Vol. 33, No. 3INFECTION AND IMMUNITY, Sept. 1981, p. 862-8690019-9567/81/090862-08$02.00/0

Enzyme-Linked Immunosorbent Assay for Detection of TypeA Streptococcal Exotoxin: Kinetics and Regulation During

Growth of Streptococcus pyogenesCLIFFORD W. HOUSTONt AND JOSEPH J. FERRETTI*

Department ofMicrobiology and Immunology, University of Oklahoma Health Sciences Center,Oklahoma City, Oklahoma 73190

Received 9 February 1981/Accepted 3 June 1981

We describe the detection and quantitation of type A streptococcal exotoxin(erythrogenic toxin, streptococcal pyrogenic exotoxin) by an enzyme-linked im-munosorbent assay. This sensitive and specific technique detected microgramamounts oftype A exotoxin and was useful for studying the kinetics and regulationof type A exotoxin production during the growth of Streptococcuspyogenes NY5.Maximum production of type A exotoxin was observed during the mid-log phaseof growth, similar to the production of other streptococcal extracellular products.When S. pyogenes NY5 was grown at 420C, decreases in both growth and type Aexotoxin production were observed. The results obtained when we studied theinfluence of nutrient additives and metal ions on the production of type Aexotoxin led to the conclusion that none of these factors significantly affectedtype A exotoxin synthesis and that regulation was constitutive.

The group A streptococci produce a variety ofextracellular products, some of which are specif-ically called streptococcal exotoxins (23). A num-ber of synonymous terms have been used todescribe these exotoxins, including Dick toxins,scarlet fever toxins, erythrogenic toxins, andstreptococcal pyrogenic exotoxins. There are atleast three antigenically distinct streptococcalexotoxins, namely, types A (18, 40), B (18, 40),and C (40). The biological activities associatedwith the streptococcal exotoxins include the pro-duction of erythematous reactions in humanskin and rabbit skin (11, 12, 18), which may bethe result of a secondary toxic moiety (35), pyr-ogenicity (7, 22, 36, 40), enhancement of suscep-tibility to lethal endotoxin shock in rabbits, mice,and monkeys (22, 37, 40), alteration of the blood-brain barrier to endotoxin (36), blockage of thereticuloendothelial clearance of colloidal carbonin rabbits (14), immunosuppression (15), en-hancement of the antibody response to sheeperythrocytes in rabbits and mice (13, 15, 16), andmitogenic activity for human and rabbit lym-phocytes (3, 20, 21, 24, 28, 32).Most of the biological assays which have been

developed to detect the activities of the strep-tococcal exotoxins have been nonquantitative,and some of these assays are difficult to perform.A method which has been used in attempts toquantitate the streptococcal exotoxins is the he-

t Present address: Department of Microbiology, Universityof Texas Medical Branch, Galveston, TX 77550.

magglutination inhibition test (36). The Limulusamoebocyte lysate assay (8) and an immunodif-fusion assay (9) have been used qualitatively todetect streptococcal exotoxins. In this paper wedescribe the use of an enzyme-linked immuno-sorbent assay (ELISA) for the detection andquantitation of type A streptococcal exotoxin.This assay was used to study the kinetics of typeA exotoxin synthesis during the growth of Strep-tococcus pyogenes NY5 type 10, as well as todetermine whether factors such as temperature,metal ions, and nutritional additives influencethe regulation of synthesis of type A exotoxin.

(A preliminary report ofsome ofthese findingswas presented at the Seventh InternationalSymposium on Streptococci and StreptococcalDiseases, Oxford, England, September 1978[30].)

MATERIALS AND METHODSBacteria. The primary strains of S. pyogenes

known to produce type A streptococcal exotoxin andused in this study were strains NY5 type 10 (12) andT253(T12) (44). Strain T253 does not produce extra-cellular type A exotoxin. Strain K56(8627) was con-structed by S. K. Nida (S. K. Nida and J. J. Ferretti,manuscript in preparation).

Media. The standard liquid medium used for bac-terial growth was Todd-Hewitt broth (Difco Labora-tories). The medium used to grow toxigenic strainswhen detection of type A exotoxin was desired was aTodd-Hewitt dialysate medium which was preparedby dialyzing 100 ml of 10-fold-concentrated Todd-Hewitt broth (30 g/100 ml of distilled water) in 500 ml

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of distilled water for 16 h at 4.00C. The resultingdialysate was autoclaved and, when necessary, supple-mented with one of the following sterile components:2% glucose; 60% yeast extract dialysate (Difco); 5%horse serum plus 0.38% K2HPO4 and 1% CaCl2; 2%glucose plus 0.9 IM CaCl2; or 0.5 mM cysteine (allfinal concentrations). Brain heart infusion broth(Difco) was used in the nutritional experiments.Type A streptoeoecal exotoxin. The purification

of type A streptococcal exotoxin was accomplished bythe method of Cunningham et al. (9), with the follow-ing modifications. A 1-liter culture of S. pyogenes NY5type 10 which was grown to mid-log phase in Todd-Hewitt dialysate was used to inoculate 19 liters ofTodd-Hewitt dialysate medium. This batch culturewas incubated at 370C for an additional 7 to 8 h andthen rapidly cooled in an ice bath. The cells wereremoved by centrifugation, and the supernatant waspassed through a positive-pressure membrane filterapparatus (pore size, 0.45 um; Millipore Corp.). Theresulting culture filtrate was concentrated with anAmicon dialyzer/concentrator to a volume of 1 liter,dialyzed, and lyophilized; the resulting preparationwas designated the concentrated crude extract. A pro-tein determination was made at each step in theisolation and purification procedure by using themethod of Hartree (17).The concentrated crude extract (350 mg) was placed

on a QAE-Sephadex A50 (Pharmacia Fine Chemicals)column, and stepwise elution was performed with thefollowing buffers: 0.1 M imidazole-acetic acid, pH 7.0;0.1 M imidazole-acetic acid-0.1 M NaCl, pH 5.0, and0.1 M sodium acetate-acetic acid, pH 4.0. Four proteinpeaks were obtained, and most of the type A exotoxinwas in the third peak. After dialysis the protein ma-terial was subjected to preparative isoelectric focusingwith pH 4 to 6 ampholyte (Bio-Rad Laboratories).The protein sample from the pH 5 region containedthe purified type A streptococcal exotoxin.Polyacrylamide gel electrophoresis. Polyacryl-

amide gel electrophoresis was performed by themethod of Davis (10), and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure for mo-lecular weight determinations was performed by themethod ofWeber and Osborn (42). Isoelectric focusingin polyacrylamide gels with an ampholyte range ofpH3 to 10 was used to demonstrate purity and to deter-mine the isoelectric point of the exotoxin. The gelsfrom all three of these techniques were stained anddestained by the method of Weber and Osborn (42).

Anti-type A streptococcal exotoxin. Purifiedtype A streptococcal exotoxin and a preparation ofstrain NY5 crude extract were used for the productionof antibody in New Zealand White rabbits. Each typeA streptococcal exotoxin preparation was subjected toa protein determination (17) before it was mixed withan equal volume of incomplete Freund adjuvant. Afinal concentration of 350 pg of protein per ml wasadministered in each injection throughout the immu-nization protocol. The initial immunization of eachrabbit consisted of 0.5-ml subcutaneous injections intwo footpads and two 1-ml subcutaneous injections inthe pericervical area. After 2 weeks, two 1-ml intra-muscular injections were given in each hind leg. Theweek 4 and 6 injections were given subcutaneously (1

ml in each flank). After a 2-month pause in the im-munization protocol, 35 intradermal injections total-ling 1.5 to 2.0 ml of type A streptococcal exotoxin weregiven in the shaved skin of the back of each rabbit at30-day intervals. These 30-day interval booster injec-tions were continued for approximately 15 months.The rabbits were bled by arterial puncture of the earbefore the initial immunization for preimmune anti-sera and 7 days after each immunization for immuneantisera.Erythematous skin test. The backs of skin-test

rabbits were shaved carefully at least 1 h before injec-tion to prevent red coloration due to injury by the hairclippers. Volumes of 0.1 ml of the type A exotoxinmateral were injected intradermally into the skin with1-ml tuberculin syringes at specific areas on the back,which were outlined with a marking pen. A positiveskin reaction to the type A streptococcal exotoxin wasdemonstrated by an erythematous response, whichformed the basis of the test. The rabbit skins wereobserved at 24 and 48 h after injection. The samerabbits were used repeatedly for skin testing.

Neutralization of the erythematous response withanti-type A streptococcal exotoxin serum was accom-plished by a modification of the type C streptococcalpyrogenic exotoxin neutralization procedure describedby Schlievert et al. (34). TypeA streptococcal exotoxinwas diluted 1:100 with buffered saline from its originalconcentration of 250 pg of protein per ml. A 0.06-mlvolume of this type A exotoxin preparation was mixedwith one of the following volumes of antiserum in eachof three vials: an equal volume, twice the volume, andthree times the type A exotoxin volume. The mixtureswere allowed to incubate for 1 h at 37°C and thenwere incubated overnight at 40C. These mixtures,together with 0.05-ml volumes of type A antitoxin and0.5-ml volumes of 1:100 diluted type A streptococcalexotoxin, were injected at separate sites intradermallyinto the shaved backs of rabbits. The skin reactionswere observed at 24 and 48 h.REUSA. We used the microtiter plate ELISA pro-

cedure described by Voller et al (39), with a slightmodification. The type A streptococcal exotoxin prep-aration was diluted 1:50 in pH 9.6 carbonate-bicarbon-ate buffer (coating buffer), and a 200-pl volume of thetype A streptococcal exotoxin solution was added toeach well of a polystyrene microtiter plate (typeM29AR; Dynatech Laboratories). This plate was in-cubated in a humid enclosure at 4°C overnight andwashed the next morning. The washing procedureinvolved removing the contents of the wells and refill-ing them with phosphate-buffered saline-Tweenbuffer (pH 7.4). The phosphate-buffered saline-Tweenbuffer was removed after 3 min and replaced withfresh buffer. The wells of the microtiter plate werewashed three times after each step in the ELISA.Rabbit antiserum to type A streptococcal exotoxinwas diluted 1:200 in phosphate-buffered saline-Tweenbuffer, and a 200-pl volume of the diluted antiserumwas added to each well included in the assay. All typeA exotoxin samples were assayed in duplicate. Themicrotiter plate was incubated for 2 h at room tem-perature and later washed. An alkaline phosphatase-labeled goat anti-rabbit globulin conjugate (Miles Lab-oratories, Inc.) was diluted 1:1,000 with phosphate-

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buffered saline-Tween buffer, and a 200-,l volume wasadded to each well. The plate was allowed to incubatefor 2 h at room temperature and then was washedwith phosphate-buffered saline-Tween buffer. A 200-pl volume of the substrate p-nitrophenyl phosphate(1 mg/ml; Sigma Chemical Co.) dissolved in coatingbuffer was added to each treated well, and after thesubstrate was added, incubation was carried out atroom temperature for 30 min. The reaction was ter-minated by adding 50 f1 of 3 M NaOH. The contentsof each well were removed, and the absorbancy at 400nm was determined spectrophotometrically.Heat inactivation of type A streptococcal ex-

otoxin. To determine the effects of heat on type Astreptococcal exotoxin, four 0.1-ml samples of type Astreptococcal exotoxin were each subjected to one ofthe following conditions: incubation at 370C for 1 h,incubation at 500C for 1 h, boiling (1000C) for 10 min,and refrigeration (4WC). A 0.05-ml volume of each typeA streptococcal exotoxin sample was used for rabbitskin testing, and the remainder of each sample wasutilized in the ELISA.

Kinetics of type A streptococcal exotoxin. Weprepared an overnight culture of S. pyogenes strainNY-5 type 10 in 100 ml of Todd-Hewitt broth incu-bated at 370C. A 10-ml portion of this overnight cul-ture was inoculated into 100 ml of fresh prewarmedTodd-Hewitt broth in a 300-ml side-arm flask. A 5-mlsample of the bacterial suspension was withdrawn byusing sterile technique, and the side-arm flask wasplaced immediately in a 370C shaking water bath. Asan indicator of growth, the turbidity ofthe 5-ml sampleof the bacterial suspension was measured with a Klett-Summerson colorimeter equipped with a no. 54 Klettfilter. The 5-ml sample was transferred to a sterilecentrifuge tube and subjected to centrifugation at5,000 x g for 10 min. The supernatant fluid wasremoved, membrane filtered (Millipore Corp.), andlater used in the ELISA for the detection of type Aexotoxin. This procedure was followed at 1-h intervalsfor a total of 10 h. Identical experiments were per-formed using strain T253(T12) and the non-toxin-pro-ducing strain T253.Regulation of type A streptococcal exotoxin

production. To determine the effect of temperatureon type A exotoxin production, a 5% inoculum froman overnight culture of S. pyogenes NY5 type 10 inTodd-Hewitt broth was added to four flasks containing100 ml of Todd-Hewitt broth, and each was incubatedfor 8 h at one of the following temperatures: 25, 30, 37,and 420C. After the incubation period, a 5-ml samplewas removed from each flask and centrifuged at 5,000x g for 10 min. The supernatant fluids were filtered

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with Millipore membrane filters and assayed for typeA exotoxin production by the ELISA. The Klett-Sum-merson colorimeter equipped with a no. 54 filter wasused in this and the experiments described below tomeasure the turbidities of the bacterial cultures.The effects of metal ions on the production of type

A streptococcal exotoxin were tested with the follow-ing metals: MnSO4. H20, FeSO4, CaCl2, and MgC926H20. The concentration ranges of metal ions used inthis study were within the ranges of the metal ionconcentrations used in other studies (6, 33). Sampleswere obtained as described above.Two-dimensional immunoelectrophoresis.

Two-dimensional immunoelectrophoresis was per-formed by the method of Laurell (25); 0.06 M Gelmanhigh-resolution buffer (pH 8.8) was used both in elec-trophoresis and for preparing gels. The type A strep-tococcal exotoxin was reduced initially with 0.1 Msodium phosphate buffer (pH 7.2) containing 5% 2-mercaptoethanol and flushed with nitrogen beforebeing incubated overnight at 370C. The addition of0.001 M mercaptoacetic acid to the electrophoresisbuffer aided in maintaining the exotoxin in its reducedstate. Pressing, washing, drying, and staining of theslides were carried out by the method of Axelsen (1).

RESULTS

Isolation of type A streptococcal exo-toxin. Type A streptococcal exotoxin was puri-fied from S. pyogenes strain NY5 type 10 by themethod of Cunningham et al. (9), with the mod-ifications described above. Table 1 shows thepurification steps and quantitation of type Astreptococcal exotoxin. Type A exotoxin assayswere performed initially by the erythematousrabbit skin response method and later by theELISA. In the QAE-Sephadex fractionationstep, four protein-containing fractions were ob-tained. The antibody to purified type A exotoxinreacted with only fraction 3 in the ELISA,whereas the erythematous skin test was positivefor both QAE-Sephadex fractions 1 and 3. Weknew that fraction 1 contained type B exotoxin.The final purified protein gave a single bandwhen it was subjected to polyacrylamide gelelectrophoresis (2% sodium dodecyl sulfate, 5%2-mercaptoethanol). The isoelectric point oftype A streptococcal exotoxin was pH 5.2.

In an attempt to determine the molecularweight of purified type A streptococcal exotoxin,

TABLE 1. Purification and quantitation of type A streptococcal exotoxinConcn of Absorbance No. of Ttlno. of No. of

Step Vol (Ml) protein (mg/ Absorba ELISA ELISA units ELISAnml) units/mil units/mgCulture filtrate 15,000 2.85 0.110 27.5 412,500 9.65Concentrated crude extract 1,000 4.3 0.600 149.7 149,750 34.8QAE-Sephadex 300 1.2 0.236 59.0 17,700 49.2Isoelectric focusing 30 0.96 0.387 97.7 2,930 101.7'Each sample was diluted 1:50, and 0.2 ml of this dilution was used in the ELISA.

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a sample was partially reduced with 2-mercap-toethanol for 2 h at 370C and then subjected tosodium dodecyl sulfate-polyacrylamide gel elec-trophoresis. Four bands were obtained, whichhad molecular weights of 5,800, 7,800, 16,000,and 20,250. In subsequent experiments with thesame purified type A streptococcal exotoxinwhich was reduced with 2-mercaptoethanol at370C for 16 h, one band was observed in asodium dodecyl sulfate-polyacrylamide gel (Fig.la). The molecular weight of the purified type Aexotoxin was 8,500.Anti-type A streptococcal exotoxin. An-

tibody to a preparation of crude strain NY5extract and to the purified type A streptococcalexotoxin was produced in rabbits. The specificityof this rabbit antibody was confirmed by neu-tralization of the type A exotoxin erythematousactivity in rabbit skin. In later experiments inwhich the ELISA was used (Table 2), the spec-ificity of the antibody to type A exotoxin wasdemonstrated by positive reactions when type Aexotoxin was present and negative reactions toall other antigens present, including type B andC exotoxins, in culture filtrates obtained aftergrowth of organisms in Todd-Hewitt dialysate.Two-dimensional immunoelectrophoresis of theanti-type A streptococcal exotoxin and a crudetype A exotoxin preparation gave a single pre-cipitin peak (Fig. lb). Similarly, a single precip-itin peak was observed with anti-strain NY5crude extract and the purified type A strepto-coccal exotoxin (data not shown). These resultsdemonstrated the purity of the type A strepto-coccal exotoxin and the specificity of its antibodypreparation.ELISA. The availability of an antibody spe-

a b

FIG. 1. Purified type A streptococcal exotoxin. (a)Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis ofpurified typeA streptococcal exotoxin (100p,g subjected to 16 h of incubation with 2-mercapto-ethanol. (b) Two-dimensional immunoelectrophoresisof antibody to purified type A streptococcal exotoxinand a crude type A exotoxin preparation. The pres-ence of 2-mercaptoacetic acid in the buffer caused anonspecific peak just above the well.

cific for the type A streptococcal exotoxin al-lowed the use of the ELISA as a sensitive andspecific assay for type A exotoxin. A positiveELISA reaction was recognized by the develop-ment of a yellow color in the microtiter platewells due to the release ofpara-nitrophenol afterthe reaction of the alkaline phosphatase-conju-gated goat anti-rabbit antibody with the sub-strate para-nitrophenyl phosphate. Theamounts of para-nitrophenol released into themicrotiter plate wells were directly proportionalto the amounts of type A streptococcal exotoxinbound to the wells of the microtiter plate usedin the ELISA within a protein range of 2.5 to 25itg (Fig. 2). The ELISA was used subsequentlyto quantitate the amount oftype A streptococcalexotoxin present during the purification proce-dure (Table 1). When this method of purificationwas used, only a 10-fold increase in type Astreptococcal exotoxin was achieved, and, appar-ently, a significant amount of type A exotoxinwas lost at each step. Exposure of the type Aexotoxin to increasing temperatures resulted ina loss of biological activity, as determined by the

TABLE 2. Specificity of antibody to type Astreptococcal exotoxin as determined by the ELISA

Strain or Exotoxin Reactivity of antibody'antigen type(s)

present Preimmune Immune

K56b A- B C+ - -T253 A- B+ C+ - -NY5 type 10 A+ B+ C+ - +K56(8627) A+ B+ C+ - +Type B exotohinc A- B+ C- -

a +, Positive ELISA reaction to an antigen dilution of 1:400; -, negative ELISA reaction.

bSample of culture filtrate from strain grown in Todd-Hewitt dialysate.

C Type B exotoxin obtained after QAE-Sephadex isolation(fraction 1).

0.2.

a

0.1

5 10 1 5 20 25TOXIN (no prtin)

FIG. 2. Standardplot ofincreasing concentrationsof type A streptococcal exotoxin and ELISA values.A400 ,., Absorbance at 400 nm.

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erythematous skin reaction (Fig. 3). However,the antigenicity of the type A exotoxin recog-nized by the ELISA remained intact.Kinetics oftype A streptococcal exotoxin

production. The kinetics of type A streptococ-cal exotoxin production during growth of S. py-ogenes NY5 type 10 were determined by usingthe ELISA (Fig. 4). A differential plot of type Aexotoxin production per increase in growth(ELISA response/Klett unit) indicated thatmaximum synthesis of type A exotoxin occurredduring the logarithmic phase of growth, with a

sharp decrease occurring in the stationary phaseof growth. Similar results were obtained withthe toxigenic strain T253(T12); however, no ex-

tracellular type A exotoxin was detected duringthe growth of the nontoxigenic strain T253.Regulation of type A streptococcal exo-

a

< 0.4

0.2.

.6 0

1-

o._

4)

-4

O2

4 37 50 160

TEMPERATURE CC )

FIG. 3. Effect of temperature on type A streptococ-cal exotoxin, as determined by the ELISA (0) andthe erythematous skin response (0).

.001 II

I'

_ -

_

.o001

re

o 1 2 3 4 s 6 7 8 9TIME (NI)

FIG. 4. Kinetics of type A streptococcal exotoxinproduction duringgrowth ofS. pyogenes NY5. A4m ,Absorbance at 400 nm.

toxin production. The ELISA was used todetect type A streptococcal exotoxin levels ingrowth experiments under various experimentalconditions. Table 3 shows the effects of incuba-tion temperatures on the production of type Aexotoxin by S. pyogenes strain NY5 type 10 after16 h of growth. Essentially, no differences were

observed in the amount of growth and type Aexotoxin production at 24, 30, and 37°C, whereasboth growth and type A exotoxin productiondecreased at 42°C.Metal ions are known to influence the produc-

tion of toxins by certain microorganisms signifi-cantly. The levels of type A exotoxin producedby strain NY5 type 10 in the presence of variousmetal ion concentrations were determined bythe ELISA (Table 4). None of the metals sig-nificantly influenced type A exotoxin produc-tion. Variations in growth medium additives alsohad little effect on the level of type A exotoxinproduction by strain NY5 type 10 comparedwith type A exotoxin production in Todd-Hewittdialysate broth (Table 5). The cumulative re-

sults from these studies suggested that type Astreptoeoccal exotoxin is synthesized in a consti-tutive manner.

TABLE 3. Effect of temperature on growth and typeA exotoxin production in S. pyogenes NY5

Incubation temp Growth (Klett Amt of type Aeso-(OC) units) to4in(absorbance

24 212 0.10830 240 0.11237 234 0.12242 160 0.063

a ELISA value (blank corrected).

TABLE 4. Effects of metal ions on growth and typeA exotoxin production in S. pyogenes NY5'

Growth Amt of type AMetals Concn (M) (Klett esotobnn (ab-

unt8) orbance at400units) mnm)MnSO4.H20 0.9 178 0.202MnSO4.H20 90 175 0.186

CaCl2 0.9 174 0.245CaCl2 90 170 0.164

MgCl2.6H20 0.9 179 0.224MgCl2.6H20 90 174 0.233

FeSO4-7H20 0.9 177 0.192FeSO4 7H20 90 177 0.225

None 175 0.188

aAverage Klett and ELISA values from two exper-iments.

b ELISA value (blank corrected).

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TABLE 5. Effects of nutrient additives on growthand type A exotoxin production in S. pyogenes NY5

Amt of type AMedium Growth exotoxin (ab-

(Klett units) sorbance at 400nm)a

Todd-Hewitt broth 180 0.050Brain heart infusion broth 135 0.071THDb 136 0.161THD + 2% glucose 139 0.090THD + 2% glucose 131 0.211maintained at pH 7

THD + 0.5 mM cysteine 138 0.173THD + 60% yeast extract 120 0.081

dialysateTHD + 5% horse serum + 140 0.048K2HPO4 + CaC12a ELISA value (blank corrected).bTHD, Todd-Hewitt dialysate.

DISCUSSIONThis study demonstrates that ELISA is a sen-

sitive and specific method for the detection andquantitation of type A streptococcal exotoxmi.Type A exotoxin was detected in microgramamounts, and quantitation was possible duringpurification and growth experiments.Of particular interest were the kinetics of type

A exotoxin production during the growth of S.pyogenes NY5. Maximum type A exotoxin pro-duction occurred during the log phase of growthin a manner similar to the production of othergroup A streptococcal extracellular products,such as nicotinamide adenine dinucleotidase (4)and hyaluronic acid (42). The simultaneous pro-duction of both hyaluronic acid and type Aexotoxin during the mid-log phase of growthmay in part explain the association of these twocomponents with each other. Other toxigenicstrains of S. pyogenes also produced type Aexotoxin in a similar manner, albeit in lowerlevels. As determined by ELISA, the nontoxi-genic strain T253 produced no extracellular typeA exotoxin, an observation consistent with otherstudies (L. K. McKane and J. J. Ferretti, sub-mitted for publication; 30).The results concerning the influence of nutri-

ent additives and metal ions on type A exotoxinproduction led to the conclusions that none ofthese factors significantly affected type A exo-toxin synthesis and that regulation was consti-tutive in manner. However, when S. pyogenesNY5-10 was grown at 420C, decreases in bothgrowth and type A exotoxin production wereobserved. Although a number of studies haveshown that growth of S. pyogenes and extracel-lular toxin production could be influenced bythe addition of various nutrients, (19, 27, 38),none of the nutrient additives appeared to en-hance type A exotoxin synthesis. Similarly,

metal ions are known to influence the productionof Pseudomonas aeruginosa toxin A (5, 6), anddiphtheria toxin (26, 31, 33); however, we foundno significant variations in type A streptococcalexotoxin production in the presence of varyingconcentrations of Mn+, Ca, Mg2+, and Fe2".Finally, attempts to influence type A exotoxinproduction by S. pyogenes NY5 at incubationtemperatures ranging from 24 to 420C alsoshowed little variation between growth andamount of type A exotoxin produced. The pos-sibility that other factors besides bacteriophageinfluence (Nida and Ferretti, manuscript inpreparation) can affect type A streptococcal ex-otoxin production seems remote, but cannot beexcluded.

Purification of the type A streptococcal exo-toxin was essential to obtain a specific antibodyfor use in the ELISA. Our purified type A strep-tococcal exotoxin had properties similar to thosedescribed by Cunningham et al. (9); i.e., thisexotoxin had an isoelectric point of pH 5.2 anda molecular weight of 8,500. Although four pro-tein bands (molecular weight range, 5,800 to20,250) were obtained in some sodium dodecylsulfate-polyacrylamide gel electrophoresis ex-periments, incubation of the type A exotoxinwith 2-mercaptoethanol for 16 h at 370C resultedin one band with a molecular weight of 8,500.Apparently, total reduction of multimers to themonomeric 8,500-dalton form occurred underthese conditions. Type A exotoxin has beenshown to contain half-cysteine residues (9),which, when oxidized, may form disulfide bondsbetween monomers. Multimeric forms of type Astreptococcal exotoxin have been reported byCunningham et al. (9) and Nauciel et al. (29).Our data showed that the type A streptococcal

exotoxin molecule contains a heat-stable anti-genic component (as determined by the ELISA)which is distinct from the heat-labile componentresponsible for the erythematous response (rab-bit skin test). The specific antibody to the typeA exotoxin used in the ELISA was also capableof neutralizing the erythematous skin reactioncaused by the type A exotoxin; these resultssuggested that the heat-labile and heat-stablecomponents were associated in a complex man-ner, with perhaps both being required to elicitan erythematous response. Whether these com-ponents were comparable to the type A strep-tococcal exotoxin subparts described by Watsonand co-workers (2, 23, 41) was not ascertained.

ACKNOWLEDGMENTS

This investigation was supported by grants from the RogerMcCormick Foundation, Chicago, Ill., and the American HeartAssociation, Oklahoma Affiliate.

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1. Axelsen, N. H. 1973. Intermediate gel in crossed and infused rocket immunoelectrophoresis. Scand. J. Immu-nol. 2(Suppl. 1):71-78.

2. Barsumian, E. L, C. M. Cunningham, P. M. Schliev-ert, and D. W. Watson. 1978. Heterogeneity of groupA streptococcal pyrogenic exotoxin type B. Infect. Im-mun. 20:512-518.

3. Barsumian, E. L., P. M. Schlievert, and D. W. Wat-son. 1978. Nonspecific and specific immunological mi-togenicity by group A streptococcal pyrogenic exotox-ins. Infect. Immun. 22:681-688.

4. Bernheimer, A. W., P. D. Lazarides, and A. T. Wilson.1957. Diphosphopyridine nucleotidase as an extracel-lular product of streptococcal growth and its possiblerelationship to leukotoxicity. J. Exp. Med. 106:27-37.

5. Bjorn, M. J., B. H. Iglewski, S. K. Ives, J. C. Sadoff,and M. L. Vasil. 1978. Effect of iron on yields ofexotoxin A in cultures ofPseudomonas aeruginosa PA-103. Infect. Immun. 19:785-791.

6. Bjorn, M. J., P. A. Sokol, and B. H. Iglewski. 1979.Influence of iron on yields of extracellular products inPseudomonas aeruginosa cultures. J. Bacteriol. 138:193-200.

7. Brunson, K. W., and D. W. Watson. 1974. Pyrogenicspecificity ofstreptococcal exotoxins, staphylococcal en-terotoxin, and gram-negative endotoxin. Infect. Immun.10:347-351.

8. Brunson, K. W., and D. W. Watson. 1976. Limulusameboycte lysate reaction with streptococcal pyrogenicexotoxin. Infect. Immun. 14:1256-1258.

9. Cunningham, C. M., E. L. Barsumian, and D. W.Watson. 1976. Further purification of group A strep-tococcal pyrogenic exotoxin and characterization of thepurified toxin. Infect. Immun. 14:767-775.

10. Davis, B. J. 1964. Disc electrophoresis. II. Method andapplication to human serum proteins. Ann. N.Y. Acad.Sci. 121:404-427.

11. Dick, G. F., and G. H. Dick. 1924. A skin test forsusceptibility to scarlet fever. J. Am. Med. Assoc. 82:265-266.

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