Antibody response to cytomegalovirus polypeptides captured by monoclonal antibodies on the solid phase in enzyme immunoassays

0095-1137/85/040517-05$02.00/0

Copyright C 1985, American Societyfor Microbiology

Antibody

Response

to

Cytomegalovirus Polypeptides Captured

by

Monoclonal Antibodies

on

the

Solid

Phase

in Enzyme

Immunoassays

NATALIE E. CREMER,* CYNTHIA K. COSSEN, GORDON R. SHELL, AND LENORE PEREIRA

Viraland RickettsialDiseaseLaboratory, CaliforniaDepartment ofHealth Services, Berkeley, California 94704 Received6 August1984/Accepted 17 December 1984

Antibodies to different cytomegalovirus (CMV) polypeptide antigens, captured by monoclonal antibodies coated on the solid phase ofan enzyme immunoassay test, were analyzed in 42 serumpairs submitted for

serodiagnosis of CMV infection. Three CMV antigens, captured on the solid phase by three monoclonal antibodies ofdifferent specificities, designated CH92-1,CH65-1, andCH16-1, wereglycoproteins A (gA), gC, and gD, respectively; and one antigen, captured by CH23, was a polypeptide with an apparent molecular

weight of 150,000, possibly associated with the nucleocapsid. Ofthesefour CMV antigens, gA captured by CH92-1 was most effective ineliciting anantibodyresponse. Antibody tothis antigen was present inserum

samplesatahigher concentrationinprimary andreactivated infectionandpersistedlonger than did antibody totheother testedantigens. Incontrast,antibodytoantigen captured by CH23wasatalower concentration,

rosemoreslowly ininfection,andpersisted forashortertimethan didantibodytothe otherantigens. Antibody

response to gC and gD was intermediate in concentration and temporal appearance compared with the

antibody responsetogA and tothe polypeptide bound by CH23. Anenzyme immunoassayon pairedserum

sampleswiththe capturedglycoproteinsasantigenwasequalforthe detectionofcurrentinfectiontoan enzyme

immunoassay withthe wholeCMV antigen from infected celllysates. Enzymeimmunoassayswith either the CMVglycoproteinsorthewholeCMV antigen frominfectedcelllysatesweresuperiortoacomplement fixation testwithaglycineextract antigen forserodiagnosisofcurrentinfection.

With the advent of monoclonal antibodies (mABs) with

specificity for individual epitopes on polypeptides, it has

become possible to better dissect the humoral immune response of patients to viral antigens (13). The temporal appearance of antibodies of defined specificities can be

determined, and the relationship of their appearance to patterns ofdisease can be correlated. In the present study we used mABs to cytomegalovirus (CMV) to capture dif-ferent CMV antigens for use in a solid-phase enzyme im-munoassaytest (EIA)to characterize antibodiesto CMVin the seraofpatients.

MATERIALSANDMETHODS

Antigens. CMV antigens for the various tests were

ex-tracted from a CMV (strain AD 169)-infected human fetal

diploid lung cell line when the cytopathic effect was 4+.

Antigen for the complement fixation (CF)testwasextracted

from the infected cells by a 30-s sonic treatment of the infected cells suspended in 0.1 M glycine buffer (pH 9.5; probe intensity, 50%; Biosonic II; Bronwill Scientific Inc.,

Rochester, N.Y.) (8). Antigen for EIA was extracted from

infected cells suspended in 0.1 M glycine buffer (pH 9.5),

containingafinal concentration of 1.0%deoxycholate(DOC). The cell suspension wasincubated with intermittent mixing for 15 min at 5°C. Cellular debris and any residual whole

virus were removed by centrifugation at 26,384 x g for 90

min.The CFantigen isdesignatedastheglycineextract(GE) antigen, and the EIA antigen, DOC antigen, and the tests

with these antigens are designated CF-GE and EIA-DOC.

Preparations ofcontrolantigens made in thesamewayfrom thenoninfectedhuman fetaldiploid lung cellswerechecked

*Correspondingauthor.

with each serum sample to ensure the specificity of test

results. The antigenswere stored at -70°C until used. CF and EIAtests. Doubling dilutions ofseraat a starting

dilution of 1:8weretestedbyastandardizedCFtest(6). For EIA with the DOCantigen directlyonthe solidphase,trays

of cuvettes with a 1-cm light path (Gilford Instrument

Laboratories, Inc., Oberlin, Ohio) werecoated with 0.25 ml

of the DOCantigen diluted 1:150 in 0.005 M

phosphate-buff-ered saline(pH 7.2; PBS), adilution previously determined tobeoptimal by block titratiqn with standardized reference sera.Whencoating thecuvettesdirectly withDOCantigen it

was necessary to use dry, untreated cuvettes. Cuvettes, after wetting with PBS containing a final concentration of

0.05%Tween (PBS-T),asafterthe washing stepsdescribed below, no longer bound the antigen. Alternate cuvettes in thetraywerecoated withcontrolantigenatthesameprotein

concentration as the CMV antigen, as determined by the

Lowry method (5). The cuvettes were dried with a fan

overnight at 37°C. They then were packaged in air-tight polyethylene bags (Belart Plastics, Pequannock, N.J.) con-taining silica geldesiccant (Dri PackBags;Davison Chemi-cal, Baltimore, Md.) and a humidity indicator card. They

were storedat 5°C until used.

Production and characterization of mABs to CMV have

been reported previously (12) mABs CH92-1, CH65-1, CH16-1, and CH23, used in the present study, immune

precipitatedCMVglycoproteins designated gA, gC, andgD

and a polypeptide with an apparent molecular weight of

150,000 (150K), respectively. For testing of antibodies to

different CMV antigens, trays ofcuvettes were first coated

with CMV mABs in mouseascites. The protein

concentra-tion ofthe ascites varied from 26to29mgofproteinperml. Forcoating the cuvettes, a 1:5,000 dilution of CH16-1 and

CH23in PBSanda1:10,000dilution of CH65-1 andCH92-1 517

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TABLE 1. Reactivity of paired serum samplesgrouped by results from CF-GEandEIA withDOCantigen Reactivity of paired serum samples bya:

EIA with:

Group CF-GE

DOC gD 150K pp gC gA

ST INC ST INC ST INC NEG ST INC NEG ST INC NEG ST INC

I

21b

0 21 0 19 0 2 18 0 3 18 2 1 19 2

II 0 il 0 il 1 10 0 0 8 3 0 9 2 1 10

III 7 0 0 7 1 6 0 0 4 3 0 6 1 1 6

IV 0 3 3 0 2 1 0 2 1 0 1 2 0 3 0

aCF-GE, Complement fixation test with glycine extract antigen.EIAantigens:DOC, supernatant fluid from CMV-infected cell

lysate;

gD,glycoproteinbound

byCH16-1; 150K pp, 150Kpolypeptidebound byCH23;gC, glycoproteinboundbyCH65-1; gA,glycoproteinboundbyCH92-1.ST, Standingtiterorindex indicating no evidence of a current infection; INC, significantincreasein antibody concentration between first and second serumsample ofapair(CFtiter increase, -4-fold; EIA ratio [index of second sample/index of first sample], -1.5. NEG, noantibody detected in either sample of thepair.

bNumber of serum pairs showingindicated results.

in PBS were used. These concentrations were optimal for

the effective binding of the different CMV proteins in the

DOC antigen to the mAB-coated cuvettes, asmeasured by

EIAwithreferencesera. Thecuvettes werecoated with0.25

ml ofthe mABs overnight at 37°C in a humidified

atmos-phere. The fluids thenwereaspirated, andthe cuvetteswere

washedwith eight cycles ofPBS-T(0.7 ml foreachcycle).

After drying thecoated cuvettes with a fan for4 to 6 h at

37°C, they were reacted overnight in a

humidified

atmos-phere at room temperature either with 0.25 ml ofa 1:150

dilution ofDOC antigen inPBS-Tcontainingafinal

concen-trationof 0.5% bovineserumalbumin (PBS-T-bovineserum

albumin) or with control antigen of comparable protein

concentration. From this point onward, the procedure for

EIA was performed in the same manner as with cuvettes

coated

directly

with DOC antigenorwith the mAB-antigen

combination. Antigens captured by

CH92-1,

CH65-1, and

CH16-1arereferredtoasgA,gC,andgD,respectively,and

the polypeptide bound by CH23 is referred toas the 150K

polypeptide.

All washes and additions ofreagents, exceptforthe serum

samples ofthe patients, were automatically delivered and

aspirated withaprogramablePR50processor-reader(Gilford

Instruments Laboratories). The coatedcuvettes werewashed

with eightcycles of PBS-T (0.7 mlforeachcycle)

before

use,

after reaction with the serum of the patients and after

reaction with enzyme-labeledconjugate.

The F(ab')2 fragment ofgoatanti-human immunoglobulin

G(IgG) labeled with alkaline

phosphatase

(Sigma Chemical

Co., St. Louis, Mo.) was used at a dilution of 1:2,500 in

PBS-T-bovine serumalbumin, an optimaldilution by block

titration with

reference

sera. Paranitrophenyl phosphate

(final concentration, 1 mg/ml) in

diethanolamine

buffer(pH

9.8)

containingafinal concentration of0.0005 M

MgCi2

and

0.02%Naazidewas used as

substrate.

Sera were tested in

duplicate at a 1:100 dilution in PBS-T-bovine serum

albu-min. All testswere read automatically by the PR50

proces-sorreaderwhen thereaction of a

reference

serum

reached

a

predeterminedoptical density (OD). In each run, inaddition

tothis

reference

serum, ahigh-positive serum (index, 14 to

18), a low-positive serum (index, 4 to 8), and a negative

serum (index, <1.0) were checked. The OD of thereaction

of each serum sample with the control antigen was sub-tractedfromthatwith the CMV antigen.

Determination of cutoff values for EIA. Fifty-three sera

negative for antibody to CMV by CF-GE (titer, <8) were

repeatedlytested by EIA. The mean OD + standard

devia-tion (SD) was 0.013 ± 0.018. The mean plus 3

SDs

was

0.067.Therefore,an OD of0.1 was taken as the cutoff value

forapositive reaction. Results arereported as anindex as

follows: index =

[(OD

with CMV antigen) - (OD with control

antigen)]/cut-off

OD

(0.1).

To determineacutoff

indicating

a

significant

differencein

antibody concentration between two serum samples, 40

serum pairs with standing antibody titers by CF-GE were

repeatedly

tested

by

EIA. The index of each

sample

was

calculated,

andthe ratio of the index of the secondsampleto theindex of the firstsamplewasdetermined. Themeanofall

the ratios±SDwas1.025±0.154. Themeanplus3SDswas

1.487. A cutoff ratio of 1.50 was taken as the ratio for a

significant differenceinantibody concentrationbetweentwo

samples ofapairandasindicativeofacurrentinfection.

IgM antibody. Certain serum samples were checked for

IgM antibody by

the indirect immunofluorescent

antibody

technique with fluorescein-labeled sheep anti-human

,u-chain specific antibody (Wellcome Reagents Limited,

Beck-enham, England) as previously described (3). The cellsmears

usedfor thedetection ofantibodywere preparedby

mixing

one part of cells infected with CMV with two parts of

uninfected cells which served as nonspecific controls. The

tests were read by epifluorescence microscopy with Zeiss

equipment.

Serum.Forty-twoserumpairssubmitted for

serodiagnosis

ofCMVinfectionwereselected for studybyEIA. They had

previouslybeen testedbyCF-GE. ByCF-GE 28 serumpairs

had standing CMV antibody titers and 14 serum pairs had

significantrisesin antibody titer(fourfold orgreater).

RESULTS

Results on the serum pairs were evaluated by several

differentcriteria. It was firstdeterminedwhether resultson

each serum pair indicated the occurrence of current infec-tion or pastinfection. On the basis of CF-GE andEIA-DOC

results, the serumpairs were dividedinto fourgroups. For

group I no change in antibody concentration between the first and second serum samples was noted by either test, indicating that there was no evidence of current infection.

Forgroup II asignificant increase inantibodyconcentration betweenthe first and second samples was noted by CF-GE

(-4-fold rise in titer) andby EIA-DOC (ratio ofindexes of

second sample to first sample, -1.50). For group III a significant increase in antibodyconcentrationwas notedby

EIA-DOC and standingtiterswere obtained by CF-GE. For group IV significant increases in CF-GE titer and standing indexes were found by EIA-DOC.The results by the

EIA-mABtechniqueon the serumpairs of each group then were

placed in those same groups. A significant change in

anti-bodyconcentrationwas determined in 18 of 42 serum pairs

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TABLE 2. Antibody concentration to different CMV antigens in serum samples frompastinfections'

EIA Antibodyconcn No. of

antigens Mean

index

± SD Median Upper Lower negative

range range samples'

DOC 15.59± 6.36 17.40 27.30 4.26 0

gD 4.31 ± 3.27 3.54 12.90 0.43 2

150K pp 2.73 ± 1.90 2.06 8.05 0.22 3

gC 4.37 ± 2.58 4.30 10.40 0.36 2

gA 16.27 ± 7.33 17.20 28.20 1.96 0

a Samples are the first serum samples from 18 patients with standing antibody titers.

bThere was no evidence of current infection by any of the tests.

'DOC, Supernatant fluid from CMV-infectedcell lysate; gD, glycoprotein

bound by CH16-1; 150K pp, 150K polypeptide bound by CH23; gC, glycopro-teinbound by CH65-1; gA, glycoprotein bound by CH92-1.

d Index = [(QD of test antigen)- (OD of controlantigen)l/ODofcutoff (0.1).

'Index is less than 1.0.

byEIA-DOC and by EIA-CH92-1, in 19 of 42 serum pairs by

EIA-CH65-1,in 17 of 42 serum pairs by EIA-CH16-1, in 13

of42 serumpairs by EIA-CH23, and in 14of42 serumpairs

by CF-GE (Table 1). Ofinterest were the three serum pairs

in group I which showed a significant change in antibody

concentrationto gA and gC bound by CH92-1 andCH65-1,

respectively, and no significant change in concentration by

CF-GE and EIA-DOC.Oneofthe three serum pairs showed

anantibody increase with both gC andgA,andthe other two

pairs showedan increase with eithergA orgC.These three

serum pairs were checked and were positive for IgM

anti-bodyto CMV. Theypossibly representeithera recentpast

infection sinceIgM maypersist formonthsafter infection(4,

9, 10, 14) or areactivated infection, asindicatedby thehigh

antibody concentration in the first serum samples by both

CF-GE and EIA-DOC (CF-GE titers, 256, 2048, and 512;

EIAindexes, 9.3, 13.7,and 11.5,respectively).

TheEIA results on thefirstserum sampleofeach serum

pair in group I which showed no significant change in

antibody concentration byany of the tests(18 serum pairs)

andthus noevidence ofcurrentCMVinfectionwere

evalu-ated as to the mean, median, and range of values to

determine which antibodies persisted and which predomi-natedasaresultof previous infection (Table 2). Antibodies togApredominated, and the indexesweresimilar in valueto

theindexes of theantibodies reactive with the DOC antigen. The lowest index values occurred with antibodies to the 150K polypeptide. Two serum pairs in group I had no

antibodies in their first serum sample to gC, gD, and the 150K polypeptide, and one lacked antibody to the 150K polypeptide only. There was considerable variation in the antibody indexes among the various patients, as would be

expected since the date of theirpastinfectionorthe level of

theoriginal response isnotknown.

All of the remaining serum pairs (24 pairs) showed a

significant increase in the ratio of the indexes of the paired samples (.1.5) byone ormoreof thetestsand thus showed evidence ofcurrent infection. Five pairs were from

trans-plant patients. In such patients a serum sample is taken

beforetransplant forabase-line antibody value and another

sample is taken after transplantationatthetime of infection, should one occur. Data on these five patients are given in Table 3. Basedontheresultsonthe firstserumsamples,two

of thepatients (patients 1 and2) developedaprimary CMV

infection and threepatients experiencedareactivated

infec-tion. The antibodies in theprimary infection ofcase 1 that reacted with antigens captured by gC, gD, and the 150K polypeptide rose more slowly andwere ata lower concen-tration than were the antibodies detected with the DOC

antigenorgA. In the primary infection ofcase2, antibodies togC and the 150K polypeptide hadnotas yetappeared at

the time of the collection of the second serum sample. In

reactivated infections antibodies to all the antigens were presentin the firstserumsamples oftwoof the threepatients but antibodiesto the150K polypeptide and gC wereabsent

inthe firstserumsample ofonepatient. Antibodiestoall the

antigenswerepresentin the secondserumsamples. Indexes togAwereclosestinvaluetoindexes of antibodies detected by DOC antigen.

The temporal appearance of the antibodies reactive to

antigens captured by the mABs was evaluated with serum

TABLE 3. Antibodyresponseby transplant patientstoCMVantigens Antibodyresponse' toEIAantigens' Time ofserum

Case no. collection' DOC gD 150K pp gC gA by

CF-GE Primary infection

1 Attransplant 0.17 0.03 0.00 0.00 0.00 <8

1 3.5molater 10.40 3.36 1.73 1.70 13.80 256

2 Attransplant 0.05 0.00 0.00 0.00 0.00 <8

2 1.25 molater 6.28 2.33 0.40 0.92 3.02 256

Reactivated infection

3 Attransplant 10.70 2.87 1.72 3.83 11.20 32

3 2.5 molater 22.70 9.30 6.62 7.48 18.20 128

4 Attransplant 5.52 3.22 0.68 0.75 1.80 64

4 3.25molater 11.90 4.63 1.18 1.82 13.00 256

5 Attransplant 14.70 5.80 4.12 3.76 13.00 32

5 4.0molater 20.50 7.96 5.97 6.90 18.60 256

aThefirstserumsamplewastaken at thetime oftransplant,andthesecondsamplewastakenatthetime of CMVinfection.

bSee Table2,footnoted, foradescriptionof theindex.Anindex of1signifiestheleastamountofantibodydetectedbythe EIAmethod. An index of <1.0

indi-catestheabsenceofantibody. C SeeTable2,footnotec.

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TABLE 4. Temporal appearance ofantibodies reactivewithantigens captured by mABs Temporalappearance of antibodiesreactivewithfollowingEIAantigensd

Days" DOC gD 150Kpp gC gA

3 9-12 2.16 ± 1.65 0.51 ± 0.50 0.17 ± 0.20 0.36± 0.43 0.53 ± 0.28

4 14-16 3.16 ± 0.76 0.84 ± 0.36 0.35 ± 0.21 0.59± 0.49 1.10 ± 0.59

4 21-26 7.85 ± 1.50 1.43 ± 0.88 0.58± 0.33 1.09± 0.81 3.02 ± 1.49

3 28-29 11.71 ± 4.25 2.84 ± 0.71 0.68 + 0.87 1.86± 1.20 5.05 ± 1.98

2 50-61 14.45 ± 4.05 4.28 ± 1.64 2.31± 0.80 4.80 ± 2.40 10.90 ± 2.90

a Number of serum samples. Serum samples were the first and second samples from eight patients.Theresultsaregroupedaccordingtodaysafteronsetof symptoms that samples were collected.

bDayfrom onset of symptoms.

CResults are the mean of indexes ±SD. SeeTable 3,

footnote

b for a description of the index. dSeeTable 2,

footnote

c, foradescription of the EIA antigens.

pairs which had lowor no

antibody

toany of theseantigens

inthe firstserumsample andwhichwerefrom

patients

with

known dates of onset ofsymptoms and

sample

collection.

Eight serum samples were so identified. The results were

groupedaccordingto thedate afterthe onsetofsymptomsat

which serum samples were collected (Table 4 and Fig. 1).

Thefirstserumsamplefromsevenpatients had been taken9

to 16days afterthe onsetofsymptoms andfromonepatient

26days afterthe onsetofsymptoms. Thesecond

samples

of

the pairs were taken 21 to 61 days after the onset of

symptoms. Antibody to gA increased more

rapidly

than

antibodytootherantigens. It wasdetected inonepatientat

alow level(index, 1.95) by14days andinall

patients

by21

to 29 days. Antibody to gD was present in three patients

(indexes,

1.10, 1.02, and 1.16) by 11 to 14 days and in all

patients by21 to29days. However, it increasedmore

slowly

thantheantibodytogA. AntibodytogCwaspresent in one

patient by 16days(index, 1.19)andin fivepatients

by

21 to

29 days.Antibodyto the 150Kpolypeptidewasnotdetected

until21to29days after theonsetofsymptoms and thenonly

in twopatients.

I5.0

DOC

13.0

11.0 / 92-1

9.0 n 7.0

5.0 65-1

16-1

3.0-23 I.c

9-12 14-16 21-26 28-29 51

Dayafteronset ofsymptoms

FIG. 1. Temporalappearance of antibodiestoCMVantigensin primary infection withCMVas measured by EIA. Data on first and second serumsamplesfromeight patients are grouped according to the time of sample collection after the onset of symptoms. The horizontal line marks the cutoff index of 1.0 for presence of antibody. DOC isthesupernatantfluid from theCMV-infectedcell

lysateusedforacompleteviralantigen. Numbers to the right of the curvesrepresent monoclonal antibodiesCH92-1,CH65-1,CH16-1, and CH23 used to capture glycoproteins, gA, gC, gD, and a

15OK-molecular-weightpolypeptide, respectively, for use as subunit

viralantigens.

DISCUSSION

Antibodies produced by hybridoma clones

CH92-1,

CH65-1, and CH16-1 reacted with antigenically distinct

CMVglycoproteins (lOa, 12). The CMV glycoproteins

iden-tified by these and other mABs were classified into four groups and were given alphabetical letter designations, A through D, according to guidelines established for

glycopro-teins ofherpessimplex virus (lOa). By using this

classifica-tion, the different glycoproteins identified by

CH92-1,

CH65-1, and CH16-1 were placed in groups A, C, and D,

respectively. Each glycoprotein, immunoprecipitated with the relevantmAB, showed electrophoreticpolymorphismon sodiumdodecyl sulfate-polyacrylamide gel electrophoresis.

CH92-1 precipitated seven antigenically related

glycopro-teins from infectedcelllysates varying in apparent molecular weights from 160K to 56.5K, CH65-1 precipitated four

antigenically related polypeptides varying in apparent

mo-lecularweights from 66K to 46K, and CH16-1 precipitated

four antigenically related polypeptides varying in apparent

molecular weights from 49K to 25K. CH23 reacted with a polypeptide with an apparent molecular weight of 150K,

most likely the nucleocapsid protein (12). Only CH92-1 of the four mABs used in the present study was capable of neutralizing viral infectivity, and all mABs efficiently cap-tured specific CMV antigens for use in EIA.

We previously showed that coating a solid phase with

mAB to the hemagglutinin of measles virus was a feasible way to capturespecificantigenfrom acrude lysateforuse as antigen in EIA for the determination of antibody to the hemagglutinin (8a). We now have used the same technique to evaluate the antibody response in CMV antigens. For diagnosis ofcurrentinfectionby EIA there was little advan-tage in using the captured antigens over the soluble cell lysate. A significant increase in antibody concentration between paired serum samples indicative of current infec-tion was found in 18 cases with the DOCantigen andin 17, 13, 19, and 18 cases with antigens captured by CH16-1,

CH23, CH65-1, and CH92-1, respectively. EIA with the DOC antigen or with CMV glycoproteins was better at detecting significant changes inantibodyconcentration than was CF-GE(Table 1).

Ourresultsindicate thatgA bound by CH92-1 is a

domi-nantglycoproteininelicitingahumoralantibodyresponse as

measured by EIA. Antibody to gA appeared earlier in

primary infection and increased in concentration more

quicklythandidantibodyto theotherantigens (Tables 3 and

4 and Fig. 1), and it persisted at a higher concentration

(Table2).Antibodyresponse to gC and gD wasintermediate

in concentration and temporal appearance compared with

theantibodyresponse to gA, the mosteffectiveantigen, and

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the 150K polypeptide, the least effective antigen of the antigens tested. In the small sample of eight patients checked, antibody to the 150K polypeptide did not appear until 3 to 4 weeks after the onset of symptoms and never reached the antibody levels elicited by the other antigens. These findings aresimilar to those of a previous study in ourlaboratory in

whicha radiolabeled 150K polypeptide was either absent or

present at lower concentrations in immune precipitates, formed with sera from patients with current or past infec-tions, than were CMV antigens of lower molecular weight (11). In that study radiolabeled antigens which were precip-itated ingreatest amounts from detergent-treated, infected cell lysates by human sera had apparent molecular weights of66K, 50K, 34K, and 32K. Polypeptides invariable amounts

in theimmune precipitates had apparent molecular weights

of 130K, 96K, and 25K. Polypeptides present in trace amounts inthe immuneprecipitates had apparent molecular weights of 150K, 76K, 74K, 45K, and 40K. Similarpatterns

of antigens also were found in immune precipitates of

antigens from infected cells extracted with glycine buffer

(11). In studies on sera from children with congenital and

perinatal CMV infections it was shown that at least 11

polypeptides were immune precipitated (13). Among these

were aprotein with an apparent molecular weightof 150K

andCMVglycoproteins subsequently designatedasgA,gC,

andgD (lOa).

The temporal appearance of antibodies with different

specificities and the continued production ofsome

antibod-ies in infections with Epstein Barr virus (7) or hepatitis B

virus (1, 2) have been of value in evaluatingthephase and

outcomeof the clinicalcourseof these infections. The data

from our presentstudy suggest that as more CMV antigens

are identified and as the antibody response to them is

analyzed apattern ofresponse mayevolve that will be as

helpful in following and monitoring the clinical course of

CMV infection as that used for evaluating the

aforemen-tioned viral infections.

ACKNOWLEDGMENTS

We thank DanaGallo for theperformanceof the indirect fluores-centantibodytestsforIgMantibodytoCMV, MarjorieHoffman for

preparation ofmouse ascites, and Judith Wilber forsome of the serumpairs.

ThisstudywassupportedinpartbyPublic Health Service grant AI-19257 from the National Institute of Allergy and Infectious Diseases.

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