JOURNAL OF BACTERIOLOGY, Mar., 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 3Printed in U.S.A.

Serological Studies of Types A, B, and E BotulinalToxins by Passive Hemagglutination and

Bentonite FlocculationH. M. JOHNSON, K. BRENNER, R. ANGELOTTI, AND H. E. HALL

Milk and Food Research, Division ofEnvironmental Engineering and Food Protection, Robert A. TaftSanitary Engineering Center, U.S. Public Health Service, Cincinnati, Ohio

Received for publication 5 November 1965

ABSTRACT

JOHNSON, H. M. (Robert A. Taft Sanitary Engineering Center, Cincinnati,Ohio), K. BRENNER, R. ANGELOTTI, AND H. E. HALL. Serological studies of typesA, B, and E botulinal toxins by passive hemagglutination and bentonite floccula-tion. J. Bacteriol. 91:967-974. 1966.-Formalinized sheep red blood cells (SRBC),sensitized with types A, B, and E botulinal toxoids and toxins by bis-diazotizedbenzidine (BDB), were tested against A, B, and E antitoxins prepared in horsesand rabbits. Type B antitoxin cross-reacted with A toxoid SRBC, but the reciprocalcross-reaction was not observed. E toxin SRBC were specifically agglutinated by Eantitoxin. Flocculation of antigen-sensitized bentonite particles was less sensitivein titration of antitoxin than hemagglutination. Also, reciprocal cross-reactionswere observed between types A and B antitoxins. Cross-reactions in both serologi-cal tests were eliminated by titration of antitoxins in the presence of the heterologousantigens, with no inhibitory effect on the homologous antitoxins. Generally, equineantitoxins were less suitable for agglutinations, especially of antigen-sensitizedbentonite particles. Types A, B, and E antitoxins were specifically inhibited by 43,39, and 245 mouse LD5o of their respective homologous toxins in the hemaggluti-nation-inhibition test. A, B, and E antitoxins were specifically inhibited by 500,950, and 1,500 mouse LD5o of their respective homologous toxins in bentoniteflocculation inhibitions. Formalinized SRBC sensitized with rabbit types A and Bantitoxins by BDB were respectively clumped by as little as 0.75 to 1.3 mouse LD5oof A toxin and 2.3 LD5o of B toxin, whereas bentonite particles sensitized by the sameantitoxins were specifically clumped by 150 LD5o of A toxin and 630 LD50 of B toxin.E antitoxin sensitization of SRBC or bentonite particles was not successful. Evi-dence is presented that indicates that the serological procedures are applicable tothe detection of botulinal toxins in food.

There are at least six types of antigenically dis-tinct Clostridium botulinum toxins distinguishableby animal protection tests. These botulinal toxinsare designated A, B, C, D, E, and F (7). The anti-genic specificity of these toxins may not be asclear and distinct when in vitro serological testsare used. Studies by Lamanna and Lowenthal (11)have shown, for example, that cross-precipitationoccurs between type A botulinal toxin and type Bantitoxin. Also, antiserum produced to type Atoxin neutralizes the hemagglutinin of type Btoxin. Conversely, antiserum to type B toxin neu-tralizes the hemagglutinin of type A toxin. Re-ports by Rycaj (14), Yafayer and Chepeler (18),and Sinitsyn (15) indicate that passive hemagglu-

tination of botulinal antitoxin-coated, tannedsheep red blood cells (SRBC) by toxins is as sensi-tive in detecting botulinal toxins as animal assay.Rycaj, however, worked only with type A toxin,whereas Yafayer and Chepeler, working with bothA and B toxins, could not distinguish betweenthem. Sinitsyn was able to distinguish between thetwo toxins, but the potencies of the toxins usedin his studies were much lower than those usuallyfound with botulinal cultures. None of these in-vestigators used antigen-sensitized SRBC in theirinvestigations. A recent review by Petty (12) in-cludes references to the passive hemagglutinationprocedures just described.The present study was initiated in an effort to

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increase understanding of the in vitro serologicalproperties of types A, B, and E botulinal toxins,and with the hope of development of an in vitroserological test for the specific detection of botu-linal toxins in cultures and contaminated foods.The serological tests used for these studies werehemagglutination and bentonite flocculation.

MATERIALS AND METHODSReagents. Trypsin used in these studies was Difco

1:250. Fisher reagents were used to prepare 0.15Mphosphate-buffered saline, pH 7.2 (PBS; 16). Fisherreagents were also used to prepare 0.15 M phosphatebuffer, pH 7.3 (PB; 10). Bovine serum albumin frac-tion V powder (BSA) was obtained from NutritionalBiochemicals Corp., Cleveland, Ohio. Solutions of1 and 0.25% were prepared in PBS. Bis-diazotizedbenzidine (BDB) was prepared according to themethod of Gordon et al. (10).

Sources of purified toxins and toxoids. Purifiedcrystalline type A botulinal toxin was obtained fromEdward J. Schantz, U.S. Army Biological Labora-tories, Fort Detrick. Botulinal toxoids types A, B,and E, in approximately 0.5% formalin, were sup-plied by Matteo Cardella, U.S. Army BiologicalLaboratories. Toxin and toxoids were exhaustivelydialyzed against PBS before immunization of rabbitsand use in serological tests.

Preparation of partially purified type E toxin forsensitization of erythrocytes and bentonite particles.The VH strain of C. botulinum type E was grown in acellophane bag that was constructed as described byBoroff (1). The toxin-production medium was pre-pared as described by Gordon et al. (9). The inoculum,2 ml of an 18-hr culture, was added to 100 ml ofphysiological saline in the cellophane bag. The culturewas incubated at 30 C for 7 days, after which thecellophane bag was removed from the medium andthe contents were centrifuged at 15,000 rev/min for1 hr in the International BD-2-ultracentrifuge (970rotor). Six volumes of a solution of saturated(NH4)2SO4 were added to 4 volumes of toxic super-natant fluid. After the toxin had been allowed toprecipitate overnight at 4 C, it was collected by cen-trifugation at 15,000 rev/min for 1 hr in the BD-2ultracentrifuge, dissolved in 10 ml of physiologicalsaline and exhaustively dialyzed against saline, thenrecentrifuged at 15,000 rev/min for 1 hr. The super-natant liquid was removed, assayed for toxicity, andused for sensitization of erythrocytes and bentoniteparticles. The specific toxicity of the preparation was6 X 10' mouse LD6o per mg of protein.

Toxic cultures. Types A (Hall strain), B, and E(VH strain) botulinal cultures were supplied by Mat-teo Cardella, U.S. Army Biological Laboratories.The organisms were cultured for toxin production asdescribed by Duff et al. (8) and Gordon et al. (9).Types A and B botulinal toxins were acid-precipitatedfrom cultures by adjustment of the medium to pH 3.2with 1 N HCl, and were washed once with distilledwater. Type E toxin was precipitated by mixing 6volumes of a solution of saturated (NH4)2SO4 with4 volumes of the supernatant fluid of the centrifuged

culture. The precipitated toxins were dissolved inPBS to one-tenth the original volume, exhaustivelydialyzed against PBS, and centrifuged at 15,000 rev/min for 1 hr in the International BD-2 ultracentrifuge(969 rotor). The supernatant fluids were then assayedfor toxicity and employed in inhibition of antitoxinsand in agglutinations of antitoxin-sensitized erythro-cytes and bentonite particles.

Cultures other than C. botulinum were from thestock culture collection of the Food Microbiologysection of Milk and Food Research, Robert A. TaftSanitary Engineering Center.

Mouse LDb0 determinations ofbotulinal toxins. TypesA and B botulinal toxins were diluted in phosphate-buffered gelatin (pH 6.5). The buffer-gelatin solutionwas prepared as described by Crisley (6). Six mice,weighing 20 g each, were injected intraperitoneallywith 0.5 ml of each dilution of toxin. Deaths wererecorded at the end of 4 days, and LD50 values weredetermined by the method of Reed and Muench (13).Type E toxin was diluted 1:10 in 0.1 M phosphatebuffer (pH 6.5) containing a final concentration of0.1% trypsin. The toxin and trypsin were incubatedat 37 C for 60 min, after which the trypsin-treatedtoxin was diluted in phosphate-buffered gelatin (pH6.5) and injected into mice.

Botulinal antitoxins. Types A, B, and E equineantitoxins were obtained from the CommunicableDisease Center (CDC), Atlanta, Ga. The stock anti-toxins contained 10 international units (IU) of anti-toxin per ml (2). Type E equine antitoxin was alsoobtained from the Microbiological Research Estab-lishment, Porton, England. The Porton antitoxincontained 1,500 IU of antitoxin per ml.

Types A, B, and E antitoxins were also produced inrabbits by repeated intraperitoneal injections of A, B,and E botulinal toxoids in complete Freund's adju-vant. Two rabbits were injected with 2 mg of eachtoxoid biweekly for 13 weeks. The rabbits were bled3 weeks after the final injection by cardiac puncture.Samples of the sera were stored at -20 C. Prior touse in serological tests, the sera were inactivated at56 C for 30 min. Before use in hemagglutination pro-cedures, all sera were absorbed with equal volumes ofpacked SRBC. Antisera to the individual toxoids hadsimilar potency in in vitro tests. The potency of the Eantitoxin produced in one rabbit was found to be 18IU per ml, with Porton equine antitoxin as reference(2). Standard antitoxins to A and B toxins were notavailable; therefore, the potencies of rabbit types Aand B antitoxins were not expressed in terms of inter-national units.

Formaldehyde treatment of erythrocytes. Themethod of Butler (5) was employed for formalinizationof SRBC. The formalinized SRBC were stored at 4 Cas a 50% suspension in PBS. The final cell suspensioncontained 0.01% thimerosal.

Coupling ofantigens toformalinized SRBC by BDB.Toxoids and toxins were coupled to SRBC by modi-fication of the procedure described by Butler (5).A 0.1-ml amount of a 50% suspension of formalinizedSRBC was added to toxoid (or toxin) contained in3 ml of 0.15 M PB. BDB was thawed, diluted 1: 15 withPB, and 0.75 ml of the diluted BDB was added to the

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VOL. 91, 1966 SEROLOGY OF BO]

mixture of toxoid and SRBC. The mixture was incu-bated at room temperature for 12 min, followed bycentrifugation of the mixture at 2,500 rev/min forseveral minutes to sediment the cells. The cells were

washed twice with PB and resuspended to 2.5 ml with0.25% BSA. Thimerosal was added to a final concen-tration of0.01% if the cells were not used immediately.The optimal concentrations of types A and B toxoidsfor sensitization of cells were, respectively, 100 and200 MAg of toxoid per ml. Partially purified type Etoxin at a concentration of 100,000 LD6o per ml wasused for sensitization of formalinized SRBC forhemagglutination by E antitoxin. Type E toxoid wasnot used for sensitization of SRBC because of con-tamination with trypsin. The toxoid was preparedaccording to the method of Gordon et al. (9), whichincluded trypsin activation in the purification pro-cedure. Rabbits immunized with the toxoid produced,along with antitoxin, potent antibodies to the trypsin,which masked the toxin-antitoxin reaction. The toxinsand toxoids used for sensitization of cells were devoidof hemagglutinating activity at concentrations used.

Hemagglutination of antigen-sensitized formalinizedSRBC by antitoxin. Hemagglutinations wese carriedout by serial twofold dilution of antisera in 0.5-mlvolumes in 0.25% BSA. To each dilution of antiserumwas added 0.05 ml of antigen-sensitized formalinizedSRBC. The mixture was shaken, incubated at roomtemperature for 4 to 18 hr, and then read for agglutina-tion by pattern formation. The titer was considered asthe highest dilution of antitoxin that produced un-

equivocal clumping of the cells.Hemagglutination-inhibition tests. Hemagglutina-

tion-inhibition tests were performed by mixing variousconcentrations of toxin from toxic cultures in 0.25-mlquantities in 0.25% BSA with 0.25 ml of antitoxinthat had been diluted with 0.25% BSA to contain athree-plus hemagglutinating dose of antitoxin. Afterthe mixtures had incubated for 20 min at room tem-perature, antigen-sensitized formalinized SRBC wereadded. The contents were mixed, incubated at roomtemperature for 4 to 18 hr, and read for pattern de-velopment.

Preparation of antitoxin-sensitized formalinizedSRBC. To 1 ml of rabbit antitoxin that had beendiluted to 10 ml with distilled water was added 5 mlof a saturated solution of (NH4)2SO4. The globulinwas collected by centrifugation at 2,400 rev/min for15 min, after which it was dissolved in 5 ml of PBSand dialyzed against several changes of PBS. Thedialyzed antitoxin globulin was then cleared of particu-late matter by centrifugation at 15,000 rev/min for30 min. The cleared antitoxin globulin was measuredin a Beckman DU spectrophotometer at a wavelengthof 278 m,. The antitoxin globulin was coupled toformalinized SRBC by 1.5 ml of diluted BDB as de-scribed for antigen coupling. The antitoxin-sensitizedcells were suspended in 2.5 ml of 0.25% BSA.

Hemagglutination of antitoxin-sensitized formal-inized SRBC by botulinal toxins. Crystalline type Abotulinal toxin and botulinal toxins removed fromcultures by acid or (NH4)2SO4 precipitation were

serially diluted in 0.5-ml volumes in 0.25% BSA. Toeach dilution of toxin was added 0.05 ml ol antitoxin-

TULINAL TOXINS 969

sensitized formalinized SRBC. The tubes were incu-bated and the test was read as described for hemag-glutination of antigen-sensitized cells by antitoxin.Controls consisted of a tube of the highest concen-tration of toxin used in the test with untreated for-malinized SRBC (2.5% solution) and a tube contain-ing antitoxin-sensitized formalinized SRBC and0.25% BSA.

Flocculation ofantigen-sensitized bentonite particles.Stock bentonite (Volclay, American Colloid Co.,Skokie, Ill.) was prepared by the method of Bozicevichet al. (3). Bentonite particles were sensitized withtypes A and B botulinal toxoids and partially purifiedtype E toxin by a modification of the method of Wolffet al. (17). To 10 ml of stock bentonite suspensionthat had been concentrated to 1 ml in distilled waterby centrifugation was added 1 ml of toxoid or toxincontaining 500 to 1,000 Ag of types A and B toxoidsand 100,000 LDbo of E toxin. After incubation ofbentonite and antigen for 1 hr at room temperature,1 ml of 1% BSA, 15 ml of distilled water, and 0.1 ml of1% methylene blue were added. The antigen-sensitizedbentonite particles were centrifuged and washed twicewith 1 ml of 1% BSA and 15 ml of distilled water.The bentonite particles were suspended in 1% BSA toa final concentration of 150,000 particles per mm3.For the test, serum was serially diluted in 0.1-mlvolumes in PBS on microflocculation slides. To eachdilution of serum was added 0.05 ml of antigen-sensitized bentonite. The mixture was rotated at 120rev/min on a variable-speed rotator (Yankee-typefrom Clay-Adams Inc., New York, N.Y.) for 20 min,after which the antigen-coated bentonite particleswere examined for clumping under a microscope at100 times magnification.

Bentonite flocculation inhibition. Bentonite floccula-tion inhibitions were performed by serial dilution ofthe acid- and (NH4)2SO4-precipitated toxins in 0.1-mlvolumes in tubes (10 by 75 mm). To each dilution oftoxin was added 0.1 ml of antitoxin. The antitoxin hadbeen diluted to contain a three-plus agglutinating doseof antitoxin in 0.1 ml after having been mixed withthe toxin. After incubating at room temperature for20 min, 0.1 ml of each dilution of the mixture ofinhibitor and antitoxin was added to microfloccula-tion slides, and bentonite flocculation was carried outas described above.

Flocculation of antibody-sensitized bentonite parti-cles. Bentonite particles were sensitized with varyingamounts of (NH4)2SO4-precipitated antitoxins ac-cording to the method of Bozicevich et al. (4), exceptthat 1% BSA was used as the stabilizing agent. Toxinswere diluted in 0.1-ml volumes. To each dilution oftoxin was added 0.5 ml of antibody-sensitized bento-nite particles, and bentonite flocculations were carredout.

Preliminary tests on culture supernatant fluids andfoods for botulinal toxins. Supematant fluids fromcultures of C. botulinum types A, B, and E, acid pre-cipitates of canned corn and beans that were inocu-lated with C. botulinum type A, along with a specimenof food that had been implicated in a case of C.botulinum type B intoxication, were examined for

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botulinal toxins by some of the serological techniquesdescribed.

RESULTS

Data on passive hemagglutination and bento-nite flocculation with types A and B toxoids andtype E toxin, and their corresponding antitoxins,are presented in Table 1. Types A and B antitox-ins are of equine origin (CDC). The E antitoxinis of rabbit origin. A strong cross-reaction oc-curred between type B antitoxin and A toxoidSRBC, whereas A antitoxin did not react with Btoxoid-sensitized SRBC. Neither A nor B anti-toxins cross-reacted with E toxin-sensitizedSRBC. Similarly, E antitoxin did not cross-reactwith A or B toxoid SRBC. A pattern of reactivitysimilar to that of passive hemagglutination wasalso observed in bentonite flocculations, exceptthat A antitoxin strongly cross-reacted with Btoxoid bentonite particles. The sensitivities of thehomologous bentonite flocculations for A, B, andE antitoxins were, respectively, 21-, 4.7-, and 4.5-fold less than in passive hemagglutination. BothCDC and Porton type E equine antitoxins failedto clump E toxin bentonite particles. CDC equineantitoxin also failed to agglutinate E toxin SRBC,but the Porton anti-E reacted with E toxin SRBCto a titer of 0.0368 IU of antitoxin (1:40,960 di-lution) per ml. In both the hemagglutination andbentonite flocculation tests, the cross-reactionswere eliminated by titration of antitoxins in thepresence of the cross-reacting antigens, with noreduction in titer of the homologous reactions.For this, 20 ,ug of the appropriate A or B toxoid,contained in 0.1 ml, was added to each dilution ofantitoxin. The two were mixed and incubated at

room temperature for 20 min, followed by theaddition of antigen-sensitized SRBC or bentoniteparticles to each tube and the performance of thepassive agglutination test.

Results of hemagglutination and bentoniteflocculation of A and B toxoid-sensitized SRBCand A and B toxoid-sensitized bentonite particlesby rabbit types A and B antitoxins are presentedin Table 2. The reactions were similar to those ofequine antitoxins (Table 1), except that titers wereconsiderably higher than with the equine sera.Type A antitoxin did not cross-react with B tox-oid-SRBC, but did agglutinate B toxoid bentoniteparticles. Rabbit B antitoxin cross-reacted with Atoxoid in both hemagglutinations and bentoniteflocculations.Data on hemagglutination inhibition with

types A, B, and E rabbit antitoxins by A, B, andE toxic cultures are presented in Table 3. Therabbit antitoxins are the same as those used in thehemagglutinations reported in Tables 1 and 2The acid- and (NH4)2SO4-precipitated toxins wereemployed against a three-plus hemagglutinatingdose of antitoxin. Types A, B, and E antitoxinswere specifically inhibited by 43, 39, and 245mouse LD50 of the respective toxins. Similar resultswere obtained when equine antitoxins were usedin the inhibitions. Results of bentonite floccula-tion inhibitions with types A, B, and E antitoxinsby A, B, and E toxic cultures are summarized inTable 4. Antitoxins that were employed in thebentonite flocculations of Table 1 were used. Al-though the inhibitions were specific, they were notas sensitive as the hemagglutination inhibitions.Prior treatment of type E toxin with a final con-centration of 0.1% trypsin did not enhance its

TABLE 1. Passive hemagglutination and bentonite flocculation with types A, B, and E botulinal toxoidsand their corresponding antitoxinis

Antitoxin (international units per mI)aSerological test Toxoid _--____|___

Ab ]~~~~Bb EC

Passive hemagglutina- A 0.0007(12800) 0.0125(800) >0.18 (100)tion B >0.1 (100) 0.0015(6400) >0.18 (100)

Ed >0.1 (100) >0. 1 (100) 0.002(8192)

Bentonite flocculation A 0.015(640) 0.03(320) >9 (2)B 0.06(160) 0.007(1280) >9 (2)Ed >1 (10) >1 (10) 0.009(2048)

a The values denote smallest number of international units of antitoxin per milliliter that producedunequivocal clumping of the antigen-sensitized SRBC and bentonite particles; >0.1, >0.18, >1.0, and>9 indicate a negative reaction with the highest concentration of antitoxin used. Values in parenthesesindicate reciprocal of serum dilution.

b Equine antitoxins from Communicable Disease Center.c Anti-E produced in rabbits.d Partially purified E toxin.

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TABLE 2. Passive hemagglutination and bentoniteflocculation with types A and B rabbit

botulinal antitoxins

AntitoxinTox- (reciprocal of dilution)

Serological test oid -

A B

Passive hemag- A 20,900,000 650,000glutination B <1,000* 1,300,000

Bentonite floccula- A 204,800 6,400tion B 25,600 102,400

* Indicates negative reaction at 1:1,000 dilu-tion.

TABLE 3. Hemagglutination inhibition with types A,B, and E botulinal antitoxins

Antitoxina Inhibitor Amount of inhibitor PassiveAntitoxin!2I(mouseitoorhemagg"lu-tination"

Type A A toxin 43B toxin 2,450 3E toxin 1,570 3

Type B A toxin 5,500 3B toxin 39E toxin 1,570 3

Type E A toxin 5,500 3B toxin 2,450 3E toxin 245

a Antitoxins produced in rabbits.b Values represent the smallest amount of toxin

(in 0.25 ml) in terms of LD50 that completely in-hibited a three-plus agglutinating dose of anti-toxin or the largest amount of toxin that had noeffect on antitoxin.

c Hemagglutination patterns were recorded asdescribed by Butler (5). This scheme of notationalso holds for hemagglutination recordings inTables 5 and 7.

inhibitory properties in either the hemagglutina-tion or bentonite flocculation procedures.The toxins that were used for inhibitions in

Tables 3 and 4 were also used for hemagglutina-tion of types A and B antitoxin-sensitized SRBC(Table 5). The rabbit antitoxins that were em-ployed in the hemagglutinations and bentoniteflocculations summarized in Table 2 were alsoemployed for sensitization of formalinized SRBC.Equine antitoxins from the CDC were unsuitablefor antibody sensitization of SRBC. SRBC weresensitized with 100 and 200 ug of antitoxin globu-lin per ml. Type A botulinal toxin was detected inquantities as low as 1.3 mouse LD50 per 0.5 ml by

both 100 and 200 Mg of A antitoxin SRBC. Aantitoxin SRBC did not cross-react with B toxin.Type B botulinal toxin was detected in quantitiesas low as 9 LD0o per 0.5 ml for 100 Mig of B anti-toxin SRBC and 2.3 mouse LD5o for 200 Mg of Bantitoxin SRBC. Strong cross-reactions of Atoxin with B antitoxin SRBC also occurred. Re-sults of similar studies with antibody-sensitizedbentonite particles are presented in Table 6. Thespecificity of the reactions depended on theamount of antitoxin globulin used for sensitiza-tion of the bentonite. For example, strong cross-reactions occurred in both systems when 5,000 Mugof antitoxin was used for sensitization of thebentonite particles. When 500 Mug of antitoxinglobulin was used for sensitization, type A toxincould be specifically detected in quantities as lowas 140 mouse LD5o per 0.1 ml and type B toxin at630 LD5o per 0.1 ml. Supernatant fluids from cul-tures of an enterococcus, Micrococcus, and Bacil-lus species, and from one strain each of C. botu-linum type E, C. perfringens, C. sporogenes, C.bifermentans, C. butyricum, C. multifermentans, C.putrefaciens, and C. tetani did not clump eitherA antitoxin- or B antitoxin-sensitized SRBCor bentonite particles. The antitoxin-sensitizedSRBC and bentonite techniques were not success-

TABLE 4. Bentoniteflocculation inhibition with typesA, B, and E botulinal antitoxins

AntiOxinInibitr Amuntof ihibior,BentoniteAmouseo inibto,b floccula-Antitoxin" Inhibitor mouse SD60 focua

Type A A toxin 1,000A toxin 500 1B toxin 6.3 X 104 3E toxin 6.3 X 103 3

Type B A toxin 1.4 X 105 3B toxin 1,900B toxin 950 1E toxin 6.3 X 103 3

Type E A toxin 1.4 X 105 3B toxin 6.3 X 104 3E toxin 1,500

a Types A and B antitoxins are of equine originand were obtained from the CDC. Type E wasproduced in rabbits.

b Values represent the smallest amount of toxin(in 0.1 ml) in terms of LD50 that completely orpartially inhibited a three-plus agglutinating doseof antitoxin or the largest amount of toxin usedthat had no effect on antitoxin.

c Bentonite flocculations were recorded as de-scribed by Bozicevich et al. (3). This scheme ofnotation also holds for flocculation recordings inTable 6.

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ful with E antitoxin from either rabbit or equinesources.Data on the sensitivity of the antitoxin SRBC

technique in detecting A toxin in a preparation ofcrystalline type A botulinal toxin is presented inTable 7. The test could detect as little as 0.75mouse LD50 of toxin in 0.5 ml, corresponding to0.000058 ,ug of toxin. Hemagglutination inhibitiondetected a minimum of 50 LD50 of toxin, corre-sponding to 0.00375 ,gg.

Preliminary studies with the serological tech-niques just described indicate that A, B, and Etoxins can be specifically detected in supernatantfluids from types A, B, and E C. botulinum cul-tures. Toxins acid-precipitated from canned cornand beans that were inoculated with a C. botu-linum type A culture, as well as from an outbreakfood (home-canned fruit obtained from the CDC)containing type B toxin, also gave specific positiveresults when tested by hemagglutination inhibi-tion and hemagglutination of antitoxin-sensitizedSRBC. The sensitivity of all of these tests was

comparable to that obtained with toxins acid- and(NH4)2SO4-precipitated from cultures. In addi-tion, tests on acid precipitates of a variety of foodsnot contaminated with botulinal toxins indicatethat the food components do not interfere withthe serological techniques. Detailed studies arein progress to determine further the applicabilityof these serological techniques for the detection ofbotulinal toxins in¶foods.

DISCUSSIONThe BDB hemagglutination procedure was

considerably more sensitive in titration of anti-toxins than was bentonite flocculation. Also, sur-prisingly, cross-reactions were less in the hemag-glutination test, since no cross-reactions occurredbetween type A equine or rabbit antitoxin and Btoxoid SRBC, whereas strong cross-reactions oc-curred in the bentonite flocculation procedure.Cross-reaction of type B antitoxin with A toxoidoccurred in both tests. Perhaps B toxoid is at-tached to SRBC by BDB in a manner that steri-

TABLE 5. Passive hemagglutination of antibody-sensitized formalinized SRBC by types A and Bbotulinal toxins

Amt (jig) of Reciprocal of dilution of toxins in thousandsToxin (LDso Antibody- globulinper05ml) sensitized per mlper0.5ml) SRBC used for

sensitization 20.5 41 82 164 328 656 1,312 2,624 5,248 10,496

A (7 X 106) Anti-A 100 3 3 3 3 3 3 3 2 1 -

Anti-B 3 3 1 1 -Anti-A 200 3 3 3 3 3 3 3 2 2 -

Anti-B 3 3 3 2 1 - _

B (3.1 X 106) Anti-A 100.3 -3 3 2Anti-B 3 3 3 3 2-Anti-A 200 _ _ __...Anti-B 3 3 3 3 2 1 1 -

TABLE 6. Benitonite flocculation of antibody-sensitized particles by types A and B botulinal toxins

Toxin (LD5oper 0.1 ml)

A (1.4 X 106)

B (6.3 X 105)

Antibody-sensitizedbentonite

Anti-AAnti-BAnti-AAnti-B

Anti-AAnti-BAnti-AAnti-B

Amt (jAg) ofgluobulinused forsensiti-

zation of20 ml ofstock

bentonite

5,000

500

5,000

500

Reciprocal of dilution of toxins

20

3

24

2

40

42

4

4

4

80

421

34

4

160

433

4

4

320

444

3

4

640

434

1

3

1,280

3+4

_

2,560

2

4

5,120

3

10,240

1

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TABLE 7. Passive hemagglutination ofA antitoxin-sensitized formalinized SRBC by crystalline

type A botulinal toxin

Amt (,ug) BDBMouse LD50 of toxin per 0.5 ml of toxin per hemagglu-

0.5 ml tination

200 0.015 3100 0.0075 350 0.00375 325 0.00188 312.5 0.00094 36.25 0.00047 33.125 0.000235 31.5 0.000117 20.75 0.000058 10.375 0.000029

Toxin control* 0.015

* Untreated formalinized SRBC were used.

cally blocks the heterologous reaction. All of thesecross-reactions were eliminated by titration ofantitoxins in the presence of the heterologoustoxoids, with no reduction in titers of the ho-mologous antitoxins. The nature of the moiety ofthe toxins that is responsible for the cross-reac-tions is not known with certainty, but previousstudies by Lamanna and Lowenthal (11), alongwith preliminary data obtained in our laboratoryby partial separation of the toxic moiety of typeA from the hemagglutinin by density gradientultracentrifugation, suggest that the hemagglu-tinin of the toxins is involved. Apparently thehemagglutinin is serologically active at dilutionsin which hemagglutination does not occur.The use of formaldehyde-treated SRBC pro-

vides a stable hemagglutination system. Also, asshown by Lamanna and Aragon (Bacteriol. Proc.,p. 94, 1956), formaldehyde destroys red blood cellreceptors for the botulinal hemagglutinin, thusmaking it unlikely that the hemagglutinin of thetoxin and toxoid can of itself cause hemagglutina-tion of formaldehyde-treated SRBC.

Hemagglutination inhibition and bentoniteflocculation inhibition appear to be quite suitablefor the rapid and specific detection of botulinaltoxins types A, B, and E in cultures and foods.Hemagglutination inhibition detects toxin atlevels well below that which is fatal to humanbeings.The antitoxin-sensitized SRBC technique is ex-

tremely sensitive, detecting toxins at levels of lessthan one to several LD50 per 0.5 ml. The strongcross-reaction of B antitoxin-SRBC with A toxindoes not hinder the differential diagnostic poten-tial of this test, since no cross-reaction occurredbetween A antitoxin SRBC and type B botulinaltoxin. With the use of the same antitoxins, anti-

toxin-bentonite flocculation was considerably lesssensitive than hemagglutination, detecting 140mouse LD5o per 0.1 ml of A toxin and 630 LD50 ofB toxin. The potency of CDC equine antitoxins,in terms of serum dilutions, was considerably lessthan that of rabbit antitoxins types A and B.This lower potency may account for the inabilityof CDC equine antitoxins to sensitize SRBC andbentonite particles. The inability of rabbit typeE antitoxin to work in the antitoxin SRBC testmay also be due to low antibody content of theserum. The hemagglutination titer of rabbit typeE antitoxin was considerably lower than that ofrabbit types A and B antitoxins.The diagnostic equine antitoxins presently

available to botulinal toxins may not be the mostsuitable for in vitro serological studies of botu-linal toxins. This observation is based on the un-suitability of equine types A and B antitoxins(CDC) to sensitize SRBC and bentonite particlesand the inability of equine type E antitoxin (CDC)to clump either antigen-sensitized SRBC or ben-tonite particles.

LITERATURE CITED

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