Evaluation of enzyme linked immunosorbent assay for the serodiagnosis of amebiasis

0095-1137/79/12-0778/08$02.00/0 Vol. 10,

Evaluation of Enzyme-Linked

Immunosorbent

Assay for the

Serodiagnosis

of

Amebiasis

JAMESYANG* ANDMARIE T. KENNEDY

OntarioMinistry of Health,LaboratoryServicesBranch, Serology Section, Toronto, OntarioM5W 1R5, Canada

Receivedforpublication20August1979

This report describes the development and evaluation ofan enzyme-linked

immunosorbent assay (ELISA) for the detection of antibodies to Entamoeba

histolytica. Highly sensitiveand reproducibleresultswere obtained in

antigen-coated plates prepared by air-drying at 370C. Comparison of the ELISA with indirect fluorescent antibody and indirecthemagglutination techniques showed that the formerwas slightly more sensitive than the two latter methods. The specificitywasevaluated by testing specially chosen populationgroups. ELISA was negative in 96.4% of 693 normal adults and children and in 96.6% of377

patients with various parasitic,bacterial,mycotic, andother clinical diseases. The

assay waspositive in 26% of461patients with suspected amebiasisand inallof53

patients with amoebic liver abscess. The ELISAwasfoundtobeaspecific, highly

sensitive, and reliable procedure for detecting anti-E. histolytica antibodies in humans.

The enzyme-linked immunosorbent assay

(ELISA) developed independently in 1971 by

Engvall and Perlman (3) andbyVan Weeman

andShuurs(14) is based on the sameprinciples

asradioimmunoassay, except thatanenzymeis

used as a label for the antigen or antibodies

instead of a radioisotope. In the past several

years, the ELISA has beensuccessfully applied

tothe detection ofa

variety

ofantibodies and

antigens inbacteriology, virology, parasitology,

and clinical chemistry (11, 16, 18). More

re-cently, the usefulness ofthe ELISA for the

im-munodiagnosis of amebiasis has been reported

(2, 4),and thesensitivityoftheassay wasfound

tobecomparabletothat of the

radioimmunoas-say(17).

We have

investigated

the

application

of the

ELISAin thediagnosisofamebiasis. After

ana-lyzingthe parameters oftheassay and

develop-ing a standard micro-ELISA system, we com-pared the sensitivity of the assay with that of theindirect fluorescentantibody (IFA) and in-directhemagglutination(IHA) tests.

Specificity

was evaluated on specially chosen population groups. This paper describes the standard micro-ELISA technique and itsreliability and appli-cationfor the detection of antibodiestoE. his-tolytica.

MATERIALS AND METHODS

Antigen.The strainofEntamoebahistolyticaused in this study was IP-106 (5) obtained through the courtesy ofE. MeerovitchfromtheInstituteof

Par-asitology, McGillUniversity, Montreal, Canada. The strain was axenically grown in Diamond TPS-1 me-dium. Forpreparation of soluble antigen the amoebae werewashed three timeswithphosphate-buffered sa-line (pH 7.2). After the final washing, about5 mlof distilled water wasaddedtoeachml of packed orga-nisms, and the amoebae were disrupted for 3 to 5 min inanicewaterbath withasonicator cell disruptor W-375 (Ultra Sonico Inc.,Plainview, N.Y.) withan op-erating frequency of20kHz at an output of 40 W.

Finally,solubleantigenwasseparatedby centrifuging the sonic extract at 9,000 x gfor 1 h at 4°C. The soluble antigen was stored in 0.5-ml quantities at -70°C. Theproteincontentofthesoluble antigenwas determined by the method of Lowryet al. (7). The samestrainwasusedasantigen in the IFAtest.

Sera from clinicalcasesofamebiasis(53).The sera ofpatientssufferingfrom amoebic liver abscess wereobtainedthroughthe courtesy ofinvestigators in

Taiwan,Thailand,SouthAfrica, Mexico,andToronto GeneralHospital,Toronto.

Sera fromcasesofsuspectedamebiasis(461). In thisgroup,172sera wereobtainedfrom ahospital laboratoryinToronto,and theother289wereselected from thosereferred for amebiasistestingto areference laboratory. The sera had been tested by the IHA techniqueattherespective laboratories.

Sera frompatients with other diseases(377). Thisgroup hadpositive serological tests for various infections and conditions. There were 132serafrom patients withparasiticinfections(toxoplasmosis,

ma-laria, trichinosis, echinococcosis), 103from bacterial infections (streptococcemia, brucellosis, whooping

cough, syphilis, gonorrhea, fungi), 92 from cases of rheumatoidarthritis, and50fromcasesof hypergam-maglobulinemia.

778

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VOL. 10, 1979

Serafrompresumedhealthypersons(693). In

this group, 150 sera were obtained from pregnant women examined for prenatal Rh factor, 255 from both sexes whowerescreened routinely forsyphilis, 104 from young women (17 to 25 years) who underwent routinecheck-up forgonorrhea, and86employees of bothsexes.Inaddition, 98 serafrom children (1to 9 years)werealso examined.

Antigen carriers. Four types of microtiter plates andonetypeof tubeweretestedasantigen carriers. They were: Linbro, no. IS-FB-96S, polystyrene flat plate(Flow Laboratories,Rockville, Md.); Linbro, no. IS-MRC-96, polystyrene Uplate (Flow Laboratories); Cooke, no. 1-220-25Apolystyrene Uplate (Dynatech Laboratories, Alexandria, Va.); Cooke, no. 1-223-29 polystyrene flatplate (Dynatech); disposable

polysty-renetube,12by75 mm(Falcon2038,FalconPlastics, Oxnard,Calif.).

Conjugate.Horseradishperoxidaselabeledtogoat anti-human immunoglobulin G, Fab fraction (Miles Laboratories,Elkhart,Ind.), was used as a conjugate. A working dilution of theconjugatewasdetermined by the boxtitration method. Optimal dilution varied frombatchtobatch, ranging from1:500 to 1:2,000of theconjugate dilutions.

Substrate solution. Eighty milligrams of

5-ami-nosalicylicacid(K& K Laboratories, Plainview, N.Y.) wasdissolved in 100ml of hotdistilled water (65 to 70°C) and kept at 4°C. The 5-aminosalicylic acid solution wasbrought to roomtemperature, and

im-mediatelybeforeusethepHwasadjustedto 6.0with 1NNaOH. To 9.9 ml of the5-aminosalicylic acid, 0.1 ml of 0.5%H202wasadded.

ELISA. Unless otherwise stated, thefollowing pro-cedurewasusedthroughout this investigation.

(i) Antigen coating. A 0.05-ml amount of antigen dilutionwasadded to eachwell of microtiter plates and air-driedovernightat37°C.Triple-distilledwater was used as an antigen coating solution. The dried antigen plates were then stored in a plastic bag at -200C.

(ii) Performance of ELISA. Before testing, the antigen plates were washed three times with 0.9% saline containing 0.05% Tween20. A0.05-ml volume ofanappropriate dilution oftest sera wasaddedto the wells and incubatedovernight (16 to 18 h) in a moist chamberatroomtemperature. Known positive andnegativesera werealwaysincluded in eachplate as controls. For the positive controls we used one strong(IFA, 1:1,280; IHA, 1:4,096) and one weak (IFA, 1:40;IHA, 1:128-256) serum. All sera were diluted in phosphate-buffered saline with 0.5% bovine serum al-bumin and 0.05%Tween20(pH 7.4).

After washingasabove,0.05ml of conjugate, diluted in the phosphate-buffered saline-0.5% bovine serum albumin-0.05% Tween20, was addedto each of the

wellsand incubated at37°Cina moist chamber for 1 h.

Thewellswere washedagain and rinsed with

dis-tilledwater.Toeachwell, 0.2 ml of substrate solution wasadded, and the plates were kept at room temper-ature.Thereaction wasstoppedafter30 min of incu-bationby adding0.025mlof1 NNaOH to each well.

Optical density (OD) of the reaction product was determined at 449 nm with a Spectro-Colorimeter,

using aself-emptying microcuvette (Beckman/Spinco 151).

ELISA results wereexpressed either as a netOD value (ODof testseraminus OD of negativecontrol), when single dilutions (1:100) of the samples were tested, or as anendpoint titer, when serial twofold dilutionswereused. Ifatitrationwasused,the end-point was thehighestserumdilution that showedan ODvalue of l0.1.

EFA test. The testwas carried out as previously described by Ambroise-Thomas and Kien-Truong (1). Thestained slideswereread underaLeitzOrthoplan UVmicroscope with fluorescein vertical illumination or aZiessmicroscope with an epi-fluorescence system.

RESULTS

Determination of optimal antigen

con-centration. For this study, plates were

sensi-tizedbyincubating overnight at 4°C with

vary-ingconcentrationsofantigenin 0.05 M

carbon-ate buffer (pH 9.6) and tested against the two positive controlsera. Best results were obtained

atbetween 7 and40

jig

ofantigen perml (Fig. 1). Athigher concentrations of theantigen there was aslightdecrease in the ELISA value.

There-fore,to conservetheantigen,aconcentration of

7

Ag

ofantigen perml was used forpreparation

ofantigenplatesinsubsequent experiments.

Selectionofantigencarrier. In this

evalu-ation,wells ofplastic platesweretreated with a

0.1-ml volume of eachreagent (antigen, serum,

conjugate) except substrate (0.2 ml),andtubes

weretreated with l.0-ml volumes of all reagents.

The Cooke polystyrene plates with flat and U

bottoms appeared to bind antigen more

uni-formly.Althoughnomarked variation inserum

titers was observed, the other types of plates

gavelesssatisfactory results duetouneven

read-ings.Good resultswereobtained with the

plastic

II

1.4/

U

1

ANTKIENCONCENTRATION,.t1J1

FIG. 1. Effect of antigenconcentrationfor optimal coating of microplates. ODat 449nmin30min. (0)

Strong positiveserum (1:20,000); (0) weakpositive serum(1:100).

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tubes, but the use of the tubes required more reagentsand handlingtime thanthe plates. In our furtherstudywetherefore choseCooke pol-ystyreneplates withflat bottoms.

Determination of maximumantigen

coat-ingprocedure. To determine maximum

bind-ingofthe amoebaantigentotheplastic surface,

optimal dilution oftheantigenwaspreparedin

triple-distilled water and 0.05 M carbonate

buffer (pH 9.6). Microplates with each of the

preparations were then either incubated

over-night at

40C

in a moist chamber (wet

antigen

plate) orair-dried overnightat37°Cin an

incu-bator(dryantigenplate).

Figure

2illustrates the

mean values obtained from six assays on the

weakpositivecontrol serum with thefourtypes

of antigen-coated plates. In the wet antigen

plates, the distilledwater was

slightly

less

effi-I.:_

0

N

-t

0.1

0

CLIN. MICROBIOL.

cient as a coating solution than the carbonate buffer, whereas in the dried plates the distilled

water was equallyeffective, with ELISA values similar to those obtained with the carbonate buffer.

However,therewasasignificant difference in

sensitivity of theassaybetween thewetand dry systemsfor sensitization of theplates. The end-point titer of the controlserumwas1:640 in the dry antigen plates, whereas it was less than 1:

160 in the wet ones. The increased sensitivity

wasobserved in further experiments with other positivesera.By using the dry antigenplates the sensitivity could be increasedtwo- toeightfold higher than with the wet antigenplates (Fig. 3). The negative serum, on the other hand,

re-mained nonreactive in bothtypesofthe antigen plates. Moreover, the dry antigen plates could

T

* I

A-A

I

I1.

Antigen coating solution; distilled water

Antigen coating solution; carbonate buffer

T

antigen plate

SERUM DILUTION

FIG. 2. Effectofcoating proceduresonthe maximumbinding oftheantigen tothemicroplates.Each line representsmeanvalueofsixtests.

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VOL. 10,1979

bestored either at-20°C or roomtemperature for a year orlonger without loss ofsensitivity

(Table1). In asubsequent study, all plates were

sensitized by using the antigen diluted in

triple-distilledwaterandair-dryingovernight at37°C

in anincubator.

Effect ofreagentvolume in the ELISA.In

thisexperimentELISA wasperformed byusing

0.05ml and0.1ml ofallreagents (antigen,serum,

conjugate), except the enzyme substrate, which waskeptconstant at 0.2ml. Although OD values

obtained withthe0.05-mlvolume were

approx-imately half of those with the 0.1-ml volume,

serumendpointsdifferedlittle(Fig.4).

Fromtheseresults an initial serum dilutionof

1:100and 0.05-mlreagentvolumeswerechosen,

becausethe differencein OD values betweenthe

weakpositiveandnegativeseracouldeasily be

differentiated withthissystem.

Reproducibility. Figure 5 shows the

repro-ducibility oftheELISA,which wasdetermined

by testing thetwopositivecontrols on 24

differ-entdaysover aperiodof 6 months.The endpoint

titers of the weakpositiveserumranged from 1:

200to 1:400,and thestrongpositiveserumtiters rangedfrom 1:6,400 to1:12,800 between day-to-daytests. When allOD readings obtainedfrom

single dilutions of the positive (1:100 for weak

and 1:3,200 for strong) and one negative sera

wereplotted, variations of the OD valueswere

within95%confidence limits duringthe 6-month

testingperiod(Fig. 6).

ELISA on sera from normal subjects.

Afteroptimal conditions fortheELISAsystem

j00 oS 4L2S 5!r 11O3)00320 400i 12000 a

-

-f ..

.d

O"

4L,

3;

: f ,>w

_-ELISA FOR AMEBIASIS DIAGNOSIS

781

had beenestablished, the assay wasperformed

on 693 serafrom groupsofpresumably normal persons. All specimenswere screened atsingle dilutionsof1:100.

Ofthe seratested,668(96.4%)gaveODvalues of50.1, 19(2.7%) gave values of between 0.1 and 0.19, 4 (0.6%) fell between 0.2 and 0.29, and 2

(0.3%) gaveOD values of between0.3 and0.39.

When theIFA test wasperformedonthe25 sera

thatgave OD values of>0.1 andon a random

sample of50 serawhich showed OD values of

<0.1, 23of the formerspecimens werereactive

with the titersranging from1:40 to 1:80, whereas

all of the lattersampleswerenonreactive.Based

ontheseresults,anOD value of0.1 wasusedas

the lower limit for the presence of antibody.

Table 2 shows the positive findings among

groups of normal subjects. The positive rates

TABLE 1. Stabilityof antigen-coated platesafter

storage at-20°C androomtemperature

Titer ofpositivesera

Period of

-200C

Room temp

storage

(months) Serum Serum Serum Serum

1 2 1 2

0.1 320 51,200 320 51,200

0.5 320 25,600 320 51,200

1 320 51,200 320 25,600

4 400 51,200 400 25,600

5 400 25,600 400 51,200

12 400 >32,000 400 >32,000

15 NDa ND 400 51,200

aND,Not done.

*00 u3 400 800 16003'001)i10]11'000

*- v.-v*~EF. c

*@A.-O ?) )

)

)af

*----Q l)) b

iJ

***** @ J J d --~~

FIG. 3. Resultsof ELISAtestswith variousantigen coating procedures. Thefirst six rows of plate A (wet antigen) andplate B (dry antigen) were filled with antigen dissolved in triple-distilled water, and the remainingsix rows werefilledwith antigen in carbonate buffer. Row C, Negative serum;remainder of rows

werepositivesera.

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782 YANG AND KENNEDY

E

0E

c

SERUNI DILUTION

FIG. 4. Effect ofthereagentvolumesonthe sensi-tivity of ELISA. The assay wascarriedoutwith0.1 ml(0) and 0.05 ml(0) each ofantigen, serum, and conjugate, keeping substrateconstant at 0.2ml.

amongthegroups varied

little, ranging

from2.7

to 4.5%, but ELISA values in these sera were

usuallylow (OD0.1 to0.39,average0.18).

Comparison of

ELISA, IFA,

and IHA

tests. When ELISA and IFA tests were per-formedon289 serafrom

patients

with

suspected

amebiasis, the

positive

findings

were67

(23.2%)

and 63(21.8%),

respectively.

Table 3shows the

correlation between OD values and IFA titers. Good correlation was observed with

regard

to

qualitativeandquantitativeresults.

Inanotherexperiment,agroup of53 serafrom

patients with amoebic liver abscess was

exam-ined in serial twofold dilutions

by

ELISA and IFA test (Table 4). Therewas excellent agree-ment between thetwotests,

although

theELISA titerswere about10 times

higher

than the IFA titers.

TheELISAwasperformedon122codedsera

from patients with suspected

amebiasis,

which had been

previously

tested

by

the IHA tech-nique atlaboratory A. When the results of the two tests werecompared,

significantly

more pos-itive reactionsweredetectedbytheIHA(52.5%) thanbytheELISA

(30.3%),

andthe correlation between the two tests was poor (68%). Of 33 serum samples that were positive by the IHA

test

only,

33 were nonreactive by the IFA test

and theremaining 2showedweak reaction.On

the other

hand,

six sera which reacted only in theELISA gavepositiveIFAresults.

Because clinicalinformationon the suspected

amoebic patientswaslacking, it was difficult to

explainthediscrepancybetween the ELISA and

the IHAtest. Therefore, an additional 50 sera from another group of suspected amoebic pa-tients were coded and split into aliquots, and eachwas examined by ELISA, IFA, and IHA. The IHA test wascarried out at laboratory A

andareferencelaboratory,B.

Thecorrelationofthe ELISA with each of the

otherserologicaltestsis shown in Table 5. Both

ELISAandIFA testshowedagoodcorrelation

1.0

1.6 l Mean valueofstrongpositive 1.4 (j's+ Meanvalueof weak positive

06 I X

104 \ \

0.0.

0.6- 0.4-

0.2-Cut-off line

100 200 400 800 1600 3200 6400 12800 25600 51200 SERUMD1U117N

FIG. 5. Reproducibility of ELISA. Two positive

sera were titrated on 24 occasions over a 6-month period.

p0s,7jPsti (a-we.kpositi., 1:100; 0 strong positi,e, 1:3200)

017

0.6 _ __ _ _ _ _ _ C, _ _ _O--C- +2

0.6 0 0

-I______

_

_-__

__+

n2

0000C 0° °O 0

110.40000000000 0000

OCT. NOV. DEC. JAN. FEB. MAR.

1917 1978

DATE

2SD

*2SD

,2SD

2SD

FIG. 6. Distribution of OD values obtained from twopositive andone negative sera tested atsingle dilutionsduringthe 6-monthtestingperiod.

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VOL. 10,1979

TABLE 2. ResultsofELISAforamebiasison sera

fromnormal individuals

No. %

Posi-Category tested tive

Pregnantwomen 150 2.7

Children (1-9 years) 98 3.1

Persons screenedforsyphilisa 255 4.7

Persons screened forgonorrheab 104 2.9

Routinecheck-ups 86 3.5

Total 693 3.6

aVDRLnegative.

bSeronegative by IFA and complement fixation

tests.

TABLE 3. Correlationof ELISA OD values with IFA titers in 289serafrom patients with suspected

amebiasis

IFAtiter ELISA OD

s20 40 80 160 320 640 1,280

0-0.09 214 4 2

0.10-0.19 7 16 5

0.20-0.29 2 5 2 3

0.30-0.39 3 4 1

0.40-0.49 1 2 3

0.50-0.59 2 1 4

0.60-0.69 1 1 1

0.70-1.0 1 1 1

TABLE 4. Correlation with ELISA with IFA titers in53serafrom patientswithamoebicliverabscess

ELISA ___IFAtiter

titr -20 40 320 0 1,280 2,560 5,120 <100

100

200 1

400

8001

1,600 2 1 1

3,200 2 2 1 1

6,400 6 6 1

12,800 3 6 5

25,600 _ 1 6 3 1

51,200 __1 2

(92%)insensitivity. Good correlation (88%)was

also observed betweentheELISA and the IHA results oflaboratory B. One serum which was

reactive(1:512) by the IHAtestbut negativeby

theELISAwasalsonegative by IFAtest. How-ever, all five sera that were positive only by

ELISA gavepositive IFA titers (1:40 to 1:160). Asearlierobservations have shown,poor cor-relationwasfound between the resultsof ELISA

andIHAtest atlaboratory A (Table 5). There wasonly 60% agreementin sensitivity between thetwo tests.Of17serathatwerepositive only

by the IHAtest,2gaveweakpositivereactions

by the IFAtest.

Since the results of IHAtests performed on 289 sera at laboratory C were available, they were also compared with the ELISA resultson

thesamesamples (Table 6). Of the 33serathat werepositive by ELISA only, 29 showed positive

IFA, with titersranging from 1:40to 1:640, and

4 werenegative. On the other hand, 9 of the 11

samples which gave positive reactions only by the IHA testwerenonreactive by the IFAtest

and 2 were weakly reactive. Although the

sen-sitivity of the ELISA and IHA test agreed (84.8%), therewaslittle correlation betweenthe

ELISA OD values and the IHA titers. This is in

contrast to the IFA findings on the same sera

(Table 3).

Specificity. Because cross-reactions with

otherdiseases often influence the results of

se-rologicaltests,serumsamples frompatients with

various parasitic and bacterial infections and other clinical conditionswereassayed for

ame-biasis (Table 7). Negative ELISAwasobserved

in 97% of 132 sera from the groups of other

parasitic infections and in 96.3% of 245 from

TABLE 5. ResultsofELISA, IHA, and IFAtests on 50 serafrompatients with suspected amebiasis

ELISA

Test No. No. %

Corre-positive nega- lation

IHA testa

A) No.positive 13 17 60.0

No.negative 3 17

B) No. positive 11 1 88.0

No.negative 5 33

IFAtest

No.positive 15 3 92.0

No.negative 1 31

aIHA testwasperformed attwo different

labora-tories.

TABLE 6. Comparisonof ELISA OD values and IHA titers in289serafromcaseswithsuspected

amebiasis IHA titer ELISA OD

c32 64 128 256 512 1,024 2,048

0-0.09 203 8 6 3 2

0.10-0.19 19 5 2 2

0.20-0.29 2 4 3 2 1

0.30-0.39 2 1 3 1 1

0.40-0.49 2 1 1 2

0.50-0.59 1 2 1 2 1

0.60-0.69 1 1 1

0.70-1.00 1 1 1

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784 YANG AND KENNEDY

TABLE 7. Resultsof ELISAon serafrompatients reactivefor variousdiseasesbyotherserological

tests

Category (othertests') tested %

Posi-Toxoplasmosis (IFA+) 121 3.3

Malaria (IHA+) 5 0

Trichinosis(CF+) 3 0

Echinococcosis(CF+) 3 0

Streptococcalinfection (ASO') 18 0

Brucellosis (CF+) 2 0

Whooping cough(CF+) 5 0

Syphilis (VDRL+) 63 3.2

Gonorrhea(CF+) 9 11.1

Mycoticdiseases(CF+) 6 0

Rheumatoid (latex+) 92 4.3

Increased gamma globulin 50 4.0 (-4.0 g/100ml)

Total 377 3.4

a+,Positive. CF,Complement

fixation;

ASO, anti-streptolysin 0test;latex, latex agglutinationtest.

patients with the nonparasiticdiseases. The ob-servationswere similar to those fromthenormal

individuals(Table2).

DISCUSSION

Inthe presentstudywehave establishedthe

optimalconditions ofamicro-ELISA system for

thedetection ofantibodyto E. histolytica. Our

resultsindicate that the ELISA is a useful

ad-dition to the wide range of

currently

available

serological methods for the diagnosis of

ame-biasis.

To obtain the maximum adsorption of the amoeba antigen to theplastic surface, the pro-cedure used for theantigen coatingisimportant. Wedemonstrated that sensitization of theplates

by air-dryingcould increase the sensitivity

sev-eral times more than the wet antigen coating system(2,8),which is carriedoutby incubating

the plates at 37 or 4°C. Increased sensitivity

withgoodreproducibleresultswasalsoobtained

byourELISAmethod with

antigens

of

Trepo-nemapallidum, Toxocara

canis,

and Ascaris

suum (unpublished data).

Furthermore, the sensitized plates could be stored for a year or longer at either -20°C or room temperature without losing antigenic

ac-tivity. The use of ready-made antigen plates eliminates thenecessityfor

daily

preparation by laboratories with limited resources. It also

re-ducespossible variations of sensitizationby pi-pettingerrorwhich may arise indaily prepara-tion oftheplates.

Reproducibility of the assaywas well within

the acceptable range. When two positive sera

and one pooled negative serum were assayed

over a6-monthperiod, thetiters of the positive seraremained within±1twofold dilution. When ODvalues of the negative serum at 1:100 were '0.1, whichrarelyoccurred, all tests in the same plate were repeated, although there appeared to be no adverse effect on the positive controls.

In the presentstudy we found the ELISA to

behighly sensitive.The assay was positive in all

of 53 cases of clinically proven amoebic liver abscess, with the ELISA titers ranging from 1: 1,000 to greaterthan 1:50,000.

When the results of ELISA were compared with those of the IFA testperformed on a total of514 serafrom patients with clinicallyproven

andsuspected amebiasis,positive findings were

33.7 and 32.9%, respectively, with a correlation

of94.9%. Our observations of good correlation between the ELISA and IFA agree with the

findingsof Bos and Van DenEijk (2).

When theELISAwascompared with the IHA results of laboratories A, B, and C, it showed

varying correlations: 60, 88, and 84.8%,

respec-tively (Tables5and6).On thewhole, the ELISA

appearedtobe slightlymoresensitive than the IHA test. The variations in the results of the IHA test mayhave been relatedtothe amoebic strainused inpreparationofantigensaswellas other reagents inuse attheselaboratories.It is

alsopossiblethat the ELISA and IHAtestmay

detect differentantibodies.Otherworkers (8, 10,

13, 15)havereported that the antibody detected by IHA and latex tests may be different from that detectedbythe IFA andgeldiffusiontests.

It should be pointed out that there was a noticeable discrepancy between the results of the ELISA and the IHA test in the present study. High IHA titers (1:>512) were observed in 17 sera that were negative by the ELISA. However,noneof them showedpositive reaction in the IFAtest.On the other hand, the IFA test detectedantibodyinsevenserawhichwere neg-ativebyIHAbutpositive byELISA (1:>800 or

OD>0.5).Furtherstudies should be carriedout

todetermine whether theantibodydetected by theELISA is similartothatinvolved in the IFA

testbut differentfrom thatdetectedby theIHA test.

The presentstudyprovidesareasonable basis for thespecificityof the assay. Positive ELISA

wasobtained in 3.6% of the normal groups and 3.4% of thepatientswith variousparasitic, bac-terial, and fungal infections and other clinical conditions(Tables2and7).The lowpercentage of the positive findingsfrom the patientswith diseases other than amebiasis, which must be considered in the differential diagnosis,

corre-lated very closely with those from the normal individuals.Thus, it isprobable thatthepositive

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VOL. 10,1979

ELISA from thesepatientsmayreflecta

resid-ual antibody level fromapastinfection with E.

histolytica. That the antibody may persist in

low titers for many years without any sign of reinfection has been reported (12).

Although the sensitivity of the ELISA is

com-parabletothatof the IFA test, the formerhas

several advantages over the latter; the assay can be carriedout withsimple equipment, the

tech-nicians do not require special skill, and it can be readvisually or assessed objectively with a col-orimeter. If the ELISA results are required within aworking day, the amountsof the

anti-gen, serum,andconjugate need only be changed from 0.05 mlto 0.1ml(0.2 ml for substrate),and the serum incubation time then should be shortened fromovernightat roomtemperature to 1 h at

370C (unpublished

data). The assay

can becompleted within4 h with asensitivity

equaltothe0.05-mltestsystem.

No conclusion can be drawn in this study

regarding thesensitivity of the ELISAin

amoe-bicdysenteryorcolitis becauseonlyafewcases

with clinical evidence have been tested.

How-ever, the findings in the present study and in otherinvestigations of amebiasisamong

homo-sexuals (6) suggest that with the assay system

describedinthisreport, anELISAtiter of 1:100

wouldappear tobe specific inthe detection of

antibodiestoE. histolytica.

ACKNOWLEDGMENTS

We thank D. J. Reed for helpful advice, C. Wiley for excellent technicalassistance,and E. Meerovitch(the Insti-tuteofParasitology, McGill University, Montreal) for supply-inganaxenic strain of E.histolytica. Weareindebted to the

followinginvestigators who kindly supplied serum samples

fromamoebic patients: E. Proctor,Tropical Diseases Unit, TorontoGeneralHospital,Toronto; J. H.Cross,U.S.Naval Medical Research Unit No. 2,Taipei, Taiwan; S. Tharavanij, Faculty ofTropicalMedicine, Bangkok, Thailand.

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