CopyrightC1977 American Societyfor Microbiology Printed in U.S.A.
Use
of the
Enzyme-Linked Immunosorbent
Assay (ELISA)
and Its Microadaptation
for the Serodiagnosis
of
Toxoplasmosis
KENNETH W. WALLS,* SANDRA L. BULLOCK, AND DONNA K. ENGLISH
Parasitic Serology Branch, Center forDisease Control, Atlanta, Georgia30333
Received forpublication 30 September1976
The enzyme-linked immunosorbentassay (ELISA) has provedto bea
sensi-tive
and
specific
quantitativeprocedure for the serodiagnosis of toxoplasmosis.Using the
toxoplasma
model, severalparametersof thetest wereinvestigated.Day-to-day reproducibility
was90%withinonetwofold dilution and98%specific whentestedagainstbatteries
ofserafrom other diseases. Both the tube methodandthe
microtitration method
wereused
successfully. ELISA
resultsareequiv-alent to those
found
inthe
indirect immunofluorescence test, yet the ELISAprocedure is simpler and
morerapidtoperform.A
number of serological
tests havebeen
usedfor
the detection of antibodies
toToxoplasma
gondii. The enzyme-linked immunosorbent
as-say
(ELISA)
appears tooffer
acombination of
the
best qualities of all. Described first by
Engvall
and Perlmann (4), ELISA is a
modifi-cation
of the radioimmunosorbent technique
(RIST),
inwhich
anenzyme
issubstituted for
the
radiolabel of the antiserum. In the short
time since its
inception,
the test has been
ap-plied
tothe detection of
anumber of
metabo-lites
(7, 12)and bacterial
(3, 5) andparasitic
diseases (2, 9-11, 15). Ruitenberg et al. (9-11)
have
published numerous articles
on the use ofELISA
intrichinosis, and Voller and others
have described its
use inamebiasis (1),
schisto-somiasis (6), malaria (7), and
Chagas' disease
(4).
Recently, Voller
etal. (13)
described
aqual-itative
ELISA for toxoplasmosis.
Although
originally
described
as asingle
tube,
photometrically quantitated
procedure,
ELISA
wasreadily
adapted
tomicrotitration
and automation. In
asemiautomated
system,
Ruitenberg
etal. (11) demonstrated that
on aroutine
basis
as many as4,000
seracould be
tested
daily.
Within the limits of the
adsorbative
qualities
of the
polystyrene sorbent,
anysoluble
antigen
apparently
canbe
incorporated
into
the
test.Consequently,
itseemedlogical
that a solubleantigen
prepared
fromdisrupted
whole
T.gon-dii
organisms would
containboth thecell wall
antigen
(the
activeantigen
inthe indirect
im-munofluorescence
test[IIF]
and
themethylene
blue
dye test) and the
cytoplasmic antigen
(which
is active inthe
passive
hemagglutina-tiontest).
ELISA
withasoluble
antigen
should
give
reactions
characteristic of all the former
procedures.
We
describe here an ELISA procedure for
toxoplasmosis
inwhich an antigen
derived
from
solubilized whole organisms is used. Both
the tube
and microtitration
procedures have
been successfully used.
MATERIALS AND METHODS
Antigen.Tachyzoites of the RH strain of T.gondii
were harvested from mice infected 3 days previ-ously. The peritoneal fluid was withdrawn and mixed with at least 10 volumes of0.5%formalinized
phosphate-buffered saline (PBS), pH 7.2. After re-mainingfor1hatroomtemperature,the cellswere collectedbycentrifugationand washed three times with PBS. After the final wash, the packed cells wereresuspendedto a 1%concentration (vol/vol)in distilledwateranddisruptedin aRibicell fractiona-tor at 20,000 lb/in2 at 7°C. After the mixture had settledovernight, grossparticles wereremoved by
centrifugationat2,000 xgfor30min.The superna-tant wascollected and extracted withanequal vol-ume oftrifluorotrichloroethane (Genitron 113, Al-lied ChemicalCorp.,Morristown,N.J.).The result-ingclearantigenwasstoredas1-mlaliquantsinthe vaporphase ofaliquidnitrogen storage box.
Conjugate. Horseradishperoxidase
conjugated
togoatanti-human immunoglobulin G-Fab was
pre-pared bythemethod of Kawaoi and Nakane
(Fed.
Proc.32:840, 1973)modifiedbyRuitenbergetal. (9).Theenzymepreparation usedwashorseradish per-oxidase type VI (Sigma Chemical Co., St. Louis,
Mo.),with an RZactivity of2.68.A10-mgamountof horseradishperoxidasewasdissolvedin 2 mlof0.3 Msodium bicarbonate,pH8.1.Two-tenths milliliter of1%(vol/vol)1-fluoro-2,4-dinitrobenzene (Eastman
Kodak Co., Rochester, N.Y.) in absolute ethanol wasadded, and thepreparationwasmixed for1hat roomtemperature. A 2-mlamountof aqueous0.08M 273
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sodiumperiodatewasaddedand mixed for 30 min at room temperature. Then, 2 mlof0.16 Methylene glycol was added and mixed for 1 h. Theresulting solution was centrifuged at900 x g for 10minto remove thesmiallamountofprecipitatedimpurities and wassubsequentlydialyzedat4°C againstfour 1-liter changesof 0.01 M carbonatebuffer,pH9.5.The dialysate was mixed with 10 mg of anti-immuno-globulinfor 3 h atroomtemperatureand thenwas dialyzed against 0.15 M PBS, pH 7.2, at4°C. The conjugated materialwasseparatedfrom the uncon-jugated protein and enzymebyfiltrationon a
Sepha-dex G-200 column equilibrated with 0.15 M PBS containing 0.02%NaN3.Thepeaksthatshowed opti-caldensityactivityatboth 280nm (fortheprotein)
and 403 nm (fortheperoxidase) werecollected and concentratedto atleast1mg ofprotein per ml. The conjugate wasstored in the cold(4°C).
Testsera. Test seraconsisted of fivepoolsof hu-mansera of known levels ofreactivity in the IIF procedure for toxoplasmosis. Additional sera used for testing sensitivity andspecificitywere selected from sera submitted to theCenter for Disease Con-trolthat had titers inthe conventional serological
testsforthediseases suspected.
Immunosorbent. Disposable polystyrene tubes (11by 55 mm,PharmaciaLaboratories, Inc., Pisca-taway, N.J.) were filled with 1 ml of antigen ad-justed to 5 ,ug of protein per ml in0.1 Mcarbonate buffer, pH 9.6, containing 0.02% NaN3. The tubes were incubated in a 37°C water bath for 3 h and stored in the cold
(4WC)
untilused.Substrate. The substratewaspreparedby dissolv-ing 80 mg of5-aminosalicylic acid (Aldrich Chemical Co., Inc., Milwaukee, Wis.)in 100mlof hot distilled water (80°C). Immediately beforethe solution was used, the pH was brought to 6.0 with1NNaOH. To 9 parts of5-aminosalicylic acid solution, 1 part of 0.05% (vol/vol)
H2O2
wasadded.ELISAtube test. Themethod of Ruitenberg et al. (11) wasfollowed for the tube test, except for a few minormodifications. The day before assay, the im-munosorbent tubes were emptied by suction, refilled with2mlof 1%(wt/vol) bovine serum albuminin0.1 M sodium carbonate buffer (pH 9.6) containing 0.02% NaN3, and incubated at 4°C overnight. To begin the test, the tubes were washed with distilled waterthreetimes for 5 min each.Then, 1 ml of each dilution of test serum in PBS was added to each tube, and the tubes were incubated at 37°C for 30 min. The tubes were washed three times as before andthenincubated with1mlof 1%(wt/vol)aqueous bovine serumalbuminsolution at 37°C for 30 min. Thetubes were washed again and thenrefilled with 1 ml ofconjugate diluted to the proper dilution in PBS with 1% bovine serumalbumin. The tubes were incubated at 370C for 30 min and then washed as before. One milliliter ofsubstrate was added and allowed to react at roomtemperature for 1 h. The reaction was terminated by adding 0.1 ml of 1 N NaOH.Thebrown-colored product was measured in aColeman Jr.spectrophotometer at 450 nm.
ELISA microtitration procedure. The procedure followed for the microtitration test was that of Rui-tenberg et al. (10). Allreagents used were the same
asthose for the tube tests. Disposable
polystyrene
microtitrationtrays with 96 flat-bottom wells (FlowLaboratories,Inc., Rockville, Md.)werecoated with
antigen by adding 100 ,l of the diluted antigen
solutiontoeach well andincubatingthetraysina water bath at 37°C for 3 h. Trays containing the
antigensolutionwerethen stored at 4°C until used. Before the assay, the trayswerewashedthree times
by flooding them with distilled water and then
draining, inverting, and vigorously shaking them. Testsera werediluted withPBS,and100 ,ul of each dilution was transferred to a well ofa tray. The trays were incubated in a 37°C water bath for 30 min.They were then washed as before, and 100 ,ul of conjugate, diluted in PBS with 1% bovine serum
albumin, was added. The trays were again incu-bated at 37°C for 30 min and then were washed. Finally, 100 ,ul of substrate was added, and thetrays
wereallowedtoremain at roomtemperature for 1 h while the reaction occurred. The reaction was then terminatedbyadding 25 ,I of 1 NNaOH.Thebrown reaction product was evaluated visually. The last serumdilutionshowing darker color than the lowest dilution of the negative serum was regarded as the end point.
RESULTS
To increase
objectivity
andreproducibility,
we set50%transmittance
(T)
onthecolorime-ter as the maximum
reading
tobeconsidered
positive.
We titered all reagents to the 50% endpoint
todetermine
optimal dilution
or serumend
points.
Our
conjugate,
prepared as ananti-human
immunoglobulin G-Fab, is highly specific
and
reactive. Table 1
illustrates
atypicalconjugate titration. In the lower dilutions,excessive
background readings caused the negative se-rum to appear tobe
positive. At the
optimum
dilution in this titration there was essentially
noreactivity in the negative serum (61%T at
1:10),but the positive serum was reactive to its known IIF titer of 1:2,560 and gave a sharp end point.
Consistency
in results of serologicalproce-dures for
the diagnosis
of toxoplasmosis isex-tremely important. Since the most widely used procedure is the IIF test, which gives the same resultsasthe methylene blue dye test, we
con-sidered it important to compare the IIF and ELISAprocedures. Table 2 illustrates the
com-parative results of thesetwo tests. Because
the
ELISA test is relatively easy to perform, we
tested it in twofold
dilutions;
the IIF testwastested in
fourfold dilutions.
Thisresulted inanapparent
skewing
of thedata, but the data still exhibit a close straight-linerelationship.
The only obviousdiscrepancy
is the oneserum that is 1:16by IIF and 1:1,024 byELISA.To
determine reproducibility,
wetested onepositive serum (IIF 1:1,024) and one negative
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TABLE 1. Conjugate titration for ELISA in the toxoplasmosis system
6% T (at 450 nm) atserum dilution of: Conjugate
dilu-
Testserumation 10 40 160 640 2,560 5,120 10,240
400 Pos 11 7 5 5 13 28 31
Neg 19 20 23 30 Xb X X
600 Pos 5 9 5 9 25 42 46
Neg 35 39 35 41 X X X
800 Pos 9 29 10 19 38 48 69
Neg 43 62 50 50 X X X
1,000 Pos 10 35 15 21 50 68 76
Neg 61 66 66 55 X X X
aPos, Positivecontrol (IIF 1:1,024); Neg,negative control(IIF <1:4).
bX, Notdone.
TABLE 2. Comparisonof IIF andELISA tests for toxoplasmosis
ELISA IIF
<16 16 32 64 128 256 512 1,024 -2,048
<16 22a
16 1 3 1
64 2 2
256 2 8 9 1 2
1,024 1 1 1 3 2 2
24,096 2
a Number of sera.
serum(IIF< 1:16)on10different days. Table 3
shows that in 8 ofthe 10tests,titersof 1:512 or
1:1,024wereobtained for the positiveserum.In oneinstancethetiterwas1:256, and in onlyone
instancewasthereamajorvariation, inwhich
the titer was 1:4,096. The high titer was
ob-tained inatestthatnormally would have been
consideredinvalid because thenegative control
was stronglypositive.
Perhaps abetter measure ofreproducibility
is seen in the results of the negative control serum. Since all dilutions of the serum are
negative, only the colorimeter readings of the initial dilutionwerecompared. In 8 of 10 tests,
T varied only from 42 to 68%. In the one
in-stancethatwaslow, 28% T, thepositiveserum was similarly low. When the
negative
serum gave 81% T, there appeared to be no adverseeffect on the positive serum. (Although the
data do not show it, the titer of the positive
serumwas1:1,024onthatday.)
Determining thespecificity oftestsfor
toxo-plasmosis
iscomplicated by the high prevalence ofantibody in thenormal population. Table 4showsthe results of batteries ofserasubmitted
for theserodiagnosis of various diseases. With
the single exception of the 1:256 result in an
amebiasis serum, all sera positive in ELISA
eitherwereconfirmedby beingalsopositive by IIForhad titerstoolow to be of clinical
impor-tance. Ofsome concern is theonerheumatoid
specimen that was positive by both IIF and
TABLE 3. Reproducibility of ELISA titers for toxoplasmosis on 10 successive days
Titer Serum tested
256 512 1,024 2,048 4,096
Positive (timestested) 1 3 5 la
Negative (% T at 1:16 64, 54, 68, 64, 81,
dilution) 28,a 50, 42, 48, 45
aTested on same day.
TABLE 4. specificity ofELISAtestfortoxoplasmosis
No. of sera Serasubmittedfor: + IIF
+ELISAa +IIF -IIF +ELISA -ELISA
Amebiasis 256 2 7
Echinococcosis 32,32 3 6
Histoplasmosis 64 3 11
Cytomegalovirus 16, 16 1 8
Rheumatoid 1 12
a Reciprocal of ELISA titer for eachserum
nega-tiveby IIF but positivebyELISA.
ELISA.
Although only
1of
13 sera waspositive,
rheumatoid
serumhas
beenrecognized
as acomplicating factor
intheIIF
procedure,
sothis
finding
justifies
a morethorough
study
of
these seraby ELISA.
DISCUSSION
These
data
clearly
delineate
thesensitivity,
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specificity, and
reproducibility
of ELISA. As evaluated here withtoxoplasmosis
as the test system, thisprocedure
clearly
shows great promise as a systemforserodiagnosis
ofagreat variety ofdiseases
and conditions. We have found it to beassensitive andspecific
astheIIF
procedure and
highly
reproducible.
Although Voller et al. (13) founddiscrepancies
between resultsfrom ELISA and the
methylene blue
dye test andbetween ELISA and
theindirect
he-magglutination test, wefound excellent
agree-mentbetween
ELISAand
IIF.With the microtitration
techniques
in partic-ular, reagents are inexpensive and stable. The conjugate, asprepared,
wasdiluted
1:1,000 for use,and ithas been used for
6months
withoutdetectable deterioration. The conjugate used
in thesestudies
wasprepared
according
topub-lished techniques for
ELISA,
exceptthatNaN3
was
included
in the bufferfor
theSephadex
column.
Since these data
wereobtained,
newconjugates
have been
prepared
according
tothemethod
of Nakane and Kawaoi (8), and theNaN:,
wasomitted since it is an enzyme inhibi-tor.This change provided
superiorconjugates
that give more
dependable reactivity,
butthe
sensitivity and
specificity
must still be evalu-ated.The
high
working dilution
andlong
shelf lifeof
the conjugates make them very economi-cal reagentsfor
large-scale testing. The
stabil-ityofhorseradish
peroxidase permits prepared conjugates tobeeasilyshipped
and stored.The high degree of reproducibility is very
encouraging. Plus
or minus one twofold dilu-tion is ademanding
criterionfor
aserological
procedure and
is notattained by
mostwith anyconfidence;
yetonly
onetestfellmarkedly
out-side this
range.The
negative serumonly varied
afew
percentT and wasneverconsidered
posi-tive in any ofthe
tests.This level of
reproduci-bility
assures aprocedure that isreliable
and thatlends
itself tomodification
and refine-ment.Specificity
was well withinexpected limits.
Clinical information
withwhich
toevaluate
thetoxoplasma
reactivity ofthe
onenegative-posi-tive serum submitted for amebiasis serology
was notavailable. Whether the IIF result is a
false
negative or the ELISA result is a false positive is undecided. Of more importance,however,
is the positive rheumatoid serum. One of the major problems with the IIF test for toxoplasmosis is the reaction with sera from autoimmune diseases. We hoped that the par-tialpurification of the antigen might eliminate this cross-reaction. One of the 13 rheumatoidserareacted inboth IIF and ELISA. One ques-tions whether the reaction is a specific
toxo-plasma
reaction in both tests orsimply
anon-specific
reaction in each.Although
ELISA has been used foravariety
ofdiseases and
conditions,
it still needs to be standardized. We haveattempted
to delineatesomeof theparameters of the
technique
itself,
using toxoplasmosis
as the test model.Never-theless, optimum
conditions forvirtually
everystep
of theprocedure
still need to be defined. Allinvestigators
who havereported
sofarhave used theoriginal
method ofEngvall
andPerl-mann
(4)
orRuitenberg
et al.(10),
or minor modifications of them. Little has been done to evaluate and standardize themethodology.
Now that we have established the limits of
specificity
andreproducibility withasomewhatpurified
toxoplasma antigen, we can use thisquantitative procedure
to define moreclearly
the
optimum
conditions for ELISA.LITERATURE CITED
1. Bos,H.J.,A.A.vandenEijk, andP.A.Steerenberg.
1976.ApplicationofELISA-enzymelinked immuno-sorbent assay-in the serodiagnosis ofamoebiasis. Trans. R. Soc.Trop.Med. Hyg.69:440.
2. Bout, D.,J. C.Dugimont, H. Farag, andA.Capron.
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9. Ruitenberg, E. J., I. Ljungstrom, P. A.Steerenberg, and J. Buys. 1975. Application of immunofluores-cence and immunoenzyme methods in the serodi-agnosis ofTrichinella spiralis infection. Ann. N.Y. Acad. Sci.254:296-303.
10. Ruitenberg, E.J., P. A. Steerenberg,andB.J. Brosi. 1975. Microsystem fortheapplicationofELISA (en-zyme-linked immunosorbent assay) in the serodi-agnosis ofTrichinella spiralis infections. Medikon Ned. 4:30-31.
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12. Van Weemen, B. K.,andA. H. W. M. Schuurs. 1972. Immunoassay using hapten-enzyme conjugates. FEBS Lett. 24:77-81.
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en-zyme-immunoassay for toxoplasmaantibody. J. Clin. Pathol. 29:150-153.
14. Voller, A., C. Draper, D. E. Bidwell, and A. Bartlett. 1975. Microplate enzyme-linked immunosorbent
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15. Voller, A., G. Huldt, C. Thors, and E. Engvall. 1975. A
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