0095-1137/80/06-0675/07$02.00/0
Standardized
Quantitative Enzyme-Linked Immunoassay for
Antibodies
to
Toxoplasma
gondii
T. M.LIN,' S. P.HALBERT,`* ANDG. R.O'CONNOR2
CordisLaboratories, Miami, Florida331271; andFrancis I. ProctorFoundationfor Research in
Ophthalmology, University
of California,
SanFrancisco,California
941432
A quantitative and standardized enzyme-linked immunosorbent assay is
de-scribed which uses lyophilized antigen-coated disks for the detection of human
antibodies to Toxoplasma gondii. It doesnotrequire serial dilution of thetest
specimen, and theobjective absorbance readingsareconverted into international
unitspermillilitertraceabletothe World Health Organization's reference
stan-dardpreparation oftoxoplasma antibodies. It was shon.-<m tobe highly specific,
reproducible, and sensitive. The reagents were stable, without biohazard, and ready foruse. Studies of variousparametersin the assayindicated that thetest
conditions were relatively flexible. The enzyme-linked immunosorbent assay
results correlated satisfactorily with the methylene blue dye test, the indirect immunofluorescence test,and the passivehemagglutinationtest.
The diagnosis of human toxoplasmosis has depended heavily on serological methods for
detecting antibodies to Toxoplasma gondii. These principally include the classical
Sabin-Feldman methylene blue dye test (MBD), the indirect immunofluorescence test (IIF),and the
passive hemagglutination test (PHA). The cur-rentmethodsofchoiceforclinicalpurposes are
the MBD andIIFtests(8). However, the former
uses liveorganisms andascarce"accessory fac-tor,"andIIFrequiresafluorescent microscope and well-standardized fluorescein-labeled
anti-body preparations.
Recently, enzyme-linked immunosorbent as-says(ELISA) have been appliedto the detection
oftoxoplasma antibodies, particularlyin micro-titerplates (1, 2, 16-18). The microtiter ELISA is generally semiquantitative and may give
er-ratic results due to the nonuniform absorbent
quality of the plastic microtiter plates (3). In our laboratories, an ELISA for detecting
hepatitis B surface antigeninhuman blood
sam-ples has been developed which utilizes small
plastic disks for covalent binding of the
solid-phase reagents (6). This procedure was
incor-poratedinto adiagnostic kit (Cordia H,Cordis
Laboratories, Miami, Fla.). As reported here, this format was modified for the quantitative determination oftoxoplasma antibodies in hu-manspecimens. Inaddition, to establish a uni-form basis ofreference in thereporting of results, we measured thetoxoplasma antibody levels in international units permilliliter, providing val-ues directlytraceable to the World Health
Or-ganization's (WHO) firststandard human
anti-toxoplasmaserumpreparation (19). In the
pres-ent ELISA technique, serial dilution titrations and pretreatment of the test samples are not required;thetotal incubationtime is short (1h and 50min); and either serumorplasmacanbe used. The communication describesseveral as-pects of thegeneralprocedure and comparative results withMBD, IIF,and PHA assays.
MATERIALS AND METHODS T. gondii organisms of the RH strain were har-vested by differential centrifugation from peritoneal
exudatesof3-dayinfected mice with
heparin-contain-ingsaline (1 mg/nm). The suspensions were centri-fuged and washed three timesat 4°C (3,000 xg, 10
min). Afterbeingfrozenat-20°Catleastovernight,
theywerethawed,sonicated,andcentrifugedathigh
speed (45,000xg,30min).The supernatant (soluble
antigen) was collected and diluted with 0.1 M
NaHCO3, pH 9.6, to an appropriate concentration.
Special plasticdiskscontainingsurfaceisothiocyanate
groups(13-mmdiameter)weremixed with thisantigen solution, agitatedat 4°C overnight, washed, and ly-ophilized. These dried antigen-coateddisks
(contain-ing3 to 5ytg ofproteineach)werestoredat4°Cin the
presenceofadesiccant untiluse.Antibodiestohuman
immunoglobulinsG and Mwereseparately produced ingoats,salted outwithammoniumsulfate, and im-munospecifically purified. Theywerethenconjugated to calfalkaline phosphatase (PL Biochemicals, Mil-waukee, Wis.)withglutaraldehyde(Polysciences,Inc.,
Warrington, Pa.) (5). Thedialyzedantibody-enzyme
conjugatesweredilutedtoworkingconcentrationsin
buffer at pH 8.0 (0.0025 M
tris(hydroxymethyl)-aminomethane [Tris], 0.2% bovine serum albumin,
0.001 M MgCl2, 0.15 M NaCl, and 0.03 M sodium
phosphate)containing0.02%sodiumazide.Theywere
mixedproportionallyforuse andkeptsterile at 2 to
8°Cwhen not in use.
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676 LIN, HALBERT, AND O'CONNOR
The human positive control was a pool of eight recalcified plasmas containing relatively high levels of Toxoplasma antibodies. It served as the secondary standard and was calibrated by the present ELISA method against the WHO's international standard for anti-toxoplasma serum, human, first standard, 1959 (19). The humannegative control was a pool of recal-cified humanplasma with negligible amounts of toxo-plasmaantibodies (less than 20 IU/ml). Both controls werepreserved with 0.1% sodium azide, made sterile by filtration, andstored in portions at 4°C until use.
Foreachtest, one of the antigen-coated disks was incubated with 10
Id
ofsample diluted in 500 Ml of specimen diluent (containing 0.01 M phosphate-buffered saline [pH 8.0], 6% bovine serum albumin, 0.05% Tween 20, and 0.1% sodium azide). After incu-bation at37°C in a shaking water bath for 45 min, the samplesolution was removed by aspiration, and theantigendisk (with antibody attached if present) was
washed five times each with 2.5 ml of 0.002 M Tris-bufferedsaline, pH 7.2 to 8.0. Antibody-enzyme con-jugate(0.5ml)wasthenaddedtothedisk, which was incubated as above for 45 min. The disk was again washed five times as above, transferred to anunused vial, and incubated as above for 20 min with 1 ml of theenzymesubstrate,p-nitrophenylphosphate (1 mg/ ml in 0.028 M sodium bicarbonate buffer [pH 9.8], containing 0.01 M MgCl2).The enzyme activity was stopped with 0.1 ml of3N NaOH and read with a spectrophotometer at a wavelength of 405 nm. To facilitate the assays,equipmentused for theCordia H
procedure (Cordis Laboratories) was employed, i.e., devices fortransferring and washing the disks.
The positive control and three fivefold serial dilu-tionsof it inspecimendiluent were tested simultane-ously in each assay. The absorbance values and anti-toxoplasmaactivity(internationalunits permilliliter) wereplotted onlog-log paper to establish a standard
curvefrom which theanti-toxoplasma activity of each
testsamplewasconvertedtointernational units per
milliliter,based on its absorbancereading. Tests with the WHO standard, as well as with the secondary
positive control, revealed that thestraight-line rela-tionship between absorbance readings and the anti-toxoplasma activity covered a range from 2 to 1,000 IU/ml. Because of the linearity of the relationship, the conversionfrom absorbance valuetointernational
units per milliliter could be made easily and rapidly with minimum errorwith an inexpensive hand-held calculator (e.g., Texas Instruments TI-55).
Inaddition to bloodsamples from local volunteers
or blood donors, we obtained serum samples from three laboratories doing routinetoxoplasma antibody immunoassays for clinical purposes and assayed them
ina codedmanner (after they had been tested in the laboratories supplying them) by the PHA, MBD, or IIFprocedures. Comparisons of titers obtained by the present ELISA test and these other tests were ana-lyzed bythecorrelation coefficient ofPearson and Lee
(R) (10) after logarithmic transformation (15), with
thenegative results assuming the titers to be either 1,
or oneor moredilutionlevel(s) below the lowest level
determinedinthetitrationsystem (15).The nonpar-ametricrankcorrelation coefficient ofSpearman (R.)
(10) wasalso usedto avoid the bias due tothis
as-sumption for negativeresults, especiallywherethey coveredawide range of titers (e.g.,less than16).For the same reason, where the sample size was large enough forameaningful conclusion,results from
sam-ples with titersof16orgreaterwere alsoseparately
analyzedby R. In additiontotheconventional arith-metic calculation for standard deviation and coeffi-cient ofvariation, the geometric standard deviation andgeometricmean werealso used forcomparisonof
reproducibilityof thepresentELISA with othertest
results, asrecomme'nded bythe Center for Disease Control (15).
RESULTS
Atypical standardcurve establishedwith
se-rialfivefold dilutions of the positive control is
shown inFig. 1. It canbe seenthat a straight-linerelationshipwasobtainedon alog-logscale
and that the coefficient of correlation of the
international unitspermilliliter andabsorbance
values in the example shown was 0.998. This
coefficientwasgenerallygreater than 0.995.
Sim-ilarstraight-line relationshipswere found with
the WHO primary standard, as well as with
severalindividual high-titeredserumspecimens. The
slopes
of these linestendedtobesimilar.Numerousparametersinthe assay were
var-ied singly to study their effects on the final
absorbance values, as well as on the linearity andslopesof thestandardcurves.Thevariables included such factorsastheamount ofantigen used for coating the disks, incubation times, volumes oftestsample, volumesofconjugates, etc.Theresultsaresummarized in Fig.2.When variousamountsofantigenwereusedfor
coat-ing, the absorbance achieved reachedaplateau
at 5 ug/disk. However, it was found that the
absorbance values for the negative control and background decreased when the diskwascoated
with moreantigen,resultingingreater slopes for
CONTROLIU/ML 405NM)
s .JUNDILUTEC340 2.865
z 1:5 68 1.135
1:25 13 0.340 1:125 2.70. 145
R=O.998
z/
a
0.5-ct'
0
C,)
C,)
3 Oj
U/ML
FIG. 1. Example ofa typical standard curve of standardizedquantitativeELISAfortoxoplasma
an-tibodies. Experimental data indicate that this
straight-linerelationshipcoversarangeatleastfrom 2 to1,000IU/ml.
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QUANTITATIVE ELISA FOR TOXOPLASMOSIS 677
the standardcurves.When the concentration of
substrate wasincreased, the slope dropped
ap-preciably, although the absorbance increased slightly. Both absorbance and slope decreased
06_
~051 _ 05
2.5 5 10 20
pGANTIGN/DS
0.6_
Z0.5_
04
2 4
° CONJUGATECONC,
0
0.67
405-0.4
200
'100
\
9 ~~~LoZ
5 10 25 50 30 45 60 90 pL SAMPLE PANUTE SAMPLE
INCUBATION
200
g~~~~~~~~~~~~~
-iooS~
0-25 05 'I 2 30 45 6;0 9`0 ° ML CONJUGATE TE CO*T
300
Is
-200U. 0
*100 §
z r
0
0 5 2 4 0.5 2 4 10 20 40 80
MG SUBSTRATE/ML ML SUBSTRAE' MNUTE SUBSTRATE
INCUBATION
FIG. 2. Effect ofsomeparameterson the
absorb-ancevaluesofthepositivecontrolandonthe
regres-sion coefficient (slope) ofthe standard curve. The
latter was established with the readings from the
positivecontrol anditsthreeserialfivefold dilutions. These data represent averages from two or three
experiments eachruninduplicate. All of these
stan-dard curvesclosely fit with correlation coefficients
rangingfrom0.995to0.999,exceptfor thetwomarked
with a check. The standardprocedure used inthe
presentstudy employed thesecondcondition listed in allcases.Theconjugate concentrationwaspresented
asfoldofthatused inthestandardprocedure(#).
substantially as the volume of substrate (at 1
mg/ml)wasincreased.Increased volumes oftest
sample increased the absorbance valuesslightly
but decreased the linearity and slope of the standard curve. Varying the incubation times
with thetestsample,orwith theconjugate,had
relatively little effect onthesevalues, whereas
increasing the substrate incubation time in-creased the absorbance, but had only a minor
effect on the slope. Increasing the conjugate
volume orits concentrationtended toincrease
theabsorbance and theslope,butalso increased thebackgroundabsorbance
unacceptably.
Back-ground absorbance decreasedto a steady level asthe concentration of bovineserumalbumin in
the specimen diluentwas increased to 4%and
above. Incubation at 370C gave increased
ab-sorbance and provided better standard curves
thanatroomtemperature.In general, these data
indicate that many of the conditions for the assay are not critical and could be tailored to
meetparticular needs withinmostof the
param-etersstudied.
Assays of specimens derived from a single
bleeding from the same donors indicated that
either serum or plasma could be used in the
ELISA assay, with equivalent results. Fifteen
volunteers donated blood, and the anticoagu-lants usedwereheparin, citrate, dextrose citrate,
andethylenediaminetetraacetic acid. Five of the donors showed toxoplasma antibodies 200IU/ mlorhigher, and three showed values of 10IU/
mlorless.After correction foranydilutionfactor
duetotheanticoagulantused, itwasfoundthat thetoxoplasmaantibody levels in theserumand
variousplasmas ofanyindividual determinedby
ELISA were equivalent, i.e., they were within
theassay error(averagecoefficient ofvariation,
15%).
The reproducibility of the ELISAprocedure
wasquitesatisfactory,asshown inTable1.The "within-run"reproducibility of theassayshowed
TABLE 1. Within-run reproducibility of ELISA for Toxoplasma antibodies
ELISAobservedvalues
Testspecimens (no. of repli- Absorbance IU/mi
cates)
Arithmetic Geometric mean Arithmetic mean Geometric mean
mean (% C.
V.)X
x/. SD" (%C. V.) x/-* SDA(9) 0.42(9.2) 0.42 x/ 1.10 14(15.5) 14x/+ 1.19
B(9) 2.80(6.0) 2.80
x/÷
1.06 420(10.7) 418 X/+ 1.11C(9) 1.58 (5.2) 1.58X/÷ 1.05 152(9.2) 151
x/+
1.10D(8) 0.14 (3.3) 0.14X/. 1.03 2.1(6.0) 2.1x/. 1.06
E(8) 0.84 (5.0) 0.84x/. 1.05 49(8.9) 49x/. 1.09
F(8) 2.60 (7.0) 2.59x/. 1.07 367(12.9) 356x/+ 1.15
G(8) 0.19 (3.0) 0.19 x/ 1.03 3.5(5.3) 3.5 X/ * 1.05
Avg % C.V.or SD 5.5 1.06 9.8 1.1
aC.V.,Coefficientofvariation.
bSD, Standard deviation.
VOL. 11,1980
I
I
I
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678 LIN, HALBERT, AND O'CONNOR
an averagecoefficientof variation of 5.5% and a geometric standard deviation of 1.06 in the ab-sorbance values. These values were 9.8% and 1.11, respectively, after conversion to
interna-tional units per milliliter. Tests of day-to-day interlaboratoryreproducibility showed approxi-mately equivalent results.
The specificity of the ELISA values for
toxo-plasma antibodieswasconfirmedby absorption
experiments. In theexamples shown in Fig. 3,
six human serum specimens (5 positive and 1
negative)werepreincubatedwith T. gondii
an-tigen, Entamoeba histolytica anan-tigen,
Trichi-nella spiralisantigen, or no antigen. The latter twoparasitic antigenswerecrude extracts incor-poratedintodiagnostickitsusing immunodiffu-sion (Cordis Laboratories) and were known to possessrelativelyhigh specific antigenic activity,
i.e., sufficient for gel difflusion techniques. The T. spiralis was derived from infected mouse tissue. The preincubated specimens were then assayed as above to determine whether
neutral-ization of the antibody in the sample had
oc-curred bytheparticular antigen in question. It is clear from the results that the toxoplasma antigen was the only preparation capable of
significantly reducing the ELISA values ofthe
positive specimens, indicating the immunologi-cal specificity of the present ELISA for
toxo-plasma antibodies. Reciprocally,when the disks were coated with E. histolytica antigens and
usedin similar neutralizationexperimentswith
humanseracontaining anti-amoeba antibodies,
600.
500.
400.
300.
2200. (I)
100 w
T.G.=T.GONDII T.S. =TSPIRAUS
E.=E.HISTOLYTICA N.=NONE
N3
R
El
onlyE.histolytica antigen (not toxoplasma an-tigen) was shown to reduce significantly the anti-amoeba antibody activity (unpublished data).
Comparisonsbetween thepresent ELISA and three other immunoassays (MBD, IIF, and PHA) are summarized in Fig. 4-7. High and
significant correlationcoefficients wereobserved
in all instances. These data also indicated that specimens showing up to 30 IU/ml by ELISA weremostly negative by the other three assays. Inaddition,almost allspecimens positive bythe
otherassaysshowed greater than 10IU/ml,and mostof themwereabove 30IU/ml.Four of the 206specimens shown in Fig. 4 were nonreactive or reactive undiluted in the MBD test, but
showed high ELISA values (>50 IU/ml). Ab-sorption experiments as described above re-vealed that onlythe Toxoplasma antigen (but riot nornalmouseplasma,oramoebaor Trichi-nella antigens) wasable tosignificantly reduce thehigh ELISA valuesin thesespecimens.
TheCenterfor DiseaseControl(Atlanta,
Ga.)
distributesreferencehuman serumcontrols for the assay oftoxoplasma antibodies. One lot of
t,hese referenceserawasassayed by
ELISA,
withgoodagreement in the resultsasshown in Table 2.These valuesareinaccordwith the distribu-tionof values shown inFig. 7.
DISCUSSION
TheELISA procedure describedhere has
sev-eral
advantages
overothercommonly
usedas-MA0
04
E;LW
uwwE
QlXE;L~
sa fS 15B mBLOCKING 0O2 dW OJL2CIIiZ (.(/LiZ0
o(/uWzf
dOXi)W
ANTIGEN H-- F-H- HI- HI- .aFs:
-HUMAN ELISA E LISA U-36-1I LI-36-12 U-36-17 LI-36-18
SERUM POSITIVE NEGATIVE
SAMPLE CONTROL CONTROL
FIG. 3. Demonstrationoftheimmunological specificity of ELISAreactions for toxoplasma antibodies by
absorption studies. Six humanserumspecimens (5 positiveand1negative)werepreincubated at37°C for30 minwith solubleantigens derived from: (i) T. gondii, (ii)E.histolytica, (iii) T. spiralis,or(iv)none.After this preincubation,eachspecimenwasassayed bytheELISAprocedure. It can be seen that the solubletoxoplasma antigensdramatically blockedthe ELISA reactions in eachof the positiveserumspecimens, whereas the
otherantigens hadnosignificant effect.
J. CLIN. MICROBIOL.
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VOL. 11,1980 QUANTITATIVE ELISA FOR
679
t
S
I
o
9I
30-- 10- a 0
5-LLI ~~~~~~~~~~RsO0.80(N 206;P<0.001
R,=0.87(N=206. P<0.001 R2z-0.64 (N= 87iP<0.00)
NEGATIVE 4 16 64 256 1024 4096
(I4) M B D TITER
FIG. 4. Scatter diagram and regression lines ofELISA compared to theMBD results on 206serum specimens (bothassaysweredone in thelaboratory ofG.R.O.).R, the rank correlationcoefficient ofSpearman.
RlandR2, the correlationcoefficient ofPearson and Leeforall260specimensand those withMBD titerof
16 orgreater,respectively. TheregressionlinesareforRl andR2.
Rs=090(N=20,P<OOoi
R,=0,92(N=20,P<OO01
NEGATIVE 4
M 8 D TITER
16 64 256 1024
FIG. 5. Comparison between the ELISA and the
MBD tests on 20 human serum samples for
Toxo-plasma antibodies. MBD, run at the University of
California, San Francisco; the ELISAs were run
independently and codedattheCordisLaboratories.
RYJthe rankcorrelationcoefficientof Spearman. Rl,
thecorrelationcoefficients ofPearsonand Lee for all
20specimens. The regression line is for Rl.
saysfordetecting andmeasuring antibodiesto T.gondii.TheMBD,IIF,PHA,and most of the micro-ELISAtestsrequireserial dilutions of the
test samples for establishing the level of
anti-bodies,whereasourELISAyieldssuch
quanti-tative values on the basis of a determination
with asingle dilution oftestsample.Moreover,
t*ie
presentassaywas standardized against theWHO reference preparation, as the antibody
concentrations were reported in international
units permilliliter. Suchasinglebasis of
refer-enceshouldminimize technical variations in
dif-ferent laboratories and permit more uniform
measurements of
toxoplasma
antibodies in anylaboratory using this procedure. This problem with the other assays is underscored by the description submitted with the WHO interna-tional standard for
anti-toxoplasma
serum.Thissingle
serumpreparation
wasassayedinanum-berofqualified
laboratories,
and the titersfound rangedfrom 400 to10,000in theMBDtest,from25 to 500in the complement fixation test, and between 250and6,000 intheIIF test.
The ELISA described here was satisfactory
for serum or plasma, and sodium azide as a preservative did not interfere with the results. No pretreatment of the test specimens is re-quired, the assay is relatively rapid, and the reagents, which arereadyforuse, revealed
con-siderable stability without any biohazard. Pre-liminary data indicated that all reagents could be kept refrigerated at 2 to8°C forat least 2.0 years without noticeable effect on the test re-sults. Theantigenonthesolidphasecould even bekeptatroomtemperature(23°C) for atleast 10months without loss ofactivity.
000-, 30--n
-i ,o.
0
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680 LIN, HALBERT, AND O'CONNOR J. CLIN. MICROBIOL.
0
Rs 0.90(N-95; P<0.00I) Ri=O2 (N=95,P<O.OOI) R2 =0.60(N=23;P<0.OI)
_
I_v
&- P H A TITERFIG. 6. Comparison between the ELISA and the PHA tests on human serum samples for Toxoplasma antibodies. PHAwasrunby the Bio-sciences Laboratories, and the ELISA were run independently and coded
atthe Cordis Laboratories. R., the rank correlation coefficient ofSpearnan. Rl and R2, the correlation coefficientsof Pearson and Lee for all 95 samples and those with titers of 16 or greater, respectively, assuming the PHA titerof1for those less than16.
o 0
8
0 0
8
0 0 0
0
0
Rs=.79(N=22;P<0.001) R,=084(N=22;P<0.001)
(<16) 16 64 256 1024 4096 16384
NEGATIVE
F TITER
FIG. 7. Comparison between theELISA and theIIFtestson22humanserumsamples for Toxoplasma
antibodies.IIFwas runbytheParasiticSerology Laboratories,CenterforDiseaseControl, Atlanta;and the
ELISAwererunindependentlyandcodedattheCordisLaboratories. R.,the rank correlationcoefficient of
Spearman.Rl, the correlationcoefficient ofPearson andLee, assumingtheIIFtitersof4forthenegative
results. Theregressionline isforRl.
Arathergood correlationwasfound between MBD and IIF. In a separate, ongoing clinical
the ELISA values(international unitspermili- research study with 34 specimens with MBD
liter) and titers of toxoplasma antibodies found titers between 1,024 and 8,192, there was no
in several panelsoftest specimens assayed by indicationofaplateauof theELISAtestresults
I1
-i
wL
0 0 0
8
0 0
0 0 0
8
(GI6}
NEGATIVE 16 32 64
1000_
looi
30-
10-Uf)
-J
w
0
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QUANTITATIVE ELISA FOR TOXOPLASMOSIS
TABLE 2. ELISAassay results with Centerfor DiseaseControl toxoplasmosis control human seraa
Center for ELISA observed(IU/ml)b Serum DiseaseCon- Arithmetic
trol-stated mean Geometrc ter, IIF m
iS
meanx/+SDNegative <32 2.1±0.3 2.1 x/+ 1.17
Lowtiter 32-128 62±11 62x/+ 1.19
High titer 512-2,048 203±30 202x/÷ 1.17 aCatalog no.
KK0501,
lot 76-0339K.b Measured in six experimentsatdifferent
times.
'SD, standard deviation.with these high-titered samples (unpublished data). Failure to achieve even higher correla-tionsmaybe duetotwofactors. The MBD and IIF procedures allow a plus-or-minus fourfold errorintheendpoint titers (12). Thus,asample witha"truetiter"of 1:64 may reveal titers of 1: 16 or 1:256 inreplicateassays.The reproducibil-ity of theELISAisconsiderably better, showing
ageometric standard deviation of1.11in inter-national units per milliliter, which is smaller thanmostofthosereported for immunoglobulin quantitation (13, 14). The latter, in turn, has beenfoundtoprovide appreciably better
repro-ducibility than those serologicaltests using serial
titrations, which allow accuracy to within one
twofold dilution (14). In addition, the ELISA
determination yieldsresults which are presum-ablyasummationofalltheantibodies directed
against the severaltoxoplasma antigens (at least
seven) knownto induce antibody responses in
humans (4, 7, 9, 11; unpublished data, G.R.O.), since the preparations used to coat the disks weretotalextracts of the organisms prepared by verymildsonication procedures. In contrast, the
MBD and theIIF tests undoubtedly measure a morelimitedsetof antibodyresponses,
presum-ably those situatedon thesurface membrane of
thetoxoplasma organisms (9).
ACKNOWLEDGMENT
Thisworkwassupported in part by grant EY-01204 from the National Institutes ofHealth, Bethesda, Md.
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12. Sulzer, A. J., and E. C. Hall.1967.Indirect fluorescent antibody test forparasitic diseases. IV. Statisticalstudy ofvariation in the indirectfluorescentantibody (IFA)
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13. Taylor,R.N.,K. M.Fulford,and A. Y.Huong.1978.
Comparison of kinetic andend-pointdiffusionmethods for quantitating human serum immunoglobulins. J. Clin. Microbiol.8:23-27.
14. Taylor, R. N., K.M.Fuilford,and V. A.Przybyszeski.
1977.Proficiencytestingsummaryanalysis: diagnostic immunology1977I: rubella,carcinoembryonic antigen,
immunoglobulin quantitation, C-reactive protein,
sal-monella (typhoid0) agglutination.Center forDisease
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