Vol. 12, No. 3JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1980, p. 361-3660095-1137/80/09-0361/06$02.00/0

Quantitative Microtiter Cytotoxicity Assay for Shigella ToxinMARY K. GENTRY'` AND JOEL M. DALRYMPLE2

Division ofBiochemistry' and Division of Communicable Diseases and Immunology72 Walter Reed ArmyInstitute of Research, Washington, DC 20012

The cytotoxic activity of Shigella dysenteriae 1 was assayed by exposing HeLacells in microtiter cultures to dilutions of toxin. Exposure to toxin caused eitherfailure of cells in suspension to attach or detachment of cells from establishedmonolayers. Estimates of toxin potency were made by staining residual cells withcrystal violet and visually inspecting the stained plates. Quantitation of thecytotoxic effect was made possible by eluting and spectrophotometrically mea-suring the stain. The dilution of toxin causing 50% cell detachment, the endpointchosen for the assay, was estimated from plots of dye absorbance versus toxindilution. The 50% cell detachment dilution of toxin varied as a function of cellconcentration, incubation of toxin with cells in suspension or as establishedmonolayers, and the cell line used for assay. The HeLa cell line was the mostsensitive of the cell lines examined. The method was easily utilized to monitortoxin purification and to measure antitoxin neutralization of toxin activity.

The precise role of the toxin of Shigelladysenteriae 1 in the pathogenesis of Shigelladysentery has not yet been elucidated. However,studies of the toxin have given evidence forseveral different biological activities: it is neu-rotoxic and enterotoxic to animals (1, 4, 5); it iscytotoxic to cells in tissue culture (3, 9); and itinhibits in vitro protein synthesis (8). Althoughthe relationship between the cytotoxic compo-nent and pathogenicity likewise remains to beshown, development of a tissue culture assay forcytotoxicity would appear to be extremely usefulin the study of Shigella toxin. A tissue cultureassay obviates the need for the maintenance ofanimal colonies, requiring only continuing cul-tures of cells; allows the simultaneous assay oflarge numbers of samples; and yields results ina relatively short time.

Cytotoxicity induced by toxin from Shigelladysenteriae 1 to cells in culture has been dem-onstrated for several cell lines. Vicari et al. (9),using a tube culture technique, first reported KBcells and human liver cells sensitive to the toxin,and Keusch et al. (6) developed an assay tech-nique using HeLa cells in chamber-slide cul-tures.Such assays have depended on cell cultures

containing large cell numbers, a requirement formicroscopic examination of each individual cul-ture, and the investigator's estimation of theextent of cellular death and disruption for thedetermination of a toxicity endpoint. Thesetechniques can be cumbersome and time-con-suming if numerous toxicity determinations arerequired, as they are when monitoring toxin

purification or determining toxin-neutralizingactivity.

In this paper we report a simple, rapid methodfor estimating cytotoxicity with HeLa cells inmicrotiter cultures and staining after incubationwith toxin. Quantitation of the toxic effect canbe accomplished by subsequent elution andspectrophotometric measurement ofthe amountof the stain.

(This material was presented in part at theAnnual Meeting of the American Society forMicrobiology, Las Vegas, Nev., May 1978.)

MATERIALS AND METHODSBacterial strains, toxin, and antitoxin. Studies

were performed with the 60R and 3818T strains ofShigella dysenteriae 1. Shiga strain 60R was the roughmutant originally described by Dubos and Geiger (2).The history ofthe 3818T strain has also been describedpreviously (7).

Bacteria were grown in a modified Syncase broth(1% certified grade Casamino Acids [Difco Labora-tories, Detroit, Mich.], 2% glucose, 0.004% tryptophan,and 0.004% nicotinic acid) for 72 h at 37°C with aera-tion. Harvested cells were disrupted in a French pres-sure cell at 15,000 lb/in2, and toxin was partially puri-fied from such extracts (M. R. Thompson et al., Fed.Proc. 35:1394, 1976).

Antitoxin to Shiga strain 3818T (purified toxin) wasa gift from Alison O'Brien, Uniformed Services Uni-versity of the Health Sciences, Bethesda, Md. (A. D.O'Brien et al., submitted for publication).

Cells, culture conditions, and assay procedure.The HeLa cell line (from CCL2, adapted to fetalbovine serum) was obtained from Flow Laboratories,Rockville, Md. Growth medium and diluent consistedof Eagle minimum essential medium (HEM Research,Rockville, Md.) with Earle salts, supplemented with

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362 GENTRY AND DALRYMPLE

10% heat-inactivated fetal bovine serum, 2 mM gluta-mine, 100 U of penicillin per ml, and 100 ,ug of strep-tomycin per ml. Cultures were maintained at 35°C and5% C02 in 32-oz. (ca. 960-ml) glass bottles. Other celllines were obtained from the following sources: the H-Vero (African green monkey kidney) cells were ob-tained courtesy ofRobert Tesh, Pacifie Research Unit,Hawaii; LLC-MK2 continuous monkey kidney cellswere originally obtained from Robert N. Hull, LilyResearch Laboratories, and are maintained at high-passage levels at the Walter Reed Army Institute ofResearch; FRhL (fetal Rhesus lung) cells came fromLederle Laboratories (Pearl River, N.Y.); HEp-2 cells(human epidermoid carcinoma of the larynx) wereobtained from Microbiological Associates (Bethesda,Md.); L-929 (mouse connective tissue CCL-1) cellswere received from the American Type Culture Col-lection; BHK-21 (baby hamster kidney, clone 15) cellsare maintained at Walter Reed Army Institute ofResearch and originally were obtained from 0. R.Eylar, University of Maryland Medical School, Balti-more, Md.

Freshly trypsinized cells were counted, suspendedto the desired concentration in growth medium, and0.1-ml samples were pipetted into 96-well microtiterplates (Costar, Cambridge, Mass.) with a repetitivepipette (Microdoser; Oxford Laboratories, Foster City,Calif.). Monolayers were established by 18 to 20 h ofincubation at 35°C in a 5% C02 atmosphere. Serialdilutions of toxin in medium were added in 0.1-mlsamples, and the plates were incubated for an addi-tional 18 to 20 h. It was possible to shorten this 2-dayprocedure by first placing dilutions of toxin in theplates, adding cell suspensions immediately afterward,and incubating the plates for a single 18 to 20-h period.Negative controls, that is, wells containing cells notexposed to toxin, were included on each plate.

After incubation, detached cells, medium, and toxinwere removed by vigorous shaking. Remaining cellswere fixed with a 2% solution of Formalin in 0.067 Mphosphate-buffered saline (PBS) (pH 7.2) for 1 min,the fixative was removed, and the plates were stainedwith 0.13% crystal violet in 5% ethanol-2% Formalin-PBS for 20 min. Excess stain was removed by waterrinsing, and the plates were air dried. For quantitation,stain was eluted from the wells with four successive50-,d samples of 50% ethanol and diluted in 0.9 ml ofPBS. Absorbance was determined at 595 nm in a 300Nspectrophotometer (Gilford Instrument Laboratories,Inc., Oberlin, Ohio). The toxin dilution resulting in50% cell detachment (CD50), i.e., 50% dye uptake, waschosen as an appropriate endpoint for the assay.

Neutralization studies. Rabbit antisera wereheated at 56°C for 30 min and serial twofold dilutionsin tissue culture medium were incubated at 37°C for1 h in the presence of an equal volume of Shiga toxinat a predetermined dilution. Duplicate 0.1-ml samplesof the toxin-antitoxin mixtures were incubated withcell monolayers.

RESULTSA stained plate used for cytotoxicity assay of

a preparation from S. dysenteriae 1 is shown inFig. 1. Direct visual inspection of the toxin titra-

tion allowed a rough estimate of toxin concen-tration. The 50% effect was estimated visually tobe between the i0-' and 10-8 dilutions of toxin.At higher concentrations of toxin, i.e., lowerdilutions, the wells appear free of any purplestain, suggesting the total destruction of theHeLa cell monolayer. The absence of cells inthese wells was verified by microscopic exami-nation before staining. Partial or incompletetoxic disruption of the cells at dilutions ap-proaching the endpoint was reflected by an in-creased intensity of the stain; direct microscopicexamination of the wells near the 50% endpointshowed that the staining reflected the numberof cells remaining in the well rather than anydecreased capacity of these cells to be stained.Assay plates, once stained, were stored indef-

initely or subjected to a more precise estimationof the toxin potency by dye elution and quanti-tation spectrophotometrically. The curve ob-tained from applying this procedure to the assayis also shown in Fig. 1. The arrow on the ordinateindicates the zero detachment dye absorbancevalue obtained from control HeLa cell cultures.Triplicate values have been plotted for eachtoxin dilution, and the curve has been drawn byhand. The CDso has been estimated by extrap-olation to be 10-7 6.An example of the utility of the cytotoxicity

assay is shown in Fig. 2. Localization of toxinactivity in liquid chromatography column ef-fluents was a routine application of the assayduring toxin purification studies. The assayclearly identifies a peak of toxin activity in frac-tions 66 to 70 that is separate from the majorprotein peaks eluting in fractions 30 to 50.

In experiments such as the column chroma-tography toxin elution profile previously de-scribed, an estimate of relative toxin concentra-tion was adequate, and the experiment was in-ternally controlled, i.e., incubation time and cellconcentration were identical. However, vari-ables, such as cell concentration and monolayerversus suspension cultures, somewhat affectedthe absolute endpoint of the toxicity assay. Theeffect of variable cell concentration on the CD50endpoint of a single toxin preparation is shownin Fig. 3. Samples of a toxin dilution series wereadded to monolayers in wells that initially con-tained from 5,000 to 40,000 cells per well. Theincreased number of cells attached with increas-ing cell concentration added is reflected by in-creased maximum dye absorbance values on theordinate. Differences in CD50 estimates of thetoxin preparation were observed and rangedfrom 10-5.6 with 40,000 cells per well to 10-62with 5,000 cells per well. It should be emphasizedthat the cell numbers referred to in the preced-

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SHIGELLA TOXIN CYTOTOXICITY ASSAY

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FIG. 1. Shigella dysenteriae I toxin assay: photograph ofstained assay plate with the elution profile oftheplate. A cell concentration of 16,00X cellsper well was used to establish the monolayers. Dilutions (10-fold) oftoxin were added along the length of the plate. Outermost wells ofplates were not used for toxin titrationbecause of the aberrent settling patterns often observed, although these wells could be used to obtain controldye elution values. The elution profile was obtained by plotting absorbance values at 595 nm (A,9,) of theeluted dye versus the log of the toxin dilution. The estimated zero detachment (control) dye absorbance valuewas as indicated on the ordinate. The CD50 value was obtained by extrapolating one-half of this value to thelog scale of toxin dilution.

ing experiment are number of cells originallyadded to the wells. In our studies with mono-

layers, cell numbers appear to increase by ap-proximately 50% during the incubation periodused to establish these monolayers. Toxicityendpoints obtained with confluent cell mono-

layers established in advance of toxin additionare somewhat lower than those for an equivalentnumber of cells exposed to toxin in suspension(Table 1); these findings are consistent with theincrease in the number of cells during the initialincubation to establish monolayers. Conse-quently, for comparable results, we have electedto use 16,000 cells per well for monolayers and24,000 cells per well for suspension experiments.

All estimates of toxin concentration werebased on the graphic estimation of a CD50. At-tempts to define more precisely the CD50 end-point were done by comparing linear and nonlin-ear least-squares regression analyses (Table 1).Plotting dye absorbance on a logarithmic scaleversus the log of toxin dilution had the effect oflinearizing the midportion of the curve, and alinear least-squares regression analysis of the

most linear portion was used to estimate theendpoint. The log of one-half the value for neg-ative controls substituted in the linear regressionequation gave the log of the dilution for theCD50. A nonlinear regression analysis by com-puter utilized all the values of the dye elutioncurve. Since the computer estimated a negativecontrol value from the variables, it was not nec-essary to use the absorbance of the negativecontrols on the plate. As can be seen from thedata in Table 1, the graphic method yielded agood estimate of the toxicity endpoint that wasnot significantly improved by more sophisticateddata analysis.The reproducibility of the method was tested

by examining numerous dilutions of frozen sam-ples of the same toxin. A total of 19 separateassays of this toxin on different days yielded amean CD50 of 10-698 with a range of 10-652 tolo-749, with a standard deviation for the end-point of + 10027.The HeLa cell Une was initially used in these

studies because of earlier reports demonstratingthe susceptibility of these cells to Shiga toxin

VOL. 12, 1980 363

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364 GENTRY AND DALRYMPLE

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FIG. 2. Elution profile ofprotein and toxin from a Sephacryl S-200 gel filtration column with photographof toxin assay plates. Protein values (A2sq) are plotted as black circles, and cytotoxicity estimated visually isplotted with bars using a log scale. Sample dilutions for the cytotoxicity assay are indicated to the left of theplate photograph. The initial chromatography sample had previously been precipitated by (NH4)2SO4 andchromatographed on an A-50 diethylaminoethyl-Sephadex column. A 7-ml sample was applied to the 3.3- by88-cm column. The eluting buffer was 20 mM tris(hydroxymethyl)aminomethane (pH 8.0) containing 0.1 MKCl; flow rate was 0.6 ml/min; fraction size was equal to 100 drops per tube. Beginning with fraction number30, every second fraction was assayed for toxin activity. Details of the cytotoxicity assay are as described inthe text.

(Samuel B. Formal, personal communication; 3,6). A variety of other cell lines were examinedwith respect to their susceptibility to Shiga toxin(Fig. 4). HeLa cells exhibited the greatest toxiceffect with increasing toxin concentrations.BHK cells appeared refractory to the toxin un-der these conditions. The Vero cell line exhibitedvery little change over the extremes of toxindilution, and this appeared to be a consistentobservation with the other monkey cell lines,monkey kidney (LLC-MK2), and fetal Rhesuslung (FRhL) cells examined (data not shown).

In addition to the obvious requirement for arapid assay for toxin activity, the developmentof a test for the quantitation oftoxin-neutralizingactivity in serum was also a major objective ofthese studies. Rabbit antiserum to highly puri-fied Shiga toxin was examined in a cytotoxinneutralization titration (Fig. 5). The curve on

the left depicts the cytotoxic activity of theantigen used. A toxin dilution of 10-5 was arbi-tarily selected as a constant antigen concentra-tion in excess of the CD50 endpoint. Incubationof this constant toxin concentration with twofolddilutions of rabbit antiserum and subsequentassay of residual toxin activity resulted in thedye absorbance curve on the right. Antisera atdilutions of 1/400 or less were capable of pro-tecting all cells, i.e., complete neutralization ofthe toxin dose added. The neutralization effectdeclined linearly with antiserum dilution, reach-ing a lower plateau at the 10-5 toxin dose se-lected. Although neutralization tests were gen-eraily performed in this manner, i.e., constantantigen versus varying dilutions of antibody,excess toxin could be titrated in toxin-antibodymixtures. In these studies the residual toxinCD50 was obviously lower, and the toxin titration

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LOGIO OF TOXIN DILUTION

FIG. 3. Change in CD50 with increasing cell concentration. HeLa cells were trypsinized, counted, diluted tothe desired concentration, and pipetted as 0.1-ml samples into the wells. At 20 h, after monolayers had beenestablished, dilutions of Shiga toxin in a 10-fold series were added to the wells. After an additional 20 h-incubation with toxin, the cells were stained, eluted, and the dye absorbance value was determined for eachwell, as described in the text.

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LOGIO OF TOXIN DILUTIONFIG. 4. Sensitivity of various cell lines to Shigella dysenteriae I toxin. Cell suspensions of 30,000 cells per

0.1 ml were added to 0.1-ml samples of toxin diluted in 10-fold series. After 20 h, unattached cells and toxinwere removed, and attached cells remaining were stained and eluted as described in the text.

curve also exhibited a reduced slope.

DISCUSSIONThe requirement for a rapid, sensitive, repro-

ducible method for quantitating cellular cytotox-icity of bacterial toxins is obvious in any labo-ratory actively pursuing toxin purification, anal-ysis of toxin-producing bacterial strains, or se-rological screening of sera for antitoxin activity.Previous studies have shown cultured cells sen-sitive to toxin (9) and have pinpointed the HeLacell as a sensitive cell line (3, 6); however, suchassays either required more time or reagent orboth or did not allow precise quantitation be-cause of their dependence on an investigator'smicroscopic estimation of a cytotoxic dose. The

method described here is rapid, sensitive, repro-ducible, allows precise quantitation of a CD50endpoint, and the laboratory manipulations areextremely easy to perform, making possible largenumbers of simultaneous assays. The use of thismethod as a neutralization test further extendsthe possible laboratory applications.Although it was not the primary purpose of

these studies to examine the mechanism of toxincytotoxicity, the description of the test and ob-servations made during its development may aidfuture investigations with these objectives. Thecytotoxicity test is based on the observation thatcultured cells exposed to toxin either rapidlyrelease from glass or plastic culture vessels(monolayer test) or fail to attach (cell suspension

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366 GENTRY AND DALRYMPLE

TABLE 1. Comparison of methods for estimatingCDs with monolayers and ceil suspensions'

CDwMethod

Monolayers Suspensions

Graphic estimation 106s 10- 5Linear least-squares lo-6.8610-07

regressionNonlinear least-squares 10-6.7U lo-7.596

regression

aA cell concentration of 16,000 cells per well wasused. Ceils were pipetted directly into toxin dilutionsfor the suspension experiment. These same dilutionswere refrigerated overnight and used for the mono-layers once they were established. Staining and elutionwere as described.

b The 95% confidence levels for CDwo: 10-.652 to10-6.s4 (monolayers), 10-7.532 to 10-'7663 (suspensions).

0.1-

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ANTI- SHIGA-TOXIN

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LOGIO OF DILUTION

FIG. 5. Toxin neutralization study. The curve on

the left is a standard Shiga toxin assay in a 10-folddilution series. The right-hand curve was obtainedby mixing a constant dilution of the toxin with in-creasing dilutions of Shiga antitoxin (twofold dilu-tion series) and incubating this mixture with mono-

layers of ceils.

test). The physiological state of the releasedcells was not determined, but it would appearthat removing released and floating cells from a

toxin-treated monolayer would be a goodmethod for selecting cells undergoing acute toxiceffects. The condition of the remaining attachedcells has been studied indirectly in that HeLacells remaining at limiting toxin dilutions can

take up the vital stain neutral red, exclude try-pan blue, and if continued in culture, can divide,yielding a cell population as sensitive to Shiga

toxin as the original culture. It was interestingto note that a totally refractory cell line (BHK-21, baby hamster kidney cells) does exist, andvery preliminary toxin absorption studies wouldsuggest that the Shiga toxin used in these studiesdoes not bind to this cell.The toxin dilution curves very clearly ex-

hibited a direct relationship between toxin con-centration and cellular cytotoxicity. Speculationmight not allow a "single hit to kill" hypothesisconcerning the mechanism of toxin action, butthe data do suggest that toxin once committedis not available for further cytotoxicity, becauseliving cells in cultures at limiting toxin dilutionscontinue to thrive. The data presented showingreduced toxin titers with increasing cell concen-tration further support this contention.

It is anticipated that the utilization of themethodology described in this report and itsapplication to other toxins, cell, and experimen-tal objectives will facilitate the investigation ofthe mechanism of bacterial toxin-mediated celldeath.

ACKNOWLEDGMENTSWe wish to thank M. R. Thompson and Alison D. O'Brien

for providing the Shiga toxin and the antitoxin used in thiswork. Douglas B. Tang kindly furnished the nonlinear regres-sion analysis programs and helped with the computer. We alsothank Samuel B. Formal, Peter Gemski, and B. P. Doctor fortheir encouragement and advice.

LITERATURE CITED1. Conradi, H. 1903. Ueber losliche durch aseptische auto-

lyse erhaltene Giftstoffe von Ruhr-und Typhus Bazil-len. Dtsch. Med. Wochenschr. 29:26-28.

2. Dubos, R. J., and J. W. Geiger. 1946. Preparation andproperties of Shiga toxin and toxoid. J. Exp. Med. 84:143-156.

3. Keusch, G. T. 1973. Pathogenesis of Shigella diarrhea.III. Effects of Shigella enterotoxin in cell culture. Trans.N. Y. Acad. Sci. 35:51-58.

4. Keusch, G. T., G. F. Grady, L. J. Mata, and J. Mclver.1972. The pathogenesis of Shigella diarrhea. I. Enter-otoxin production by S. dysenteriae 1. J. Clin. Invest.51:1212-1218.

5. Keusch, G. T., G. F. Grady, A. Takeuchi, and H.Sprintz. 1972. The pathogenesis of Shigella diarrhea.II. Enterotoxin-induced acute enteritis in rabbit ileum.J. Infect. Dis. 126:92-95.

6. Keusch, G. T., M. Jacewiez, and S. Z. Hirschman.1972. Quantitative microassay in cell culture for enter-otoxin of Shigella dysenteriae 1. J. Infect. Dis. 125:539-541.

7. Mata, L. J., E. J. Gangarosa, A. Caceres, D. R. Perera,and M. L. Mejicanos. 1970. Epidemic Shiga bacillusdysentery in Central America. I. Etiologic investigationsin Guatemala, 1969. J. Infect. Dis. 122:170-180.

8. Thompson, M. R., M. S. Steinberg, P. Gemski, S. B.Formal, and B. P. Doctor. 1976. Inhibition of in vitroprotein synthesis by Shigella dysenteriae 1 toxin. Bio-chem. Biophys. Res. Commun. 71:783-788.

9. Vicari, G., A. L. Olitski, and A. Olitski. 1960. Theaction of the thermolabile toxin of Shigella dysenteriaeon cells cultivated in vitro. Brit. J. Exp. Pathol. 41:179-189.

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