtiter wells t net, 1:10, nd 1:100 dilutions in PBS-T. Detection of TSST-1 by Ouchterlony immunodiffusion ws exmined in 1% Noble gr (Difco Lbortories) with reference rbbit ntitoxin (1:36 dilution) in the center well. TSST-1 detection in vginl wshings of TSS ptients nd helthy controls. Vginl wshings were collected from nine the cse definition for menstrul TSS ptients who fulfilled ccording to the Centers for Disese Control nd were studied in Vncouver between August 1980 nd Februry 1985 (5). Wshings were lso collected from 14 helthy control women recruited from the University of British Columbi Student Helth Service. Informed consent ws obtined from ll subjects. Vginl wshings were obtined J. CLIN. MICROBIOL. with 10 ml of sterile pyrogen-free sline from the posterior vginl fornix, using plstic pipette nd under direct visuliztion. The wshings were immeditely centrifuged (90 x g for 10 min t 4 C), nd the superntnt ws filter sterilized nd stored t -70 C. Quntittion of TSST-1 in vginl wshings ws performed by the bove ELISA method with severl dditionl controls. Vginl wshings to be tested for TSST-1 were diluted 1:2, 1:4, nd 1:8 in PBS-T nd exmined in duplicte in 100-pi volumes. Vginl wshings giving positive results were retested with nd without 10% norml rbbit serum pretretment. Pooled vginl wshings devoid of TSST-1 from helthy women (C-0) served s negtive control, nd pooled wshings with TSST-1 dded (50 ng/ml) served s positive control (C-50). These were diluted 1:2, 1:4, nd 1:8 in PBS-T nd tested in 100-pil volumes in duplicte. A third control for specificity consisted of the C-50 control to which hd been dded the sme reference rbbit nti-tsst-1 used to cot the microtiter wells; this ws lso diluted 1:2, 1:4, nd 1:8 in PBS-T nd tested in 100-pi volumes in duplicte. Sttisticl methods. All ssys were performed in duplicte. Regression coefficients were clculted by the lestsqures method. Sttisticl comprisons were performed by Fisher's exct test for discrete vribles nd by the Mnn- Whitney rnk sum test (two tiled) for continuous vribles (16). RESULTS Stndrdiztion of ELISA procedure. The Immulon I (Dyntech Lbortories) polystyrene microtiter plte ws chosen over severl other brnds, including two other polystyrene pltes (Linbro Titertek, Flow Lbortories; nd Nunc Immunoplte t, Vngrd Interntionl, Neptune, N.J.) nd polyvinyl plte (Immulon II; Dyntech Lbortories). The Immunulon I plte ws found to be superior for its mximl specific binding of ntitoxin nd for miniml nonspecific binding of other immunorectnts (dt not shown). Binding of specific ntitoxin ws determined with got nti-rbbit IgG conjugted to lkline phosphtse (Sigm Dignostics). Nonspecific binding of immunorectnts ws determined in ssys in which the coting rbbit nti-tsst-1 ws omitted, nd pltes were incubted with coting buffer nd got nti-rbbit immunoglobulin G conjugted to lkline phosphtse. Optiml dilutions of coting ntitoxin nd conjugte were determined by series of checkerbord titrtions to chieve the gretest sensitivity nd specificity while requiring the lest mount of immunorectnts. Optiml working dilutions were 1:10,000 for the coting ntitoxin (0.33 pug of protein per well) nd 1:750 for the ntitoxin conjugte. At these dilutions of ntitoxin nd in the presence of 16 ng of TSST-1 reference stndrd per ml, constnt rte of enzymtic hydrolysis for the p-nitrophenyl phosphte substrte could be demonstrted over 90 min of incubtion t 37 C (dt not shown). Reproducibility of ELISA. A typicl stndrd curve generted with reference TSST-1 over the rnge of 0.5 to 16.0 ng/ml is shown in Fig. 1. The regression coefficient (r) obtined from 20 seprte experiments ws ± (95% confidence limits). Considerble within-dy, nd dyto-dy, vritions owing to plte-to-plte differences in binding chrcteristics were observed (dt not shown). These results indicte tht test smples nd reference TSST-1 stndrds must be determined on the sme plte to obtin relible results. The coefficient of vrition from repeted testing of the sme TSST-1 ws consistently less thn 10%, confirming the reproducibility of the ELISA.

3 VOL. 25, 1987 Specificity of ELISA. The specificity of the ELISA ws demonstrted in dditionl experiments in which microtiter wells were coted with 1:10,000 dilution of mmonium sulfte-precipitted nonimmune rbbit serum tht ws shown to be negtive for nti-tsst-1 by Ouchterlony immunodiffusion (Fig. 1). The fct tht no dose-response reltionship ws produced with this control, s ws demonstrted in wells coted with nti-tsst-1 immune serum, further demonstrted the specificity of the ELISA for TSST- 1. Other negtive controls included wells with no toxin dded nd wells with toxin tht hd been pretreted with ntitoxin. Furthermore, no flse-positive results were obtined with purified preprtions of other S. ureus enterotoxins including A, B, C1, C2, C3, D, nd E t concentrtions rnging from 10 to 1,000 ng/ml. A crude preprtion of enterotoxin D t concentrtion of 1,800 ng/ml did demonstrte flse-positive results equivlent to 1.29 ng of TSST-1 per ml (0.07%). This preprtion did not immunoprecipitte with rbbit nti-tsst-1 by Ouchterlony immunodiffusion. The flse-positive rectivity in the ELISA, presumed to be due to protein A, ws completely eliminted when the crude enterotoxin D preprtion ws pretreted with 10% norml rbbit serum. Elimintion of protein A effect with norml rbbit serum. The interfering effect of protein A nd its elimintion by pretretment of test smples with 10% norml rbbit serum were further chrcterized (Fig. 2). Stndrd concentrtions of reference TSST-1 (0.5 to 16 ng/ml, finl concentrtions) were prepred in BHI broth nd pretreted with protein A (1,000 ng/ml; Sigm P-6650), protein A plus 10% norml rbbit serum, or 10% norml rbbit serum lone. Negtive controls included BHI broth without TSST-1 dded. Quntittion of TSST-1 in these different experimentl groups ws done in prllel by our ELISA procedure s described bove nd compred with positive controls in which reference E U"% u 1.0- c: " //' -r/', TSST-1 (ng/ml) FIG. 1. Reproducibility nd specificity of ELISA. Typicl curve (-----) generted with stndrd reference TSST-1 over the concentrtion rnge of 0.5 to 16.0 ng/ml in wells coted with reference rbbit nti-tsst-1 ntibody; regression coefficient (r) = (95% confidence intervls for 20 experiments). Specificity of ELISA ws demonstrted in identicl experiments in which microtiter wells were coted with 1:10,000 dilution of rbbit serum devoid of nti-tsst-1 (-). ELISA FOR TOXIC SHOCK SYNDROME TOXIN 1 DETECTION E 1.4- c: C: 0.6 u-z 1.2 -> u r->1.0- c).0 O M1~ TSST-1 (ng/ml) FIG. 2. Comprison of TSST-1 detection by ELISA in PBS-T (O) or BHI broth (A) lone or in BHI broth nd pretreted with 1,000 ng of protein A per ml (M), 10% norml rbbit serum (E), or protein A plus 10% norml rbbit serum (Z1 ). The interfering effect of protein A ws completely eliminted by the ddition of 10% norml rbbit serum without loss of sensitivity when compred with stndrd curves generted in PBS or BHI broth lone. TSST-1 stndrds were prepred in PBS-T. The results clerly demonstrte (i) the interfering effect of protein A in the ssy, nd (ii) the complete elimintion of this effect by the ddition of 10% norml rbbit serum, without loss of sensitivity or specificity over the rnge of reference TSST-1 stndrd concentrtions studied. Detection of TSST-1 production by culture superntnts of S. ureus isoltes. The results of testing for TSST-1 production in culture superntnts of 95 S. ureus isoltes re shown in Fig. 3. Among the 60 isoltes confirmed to be negtive for TSST-1 by immunodiffusion, 59 lso hd undetectble toxin by ELISA. Among the 35 isoltes confirmed to be positive for TSST-1 by immunodiffusion (24 were isolted from TSS ptients), ll hd detectble toxin by ELISA. Negtive controls included uninoculted BHI broth treted in identicl fshion. The necessity of pretreting the culture superntnts with 10% norml rbbit serum to bsorb out protein A produced by the isoltes is clerly illustrted. Of 60 isoltes negtive for TSST-1 production s tested by immunodiffusion, 42 would be flsely positive by ELISA if culture superntnts were not pretreted with 10% norml rbbit serum to remove the interfering effect of protein A. Similrly, ll 35 isoltes tht were positive for toxin production by immunodiffusion yielded higher vlues tf TSST-1 in untreted compred with treted culture superntnts. Of interest, isoltes from TSS ptients produced significntly higher concentrtions of TSST-1 in culture superntnts compred with toxin-producing strins from non-tss ptients (P < 0.05, Mnn-Whitney two-tiled rnk sum test) (Fig. 4). Detection of TSST-1 in vginl wshings of TSS ptients nd helthy control women. A totl of 36 vginl wshings from 9 menstrul TSS ptients nd 14 helthy control women were 329

4 330 ROSTEN ET AL. E * Nontox gen ic S. ureus (n=60) 0 *. e. e. * " *- Toxigenic S. ureus * (n-35) At &A l Medi Control (n-l7).e *o -..B * Without With Without With Without With NRS NRS NRS NRS NRS NRS PRETREATMENT OF CULTURE SUPERNATANTS FIG. 3. Effect of 10% norml rbbit serum (NRS) on interference by protein A during detection of TSST-1 in culture superntnts of S. ureus by ELISA. S. ureus isoltes were previously determined by Ouchterlony immunodiffusion to be toxigenic (A) or nontoxigenic (0). ELISA results of culture superntnts were compred with those of uninoculted medium controls (M) with or without the ddition of NRS. vilble for quntittion of TSST-1 by ELISA (Tble 1). Three of four specimens collected within 3 dys of hospitliztion from three women with cute TSS hd detectble TSST-1, compred with 0 of 17 wshings from 9 TSS ptients hospitlized longer thn 3 dys (rnge, 6 to 63 dys; medin, 30 dys) (P = 0.003, Fisher's exct test) nd 1 of 15 wshings from 14 helthy control women (P = 0.016). Specific TSST-1 concentrtions detected in vginl wshings from the two women with cute TSS nd single control subject re shown in Tble 2. TSST-1 concentrtions from vginl wshings of 29-yer-old womn with typicl menstrul TSS (subject A) were highest on dy 1 of hospitliztion nd rpidly declined therefter, coincident with ntistphylococcl therpy nd elimintion of S. ureus in the vginl flor. Vginl wshings from 22-yer-old helthy control womn (subject C) were initilly negtive for TSST-1 concurrent with negtive vginl culture for isoltion of S. ureus. A subsequent wshing obtined 1 month lter ws positive for TSST-1 when her concurrent vginl culture ws lso positive for TSST-1-producing S. ureus. Her serum ntibody titer to TSST-1 ws within the 69th J. CLIN. MICROBIOL. percentile of norml nti-tsst-1 titers s determined from 87 helthy women by n ELISA method (P. M. Rosten, K. H. Brtlett, nd A. W. Chow, submitted for publiction). DISCUSSION Severl ssy techniques hve been developed to demonstrte nd quntitte TSST-1 in S. ureus culture superntnts in vitro, including nlytic isoelectric focusing in polycrylmide gels (15), Ouchterlony immunodiffusion (13), immunoblotting (21), pssive ltex gglutintion (8), rdioimmunossy (Melish, et l., Abstr. Annu. Meet. Am. Soc. Microbiol. 1983), nd competitive ELISA with TSST-1 s the enzyme conjugte (12). Anlytic isoelectric focusing nd immunoblotting re both qulittive ssys, nd the former lcks specificity (13). Ouchterlony immunodiffusion techniques, including severl modifictions such s the microslide immunodiffusion test (4), the optiml sensitivity plte method (2), nd the single gel diffusion tube method (14), cn be used for quntittion of TSST-1, but these generlly lck sensitivity, require excessive use of regents, nd cnnot be dpted for btch testing of lrge smples. Although use of rdioimmunossy should improve both sensitivity nd specificity, this method requires expensive equipment nd hndling of rdioctive mterils. A reversed pssive ltex gglutintion ssy hs been developed by Igrshi et l. (8) E e-i. c-) C.,, C,) -J o n A* LA *A- *- *- A TSS Associted Non-TSS Control S. ureus S. ureus (n=24) (n=11) FIG. 4. Quntittive TSST-1 production s mesured by ELISA mong 24 toxigenic S. ureus vginl isoltes from TSS ptients (A) nd 11 toxigenic S. ureus vginl isoltes from non-tss helthy women (,A) (P < 0.05, Mnn-Whitney rnk sum test).

5 VOL. 25, 1987 ELISA FOR TOXIC SHOCK SYNDROME TOXIN 1 DETECTION 331 nd cn detect nnogrm quntities of TSST-1. Although promising, its specificity nd possible interference by protein A, however, will require dditionl evlution. Most recently, Prsonnet et l. (12) described competitive ELISA cpble of quntitting 0.03,ug oftsst-1 per ml in S. ureus culture superntnts. This ssy ws not influenced by protein A, but did cross-rect minimlly with stphylococcl enterotoxins A, D, nd E. Furthermore, competitive ELISA with enzyme-lbeled ntigen requires purified TSST- 1 in reltively lrge mounts for preprtion of the enzymentigen conjugte. A more importnt disdvntge of this method is tht the competitive ELISA might not be redily dpted for quntittion oftsst-1 in biologic fluids or tissue extrcts. This is due to the need to incubte enzyme-lbeled TSST-1 directly with biologic fluids or tissue extrcts which themselves my contin proteses or enzyme inhibitors nd my significntly lter the kinetics nd ctivity of the enzyme conjugted to TSST-1, thus severely ffecting the ccurcy of the test (6). The noncompetitive ELISA described here hs number of dvntges over the other methods described bove. It is sensitive, specific, simple, economicl, nd does not require hndling nd disposl of rdioctive mterils. It obvites the necessity of repeted nd elborte procedures for purifiction of TSST-1. Importntly, it cn be redily dpted to quntitte TSST-1 in biologic fluids nd tissue extrcts, including urine, serum, nd vginl wshings. Its lower limit of sensitivity, 0.5 ng/ml, ppers to be dequte for TSST-1 detection in culture superntnts s well s vginl wshings. It does not cross-rect with other stphylococcl enterotoxins, nd the potentil interference by protein A is redily eliminted by pretretment of smples with 10% rbbit serum. Protein A is produced by most strins of S. ureus, but the mount relesed in culture my vry from strin to strin. Since protein A hs the unique bility to bind the Fc portion of immunoglobulin, primrily immunoglobulin G, its presence cn redily interfere with the ELISA by binding to both the coting s well s the enzyme-conjugted ntitoxin, s is demonstrted in Fig. 2. Berdl et l. (1) determined tht the presence of protein A in concentrtions greter thn 500 ng/ml could ffect the detection of stphylococcl enterotoxins A, B, nd C from culture superntnts by similr ELISA technique. These investigtors used ffinity chromtogrphy to remove protein A before the enterotoxin ssys. Our method of protein A dsorption with 10% rbbit serum ppers to be eqully effective but is much more simple. Since both the sensitivity nd specificity of our ELISA method pper stisfctory, we did not feel tht the use of monoclonl ntibodies to TSST-1 for coting or ntibody enzyme conjugtion, s ws recently described (D. E. Wells, M. W. Reeves, L. M. Grves, nd R. M. McKinney, Abstr. Annu. Meet. Am. Soc. Microbiol. TABLE 1. Detection of TSST-1 in vginl wshings of menstrul TSS ptients nd helthy control women No. positive/totl tested Subjects Women Wshings (p) (p) TSS ptients 2/12 3/21 <3 dys illness 2/3 3/4.3 dys illness 0/9 (0.045) 0/17 (0.003) Control 1/14 (0.063) 1/15 (0.016) P vlue by Fisher's exct test, compred with TSS ptients tested within 3 dys of cute illness. TABLE 2. Wshings Quntittion of TSST-1 in vginl wshings of TSS ptients nd control women TSST-1 detected (ng/ml) With NRS Without NRS TSS Subject A Dy 1b Dy Dy Subject B Dy Dy Control (subject C) April My Pretretment with 10% norml rbbit serum (NRS) to eliminte ny interfering effect of protein A. b Dy fter hospitliztion when specimen ws collected. 1985, V17, p. 391), or the use of immunoglobulin subçlsses or Fb-specific preprtions will offer ny significnt dvntge. Indeed, the use of polyclonl ntitoxin my hve provided n element of mplifiction in the sensitivity of our ssy by possibly permitting severl different enzymelbeled ntibody epitopes to bind single TSST-1 molecule with different ntigenic domins. Use of polyclonl ntitoxin lso obvited the need for tissue culture fcilities. Our finding tht toxigenic S. ureus strins isolted from TSS ptients produced significntly more TSST-1 in vitro compred with toxigenic control strins is of interest. The bility to produce lrge quntities of TSST-1 in vivo my be n importnt virulence fctor. Conversely, locl nd mucosl host fctors which permit optiml production of TSST-1 in vivo by colonizing or infecting strins of S. ureus my be importnt risk fctors of TSS. In this regrd, Mills et l. (10, 11) hve noted the importnt role of mgnesium ion on TSST-1 production in vitro. They postulted tht use of certin tmpon brnds could hve precipitted menstrul TSS since these tmpon fibers my vidly bind mgnesium ions in vivo nd cuse striking increse in TSST-1 production. Alterntively, since our studies indicte tht TSST-1 cn be detected in vginl wshings of some helthy women, the presence of neutrlizing systemic or locl ntibodies to TSST-1 my be n importnt protective mechnism in norml individuls. In support of this concept, we (Rosten et l., submitted) nd others (2, 3, 17) hve demonstrted tht nti-tsst-1 is generlly low or bsent in cute ser of menstrul TSS ptients s compred with ser of gemtched control women nd tht serologic unresponsiveness ppers to be prominent hllmrk in such ptients. It is cler tht the vilbility of simple, sensitive, specific, nd reproducible ssy s described here, cpble of detecting TSST-1 in concentrtions s low s 0.5 ng/ml both in vitro nd in vivo, should gretly fcilitte future studies which re urgently needed to further our understnding of the biologic effects of TSST-1 nd the pthogenesis of toxic shock syndrome. ACKNOWLEDGMENTS This work ws supported in prt by Medicl Reserch Council of Cnd grnt (MT7630) nd British Columbi Medicl Services Foundtion grnt (85-6). The technicl ssistnce of A. M. Goldring is much pprecited.

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