quantitative assay of diphtherial toxin and of immunologically cross

INFCTION AND IMMUNITY, Dec. 1975, p. 1392-1400Copyright (C 1976 American Society for Microbiology

Vol. 12, No. 6Printed in U.S A.

Quantitative Assay of Diphtherial Toxin and ofImmunologically Cross-Reacting Proteins by Reversed

Passive HemagglutinationRANDALL K. HOLMES* AND ROBYN B. PERLOWI

Departments of Internal Medicine and Microbiology, University of Texas Southwestern Medical School,Dallas, Texas 75235

Received for publication 31 July 1975

A reversed passive hemagglutination (RPHA) assay for diphtherial toxin hasbeen developed. Antitoxic antibodies were isolated from commercially availableequine diphtherial antitoxin by immunoabsorption using highly purified diph-therial toxin covalently linked to Sepharose 4B. Formalinized, tanned sheeperythrocytes sensitized with the purified antitoxic antibodies are specificallyagglutinated by diphtherial toxin but are not agglutinated by extracellularantigens ofCorynebacterium diphtheriae that are unrelated to toxin. The RPHAassay described can detect less than 20 pg of diphtherial toxin and is comparablein sensitivity to intracutaneous tests for toxin. The RPHA assay was shown to beat least 1,000 times more sensitive than quantitative immunological assays fordiphtherial toxin performed by single radial immunodiffusion or by one-dimen-sional double diffusion in agar gels. Fragment A prepared from purified diph-therial toxin and nontoxic mutant proteins that cross-react immunologicallywith toxin can be assayed directly by RPHA, but the sensitivity of the assay forthese proteins is less than for native diphtherial toxin. Inhibition of RPHA was

also shown to be a sensitive quantitative method for measuring diphtherialantitoxin in vitro.

Many strains of Corynebacterium diphthe-riae produce diphtherial toxin, an antigenic ex-tracellular protein that is highly toxic for sus-ceptible animals. Diphtherial toxin is one of thebest studied microbial toxins, and the extensiveliterature concerning the synthesis, structure,immunochemistry, and mode of action of thistoxin has been reviewed elsewhere (2, 3, 8, 26).Assays for diphtherial toxin can be based on itsbiological activities in animals (28, 40) or intissue cultures (32), on its immunological prop-erties (19, 24), or on the NAD:EF-2 ADPR-transferase activity associated with fragment Aof diphtherial toxin (9, 17). (Abbreviations:NAD, nicotinamide adenine dinucleotide; EF-2,elongation factor 2; ADPR, adenosine diphos-phate ribose.) The skin test, based on the pro-duction of erythema at the site of intracutane-ous inoculation of toxin in rabbits or guineapigs, is one of the most sensitive of these assays.The minimal reactive dose of United StatesStandard Diphtherial toxin in skin tests is0.000025 flocculating units (1), equivalent to ap-proximately 50 pg of diphtherial toxin. Skintests are thousands of times more sensitive

I Present address: Michigan State University, College ofHuman Medicine, East Lansing, Mich. 48823.

than flocculation tests (19) or gel diffusion tests(24), and also exceed the sensitivity of radio-immunoassays for diphtherial toxin (4, 18).Assays for NAD:EF-2 ADPR-transferase ac-tivity provide a direct and sensitive methodfor detecting fragment A, but intact diphtherialtoxin lacks this enzymatic activity (9, 17).The structural gene for diphtherial toxin,

tox, is present in the genome of corynebacterio-phage /3 (3). During studies of nontoxinogenic(tox-) mutants of corynebacteriophage /B(Holmes, manuscript in preparation), we in-vestigated and compared several quantitativeimmunological methods for detecting diph-therial toxin or immunologically related non-toxic proteins (CRMs) synthesized by strains ofC. diphtheriae harboring prophages with muta-tions in the tox gene (3, 8, 26). A reversed pas-sive hemagglutination assay for toxin or fornontoxic CRMs was developed that is compara-ble in sensitivity to the intracutaneous test fordiphtherial toxin. In addition, inhibition of thisreversed passive hemagglutination reactionprovides a sensitive method for measurementof diphtherial antitoxin in vitro. (A preliminaryreport of this work was presented at the 74thAnnual Meeting of the American Society for

1392

DIPHTHERIAL TOXIN ASSAY BY AGGLUTINATION

Microbiology, Chicago, Illinois, 12-17 May1974.)

MATERIALS AND METHODSBacteria. C. diphtheriae strain C7,(-)'0x-, here-

after called C7, and the lysogenic strains C7(,8)to°+and C7(y)Yox have been described (20). C7(,Bh t@o W')was constructed by lysogenization of strain C7 withthe recombinant corynebacteriophage 3h, tox h' de-scribed previously (20). Strains C7(f345) and C7(f830)were prepared by lysogenization of strain C7 withthe nontoxinogenic mutant phages f45 and /830 pro-vided by T. Uchida (37). C. diDhtheriae strain PW8r(P)tOz+ was provided by L. Barksdale (2). C7 strainswere cultivated in PGT medium with 2% maltosesupplement as described previously (20). In tests forproduction of diphtherial toxin by lysogenic C7strains, 25-ml samples of deferrated PGT mediumcontaining 2% maltose supplement and 0.075 ,ug ofFe per ml in 125-ml acid-cleaned Erlenmeyer flaskswere inoculated with 0.25-ml samples from over-night low iron cultures and were incubated for 17 hat 34 C with rotary shaking at 240 rpm. Cells wereremoved by centrifugation for 10 min at 12,000 x g,and the cell-free culture supernatants were assayedfor diphtherial toxin.

Chemicals. Whatman microgranular diethyl-aminoethyl (DEAE)-cellulose was obtained from H.Reeve Angel and Co., Inc. (Clifton, N.J.). SephadexG-100 and Sepharose 4B were from Pharmacia FineChemicals, Inc. (Piscataway, N.J.). Trypsin, soy-bean trypsin inhibitor, and bovine serum albuminwere from Sigma Chemical Co. (St. Louis, Mo.). Allother chemicals were reagent grade and were pur-chased commercially. The following buffer solutionswere used: buffer 1-0.01 M sodium phosphate (pH7.7); buffer 2-0.05 M sodium phosphate (pH 7.0),containing 0.02% (wt/vol) NaN3; buffer 3-0.05 Mglycine-chloride (pH 3.5); buffer 4-0.05 M glycine-chloride (pH 3.0); buffer 5-0.082 M NaCl, 0.043 MNa2HPO4-2H2O, 0.0107 M KH2PO4 (pH 7.4); buffer6-0.077 M NaCl, 0.024 M Na2HPO4 2H20, 0.0508 MKH2PO4 (pH 6.4).

Equine diphtherial antitoxins. United StatesStandard Diphtherial Antitoxin (lot A32, 6 units/ml)was provided by Edward B. Seligman, Bureau ofBiologics, Food and Drug Administration, U.S. De-partment of Health, Education, and Welfare. Massa-chusetts standard antitoxin (lot SA10, 500 units/ml)was obtained from W. C. Latham, State LaboratoryInstitute, Commonwealth of Massachusetts. Diph-therial antitoxin lot DP 1252 (11.25 units/mg, in vi-tro/in vivo ratio 1.04) was supplied as a lyophilizedsample by R. 0. Thomson, Wellcome Research Labo-ratories, Beckenham, Kent, England. DiphtherialAntitoxin, U.S.P (lot 7619 Dl, 20,000 units/vial),was the gift of A. N. DeSanctis, Merrell-NationalLaboratories, Philadelphia, Pa. Assays of toxin andof antitoxin by flocculation tests were based on themethod of Ramon as described by Glenny and Okell(19), and Massachusetts standard antitoxin wasused as the reference serum for flocculation tests.

Puriflcation of diphtherial toxin and of frag-ment A. Procedures for production and purification

of diphtherial toxin and of fragment A were modifi-cations of published methods (9, 16). Diphtherialtoxin was produced in 250-ml cultures of C. diphthe-rzae strain PW8r(P)'tx+ as described by Lampidis and,Barksdale (21). All subsequent procedures were car-ried out at 4 C. Ammonium sulfate was added to2,000 ml of pooled culture supernatants containing7.4 x 104 flocculating units (Lf) of toxin, and thematerial that precipitated between 40 and 70% ofsaturation was dialyzed against buffer 1 and appliedonto a DEAE-cellulose column (3.1 cm2 by 37 cm).Elution was performed with a 0 to 0.15 M gradient ofNaCl in buffer 1, and the toxin peak, recovered atapproximately 0.08 M NaCl, was subjected to gelfiltration on a Sephadex G-100 column (4.9 cm2 by 53cm) in buffer 1. Diphtherial toxin eluted from theSephadex G-100 column as a single peak, and thefractions containing toxin were combined and storedfrozen at - 70 C. This sample contained about 72 mgof protein and approximately 2 A.g of protein per Lfof toxin, based on an assumed extinction coefficientat 280 nm (El1m) of 10 (4). Analysis of 20-,ug samplesof toxin by discontinuous polyacrylamide gel electro-phoresis in the presence of sodium dodecyl sulfate(22) revealed a single band of protein with a molecu-lar weight of 62,000. The same result was obtainedin the presence or absence of 2-mercaptoethanol,indicating that the purified toxin was "intact toxin"and contained no detectable "nicked toxin" (13, 16).Fragment A of diphtherial toxin was prepared as

described below. Trypsin (10 j.g/ml) was added to 15mg of purified diphtherial toxin in 7 ml of 0.05 Mtris(hydroxymethyl)aminomethane-chloride buffer(pH 8.0), containing 2 mM ethylenediaminetetraace-tic acid and 5 mM dithiothreitol, and the mixturewas incubated for 10 min at 37 C. Soybean trypsininhibitor (20 Ag/ml) was added, and the reactionmixture was fractionated on Sephadex G-100 inbuffer 1 containing 1% (vol/vol) of 2-mercapto-ethanol. A small peak of protein eluting at 1.86times the void volume and containing fragment Awas collected and applied to a DEAE-cellulose col-umn equilibrated with buffer 1 containing 2-mer-captoethanol, and elution was carried out with a 0to 0.2 M gradient of NaCl in the same buffer. Frac-tions containing fragment A eluted at approximately0.10 M NaCl and were pooled. This sample wasfractionated with ammonium sulfate, and materialprecipitating between 60 and 80% of saturation wasdissolved and dialyzed against buffer 1. This finalsample contained purified fragment A and showed asingle protein band of molecular weight 24,000 (13,16) when a 10-,Ig sample was analyzed by electro-phoresis on polyacrylamide gels containing sodiumdodecyl sulfate (22).

Immunodiffusion assays for diphtherial toxin.An agar gel containing 1 g of Noble agar (DifcoLaboratories, Detroit, Mich.) and 1 g of sodium azideper 100 ml of distilled water was used for all immuno-diffusion assays. Qualitative assays for diphtherialtoxin or fragment A in fractions obtained duringpurification was performed by the double diffusionmethod of Ouchterlony (25).

For quantitative assays of diphtherial toxin bysingle radial immunodiffusion (23), antitoxin (Mer-

1393VOL. 12, 1975

1394 HOLMES AND PERLOW

rell-National Laboratories, Philadelphia, Pa.) wasadded at a final concentration of 2 units/ml to mol-ten agar at 48 C, and 2.5-ml samples were dispensedinto disposable plastic immunodiffusion slides (Im-munoplates, Hyland Laboratories, Costa Mesa,Calif.). A linear array of wells 2 mm in diameter wascut with a template, measured samples (3 Il) wereadded to the wells with calibrated micropipettes,and the plates were incubated overnight at roomtemperature. The diameters of toxin-antitoxin pre-cipitin rings surrounding the wells were measuredwith a dissecting microscope using a calibrated opti-cal reticle. Each sample was tested in quadrupli-cate, and the average ring diameter was used todetermine toxin concentrations from a standardcurve obtained with purified diphtherial toxin.

Quantitative, one-dimensional, double immuno-diffusion assays based on the method of Oakley andFulthorpe (24) were performed as described previ-ously for the assay of cholera enterotoxin (14), ex-cept that diphtherial antitoxin (Merrell-NationalLaboratories, Philadelphia, Pa.) at concentrationsfrom 2 to 10 units/ml was used as antibody in thebottom layer of agar and supernatants from culturesof C. diphtheriae were used as antigen for the liquidupper layer. Tests were performed in duplicate, andaverage values were used to determine toxin concen-trations from a standard curve obtained with puri-

INFECT. IMMUN.

fled diphtherial toxin.Purification of equine diphtherial antitoxin by

immunoabsorption. A 10-ml sample of Sepharose4B was activated with cyanogen bromide as de-scribed previously (11), washed with 200 ml of 0.1 MNaHCO3, and suspended at 4 C in 0.1 M NaHCO3 ina final volume of 20 ml. To this suspension, 12.4 mgof purified diphtherial toxin was added rapidly, andthe mixture was stirred gently at 4 C for 24 h. Thegel was washed extensively with water and thensuspended in 1.0 M ethanolamine-chloride (pH 8.0)for 1 h at 4 C. The gel was washed repeatedly with0.1 M acetate buffer (pH 4.0) alternating with 0.1 Mborate buffer (pH 8.5), equilibrated with buffer 2,and a column (0.6 cm2 by 10 cm) was prepared.A sample containing 1,000 units of equine anti-

toxin (Merrell-National Laboratories, Philadelphia,Pa.) in 4 ml ofbuffer 2 was applied to the column andwas eluted with 30 ml of buffer 2, followed by 20 mlof buffer 3, followed by 20 ml of buffer 4, and frac-tions of approximately 1.0 ml were collected (Fig. 1).Ouchterlony immunodiffusion tests demonstratedthat purified antitoxic antibodies were eluted fromthe immunoabsorbent column by buffer 4. The col-umn was regenerated by washing with buffer 2 andwas used repeatedly. The samples of purified anti-toxic antibodies were concentrated by precipitationat 4 C with ammonium sulfate at 50% of saturation,

25

20H

15 -

10 -

0.5

PurifiedAntitoxicAntibody

P00,0

00I 0q\ b

r------o

0LO-- Y-^00-6 *-r----- I

0 10 20 30 40 50 60 70|-.05 M phosphate buffer,-1-.05M glycine-CI-|-.05M glycine-CI-|

t plH 7.0 pH 3.5 pH 3.0Sample

FRACTION NUMBERFIG. 1. Purification of antibodies to diphtherial toxin by immunoabsorption. The immunoabsorbant was

purified diphtherial toxin covalently linked to Sepharose 4B. The sample applied to the column contained1,000 units of equine diphtherial antitoxin. The bars indicate fractions containing antitoxin detected by theOuchterlony gel diffusion method. The antitoxic antibody absorbed to the column atpH 7.0 was eluted atpH3.0 in 0.05 M glycine-chloride buffer.

Ec

00nN

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and the precipitates were dissolved and dialyzedagainst buffer 2. The final sample of 16.2 ml had anabsorbance of 2.48 at 280 nm and contained approxi-mately 130 Lf of antitoxin per ml.

Reversed passive hemagglutination assays.Procedures for collection of sheep erythrocytes, pres-ervation of the erythrocytes by formalinization, andtreatment of the erythrocytes with tannic acid havebeen described previously (15). The following opti-mal conditions for sensitization of tanned erythro-cytes were determined in preliminary experiments.A 10-ml sample of a 10% (vol/vol) suspension oftanned erythrocytes in normal saline solution and0.5 ml of purified antitoxic antibodies were added to40 ml of buffer 6 at room temperature and incubatedfor 1 h with frequent mixing. The suspension wascentrifuged for 15 min at 270 x g at 4 C, the superna-tant was discarded, and the cells were washed threetimes with 50-ml samples of normal saline solution.The cells were suspended at a final concentration of2% (vol/vol) in buffer 5 containing 1 mg of bovineserum albumin per ml and 0.01% (wt/vol) Merthio-late. The antitoxin-sensitized cells were distributedin 1-ml volumes in plastic tubes (13 by 100 mm) withcaps, quick-frozen in an alcohol-dry ice bath, andstored at -70 C.

All reversed passive hemagglutination tests werecarried out in disposable plastic microtiter trayswith V-bottom wells (Linbro Chemical Co., Inc.,New Haven, Conn.). The diluent was buffer 5 con-taining 0.5% (vol/vol) heat-inactivated normal rab-bit serum and 0.01% (wt/vol) Merthiolate. Negativecontrols for all experiments included antitoxin-sensi-tized cells incubated in the absence of toxin andunsensitized control cells incubated in the presenceof serial twofold dilutions of toxin. A positive controlwas provided by incubation of the antitoxin-sensi-tized cells in the presence of serial twofold dilutionsof purified diphtherial toxin. Assays were carriedout in 100-,ul volumes of diluent containing 0.1%(vol/vol) of antitoxin-sensitized sheep erythrocytesand serial twofold dilutions ofthe experimental sam-ples to be tested for antigen. Serial dilutions wereperformed with a microtiter diluter (Cooke Engi-neering, Alexandria, Va.). After preparing the reac-tion mixtures, we sealed the microtiter trays andallowed them to stand overnight at 4 C before ob-serving the agglutination patterns.

For titrations of antitoxin by inhibition of re-versed passive hemagglutination, reaction mixturesof 50 j1A containing constant amounts of purifieddiphtherial toxin and serial twofold dilutions of anti-toxin in diluent were incubated for 2 h at roomtemperature before the addition of 50-Al samples of0.2% suspensions ofantitoxin-sensitized sheep eryth-rocytes in the same diluent. After mixing the sam-ples and sealing the plates, we incubated the reac-tion mixtures at 4 C overnight before observing theagglutination patterns.

RESULTS

Under conditions optimal for toxin produc-tion, C. diphtheriae strain C7(,() produces 10 to20 Lf of toxin per ml, whereas PW8 strains may

produce 100 Lf or more per ml. Both toxicitytests in experimental animals and quantitativeassays for toxin based upon flocculation in vitroare tedious when large numbers of samplesmust be assayed for diphtherial toxin, and themaximum concentrations of toxin in crude cul-ture supernatants of the widely used strain C.diphtheriae C7(j3) are only slightly above thelimit of sensitivity of standard flocculationtests. Because a sensitive, inexpensive, andrapid quantitative assay for diphtherial toxinwould be very helpful for studies on the physiol-ogy and genetics of toxin production by Coryne-bacterium diphtheriae C7(,8) and relatedstrains, several immunological assays were in-vestigated and compared.The results of assays for diphtherial toxin by

single radial immunodiffusion are presented inFig. 2. The diameter of the toxin-antitoxin pre-cipitin ring increased in proportion to the log ofthe concentration of diphtherial toxin in thetest sample. When the antitoxin concentrationwas reduced below 2 units/ml, easily visibleprecipitin rings did not develop. When the anti-toxin concentration exceeded 2 units/ml, theminimal detectable concentration of diphthe-rial toxin was greater than 10 Lf/ml. Under theconditions illustrated in Fig. 2, using 2 units ofantitoxin per ml, precipitin rings were observedonly at toxin concentrations between 10 Lf/mland 50 Lf/ml. Assay of diphtherial toxin bysingle radial immunodiffusion is inexpensive,requires small volumes of material, and caneasily be performed with large numbers of sam-ples. In terms of sensitivity and accuracy, how-ever, this method appears to have no signifi-cant advantages over classical flocculationtests.The results of assays for diphtherial toxin

performed by the one-dimensional, double im-munodiffusion method of Oakley and Fulthorpe(24) are presented in Fig. 3. Results obtainedwith 2 and with 10 units of antitoxin per ml areshown. When the antitoxin concentration wasreduced below 2 units/ml, visible precipitinlines were not formed. Concentrations of toxinas low as 1 Lf/ml can be detected and measuredquantitatively after 48 to 72 h of incubation.This assay is inexpensive, easy to perform, andapproximately 10 times more sensitive than thesingle radial immunodiffusion assay illustratedin Fig. 2. Because this method is sensitiveenough to detect 10% or less of the extracellulartoxin produced by strain C7(,Q) under optimalconditions, it is sensitive enough to be useful instudies on the physiology of toxin production byC7 strains.

In an effort to develop a much more sensitivequantitative assay for diphtherial toxin in vi-

1395VOL. 12, 1975


E

-J

C

.x

0

0

c0

c0

5040

30F-

20

10l,2.75 3.00

FIG. 2. Quantitatitsingle radial immunctoxin-antitoxin preciptional to the logarithnate).

21

E

-i

C

c

1(

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FIG. 3. Quantitatitone-dimensional doumethod ofOakley andthe toxin-antitoxin pi

liquid interface (abscirithm of toxin conceni

ity of the assay is dettdiphtherial antitoxin:

0.5% heat-inactivated rabbit serum was pres-

ent in the diluent, but less satisfactory resultswere obtained when the concentration of serumwas reduced or when bovine serum albumin

was substituted for serum. The agglutination/ patterns were more satisfactory when the reac-

tion mixtures were incubated overnight at 4 Cinstead of at room temperature. Reproducible

0 titrations of diphtherial toxin by RPHA were

possible using either complete agglutination(an even blanket of agglutinated erythrocytes)or partial agglutination (a central button sur-

3.25 3.50 3.75 4.00 rounded by a halo of agglutinated erythrocytes))iameter (mm) as the end point. The sensitivity of the RPHA

assay was two- to fourfold greater when partialwe assay of diphtherial toxin by agglutination was used, and the quantitative

Ddiffusion. The diameter of the data presented below are based on partial agglu-

itin ring (abscissa) was propor- tination as the end point.of toxin concentration (ordi- Data documenting the sensitivity of the

RPHA assay for purified diphtherial toxin andfor fragment A are summarized in Table 1, and

0O the assay is illustrated in Fig. 4. A stock solu-

tion containing 1 ,g of purified diphtherialtoxin per ml contained approximately 64,000hemagglutinating units per ml. The sensitivityofthe RPHA assay for diphtherial toxin is there-fore less than 20 pg and is comparable to thesensitivity of the intradermal test for diphthe-rial toxin (1). Purified fragment A, which lackstoxicity but possesses NAD:EF-2 ADPR-trans-ferase activity, was also detected by the RPHAassay. The hemagglutinating activity of frag-

//O ment A is approximately eight times less on a

I I weight basis and 20 times less on a molar basis3 4 5 6 7, than that of purified diphtherial toxin. Thus,

Distance (mm) proteins that cross-react immunologically with

ue assay of diphtherial toxin by diphtherial toxin can be detected by this RPHAible immunodiffusion by the assay, but their specific hemagglutinating ac-'Fulthorpe (24). The distance of tivities (RPHA units per microgram or RPHArecipitin band from the agar- units per nanomole) may be different from thatissa) is proportional to the loga- of native diphtherial toxin. Hemagglutinatingtration (ordinate). The sensitiv- activity is independent of toxicity.ermined by the concentration of Data concerning the specificity of the RPHA

, n '. /__ I " IO, 2 units/ml; *, 10 units/ml.

tro, we investigated reversed passive hemagglu-tination (RPHA) ofantitoxin-sensitized erythro-cytes in the presence of diphtherial toxin. For-malinized erythrocytes were sensitized withpurified antitoxic antibodies and were found tobe stable at -70 C for at least 1 year. In controlexperiments without toxin, the antitoxin-sensi-tized erythrocytes formed compact buttons in"V"-bottomed microtiter trays but less satisfac-tory results were observed with "U"-bottomedtrays. In the presence of small amounts of puri-fied diphtherial toxin, agglutination of the sen-

sitized erythrocytes occurred. The agglutinatedcells were distributed in an even blanket when

TABLE 1. Specificity and sensitivity of the reversedpassive hemagglutination assay for diphtherial toxin

Sample tested Hemagglutination titer(HA units/ml)

Purified antigensDiphtherial toxin (1 ,.g/ml) 64,000Fragment A (1 ,ug/ml) 8,000

Culture supernatantsC7 (-) <40C7 (,3) 1,310,000C7 ([345) 650,500C7 ([330) 43,960C7 (y) <40C7 (3h tox h) <40

5

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INFECT. IMMUN.

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1 2 3 45 67 8910101112

B wst * **. *. . . .a * 1

C9t -~ * * * * . . .* RD (v--;*< . . . . . ..'0;*79Et4'*< *$-il-. xF ' k*-(h*-*. _ A*.*.9t

FIG. 4. Reversed passive hemagglutination assays for diphtherial toxin, for immunologically cross-react-ing proteins, and for diphtherial antitoxin. Row A contains unsensitized sheep erythrocytes. Column 1contains antitoxin-sensitized sheep erythrocytes in the absence of diphtherial toxin. Assays for antigen areillustrated in rows A through F. Samples of antigens were added to wells in column 2, and serial twofolddilutions were carried out to column 12. The antigens tested and the concentrations present in the wells incolumn 2 were as follows: (A and B) purified diphtherial toxin, O.(2Y25 Lf/ml; (C) purified fragment A ofdiphtherial toxin, 2.5 ng/ml; (D, E, and F) 1:4 dilutions ofculture supernatants ofC. diphtheriae strains C7,C7(/3), and C7(fh to~ h'). Assays for diphtherial antitoxin by inhibition ofhemagglutination are illustrated inrows G and H, in which wells 2 through 12 all contained diphtherial toxin at 0 tYXY25 Lf/ml . Samples ofanti-*toxin were adde¢d to wells in column 3, and serial twofold dilutions were carried out to column 12. The equineantitoxins were present in column 3 at 0.25 units/ml and were as follows: (0) Diphtherial Antitoxin, U.S.P.,Merrell-National Laboratories, and (H) Massachusetts Standard Antitoxin, lot SA 10.

assay were obtained by testing for antigen insupernatants from cultures of C. diphtheriaestrains C7, C7(,B), C7(S45), C7(,p30), C7(y), andC7Mh tox h') grown under low iron conditionsoptimal for toxin production. Intradermal testsin rabbits documented that the supernatantfrom strain C7(,p) contained more than 500,000minimal reactive doses of diphtherial toxin per

ml and that the toxicity could be neutralized bypassive immunization of the rabbits withequine antitoxin. Supernatants from the otherfive strains contained no toxin demonstrable bysimilar intradermal tests. As shown in Table 1,the supernatant from strain C7(,p) containedmore than 1 million hemagglutinating unitsper ml, equivalent to approximately 10 Lf ofdiphtherial toxin per ml. Strains C7(,45) andC7(,B30), which produce nontoxic amino-termi-nal fragments of toxin with molecular weightsof 45,000 and 30,000, respectively (37), both

gave positive tests by RPHA, although the titerin the RPHA assay decreased with decreasingmolecular size of the cross-reacting mutant pro-tein. No hemagglutination was observed withsupernatants from strains C7, C7(y), andC7Mh tOxI h'). These results indicate that the an-titoxin-sensitized erythrocytes reacted onlywith diphtherial toxin or with the cross-react-ing proteins CRM45 and CRM30 produced by themutant tox genes of phages I5 and p30. Noother extracellular antigens controlled by thegenomes of C. diphtheriae C7 or of phages , ory gave detectable agglutination of the anti-toxin-sensitized cells. Previous attempts to dem-onstrate that phage -/"x- controls the produc-tion of antigens that cross-react with diphthe-lial toxin have been negative. Our present obser-vations with the more sensitive RPHA assayconfirm this conclusion and also establish thatthe recombinant phage ph to- h', which carries

1397VOL. 12, 1975

-A Adpilk 'A


the tox- marker froxknown genes fromsynthesis of antigdiphtherial toxin.When serial di]

added to hemaggluing constant amoutoxin, inhibition ofexcess antitoxin waamount of antitoxihemagglutinationamount of toxin initures over a widetoxin (Fig. 5). Attested, the amounthibit RPHA was tvthe calculated amoiin neutralization oient results were cdifferent equine Etoxin standards arstandards, the am(known sample cancomparing its RPHof a reference anti

C -Ic 0

0 0

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FIG. 5. Assays fortion of reversed passiferent equine antito.States Standard Dipchusetts Standard ArResearch LaboratorieDiphtherial Antitoxir,National ,Laboratori4with various amountper assay, and the n

required to inhibit he,from the observed en

antitoxin concentratirequired to inhibit htional to the amounts

m phage y but all of its otherphage 13, fails to direct therens that cross-react with

lutions of antitoxin wereitination mixtures contain-

sensitivity of the RPHA assay, it should bepossible to use inhibition of RPHA to measureantitoxin antibodies in sera of low titer.

DISCUSSION

LInts of purified diphtherial During the 1950's and 1960's, methods for

ftRPHA in the presence of sensitizing and preserving erythrocytes for use

S demonstrated (Fig. 4). The in passive hemagglutination reactions and hem-n required for inhibition of agglutination inhibition reactions were system-was proportional to the atically explored, popularized, and applied withLcluded in the reaction mix- many different systems (5, 12, 33-35). In mostrange of concentrations of of these initial studies, erythrocytes were sensi-each concentration of toxin tized with soluble antigens. Thus, antibodiesof antitoxin required to in- could be measured directly by passive hemag-wo- to fourfold greater than glutination, whereas assays for antigen wereunt required for equivalence performed by hemagglutination inhibition tech-r flocculation tests. Consist- niques. In 1962, Richter, Cua-Lim, and Rose)btained with each of four (27) reported that erythrocytes could be sensi-antitoxins tested. Because tized with specific antibodies purified by immu-e less stable than antitoxin noabsorption and could be used for the detec-ount of antitoxin in an un- tion of minute amounts (less than 1 ng) of spe-be estimated most easily by cific antigens. The detection of specific antigensIA-inhibiting titer with that using antibody-sensitized erythrocytes hasitoxin. Because of the high been called reversed passive hemagglutination.

Assays for several important microbial toxins,including tetanus toxin (10), staphylococcal en-

terotoxin A (31), the enterotoxin of Clostridiumperfringens (38), and several immunologicaltypes of botulinum toxin (29), have successfullyemployed RPHA techniques.

. i / Although Richter and associates (27) suc-o m/ ceeded in sensitizing erythrocytes with purified

b:/ antibodies, they were not able to achieve ade-* / quate sensitization using gamma globulin frac-

tions from immune sera. In contrast, Cook (10)0* reported that immunoglobulin fractions from

0O horses hyperimmunized with tetanus toxoido could sensitize erythrocytes, although whole se-

|l rum was unsatisfactory. Purified immunoglobu--4 -3 -2 - lin G containing antibodies to Clostridium per-

iount of Toxin per Assay fringens enterotoxin was used successfully inthe studies of Uemura et al. (38), but Sakaguchilog10 (Lf/assay) et al. used purified antibodies in their RPHA

diphtherial antitoxin by inhibi- assays for botulinum toxins (29). Most equineye hemagglutination. Four dif diphtherial antitoxins available commerciallyxins were tested: El, United contain antibodies against several antigens un-)htherial Antitoxin; *, Massa- related to diphtherial toxin. In the presentrititoxin lot SA 10; U, Wellcome study, therefore, antitoxic antibodies were puri-Xs antitoxin lot DP 1252; and 0, fied by immunoabsorption before they werers, U.S.P., lot 7619D1, Merrell- used to sensitize formalinized, tanned sheepes. Titrations were performed erythrocytes for RPHA assays. As shown by thets of purified diphtherial toxin data in Fig. 4 and in Table 1, the RPHA assayninimum amounts of antitoxin 'magglutination were calculated that was developed is hlghly sensitive and ap-d points and the known initial pears to be completely specific for diphtherialons. The amounts of antitoxin toxin or for immunologically cross-reacting pro-lemagglutination were propor- teins.of toxin in the assays. The RPHA assay for diphtherial toxin has

INFECT. IMMUN.


several advantages and potential applications.Diphtherial toxin can be detected by RPHA inamounts as low as 20 pg and in concentrationsas low as 200 pg/ml. Using reagents that arestable during storage at -70 C, RPHA providesan in vitro assay for toxin antigen that is inex-pensive, rapid, and comparable in sensitivity tothe intracutaneous test for diphtherial toxin(28, 40). Thus, the RPHA assay for diphtherialtoxin could be useful both for identification oftoxinogenic strains of C. diphtheriae in refer-ence laboratories and for studies on the physiol-ogy or genetics of toxin production in researchlaboratories that use strains of C. diphtheriaelike C7(,B) that produce relatively low yields oftoxin. In our laboratory, the RPHA assay hasbeen used as a sensitive method for classifyingtox- mutants of corynebacteriophage ,B intoCRM+ and CRM- groups (Holmes, manuscriptin preparation). For this purpose, the RPHAassay has the advantage that a positive reac-tion directly reflects the presence of a cross-reacting antigen, even if the titer is low. Incontrast, radioimmunoassays based on displace-ment of radioactively labeled toxin from toxin-antitoxin complexes (4, 18) have limited valuefor detecting weakly cross-reacting proteinsthat possess few of the antigenic determinantsof native diphtherial toxin.

Methods for quantitative assay of diphtherialantitoxin in sera of low titer have been recentlyreviewed (39). The data in Fig. 4 and 5 demon-strate that inhibition ofRPHA provides an addi-tional sensitive method for measurement ofdiphtherial antitoxin. Passive hemagglutina-tion (HA) of toxoid-sensitized erythrocytes hasbeen used frequently for titration of diphtherialantitoxin in human sera (39). Discrepancies be-tween antitoxin titers obtained by HA and byneutralization have been reported in some sera(6, 7, 30, 36). In sera of low titer, determinationsof antitoxin by HA can overestimate or underes-timate titers obtained by neutralization tests.Neutralizing but nonagglutinating antibodiescould be responsible for underestimates of neu-tralizing activity by HA tests (30), whereas over-estimates of neutralizing activity by HA tests(6) could be related to the presence in test serawith low avidity of immunoglobulin M anti-bodies with a high ratio ofhemagglutinating ac-tivity to neutralizing activity. Since the prepa-rations of diphtherial toxoid used to sensitizeerythrocytes for HA tests in most publishedstudies were not purified to homogeneity, it isalso possible that antibodies to antigens otherthan toxin might contribute to the observed HAtiters but not to the neutralization titers. In thepresent study, evidence is presented in Fig. 4

and in Table 1 that the antitoxin-sensitizederythrocytes used for the RPHA assay are ag-glutinated only by diphtherial toxin or by im-munologically cross-reacting antigens. Thus,assays based on inhibition of this RPHA testshould be specific for diphtherial antitoxin. Ifthe ability of immunoglobulin M and G anti-toxic antibodies to inhibit the RPHA test weredirectly proportional to their neutralizing activ-ity, then antitoxin titers measured by neutrali-zation tests might correlate better with titersobtained by inhibition of RPHA than by HA oftoxoid-sensitized erythrocytes. If this possibil-ity were confirmed by future studies, measure-ments of diphtherial antitoxin by inhibition ofRPHA could be a valuable tool for epidemiologi-cal studies of immunity to diphtheria.

ADDENDUMIt has recently come to our attention that a re-

versed passive hemagglutination assay for diphthe-rial toxin was developed independently and was usedto test for diphtherial toxin in the serum, throatwashings, and urine of patients with diphtheria (D.D. A. Christovao and Z. L. G. Cotilla. 1966. Pesquisae dosagem da toxina difterica no sangue de pacientesde difteria por reacao de hemaglutinaq&o passiva.Arg. Fac. Hig. S. Paulo 20:223-232; D. D. A. Chris-tovao, Z. L. G. Cotillo, and J. A. N. Candeias. 1967.Prova de hemaglutinaqao passiva para a eviden-ciacao da toxina do C. diphtheriae na lesao difterica.Rev. Saude Publica 1:210-216). The reversed pas-sive hemagglutination assay described in these stud-ies utilized tanned human type 0, Rh-negativeerythrocytes sensitized with diphtherial antitoxin.The sensitivity ofthe assay for diphtherial toxin wasapproximately 0.00032 Lf/ml, but specificity of theassay for diphtherial toxin and not for other coryne-bacterial antigens was not demonstrated.

ACKNOWLEDGMENTSThis research was supported by a Brown-Hazen grant

from the Research Corporation and by Public Health Ser-vice research grant 5 R22 A111478 awarded by the NationalInstitute of Allergy and Infectious Diseases.

R. K. H. is the recipient of a Public Health ServiceResearch Career Development Award (1 K04 A100051) fromthe National Institute ofAllergy and Infectious Diseases.

The competent technical assistance of Linda Baily and ofJames McNutt is acknowledged.

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