Immunoglobulins M and G to varicella zoster virus measured by solid phase radioimmunoassay: antibody responses to varicella and herpes zoster infections

Vol. 12, No. 3 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1980,p.367-374

0095-1137/80/09-0367/08$02.00/0

Immunoglobulins

M

and

G

to

Varicella-Zoster Virus Measured

by Solid-Phase

Radioimmunoassay: Antibody Responses

to

Varicella and Herpes Zoster Infections

A. M.ARVIN' ANDC. M. KOROPCHAK

DepartmentofPediatrics, Divisionof Infectious Diseases, Stanford UniversitySchoolofMedicine,

Stanford,

California

94305

Both immunoglobulin M (IgM) and IgG antibodies to varicella-zoster virus (VZV)were detectablein asolid-phase radioimmunoassaywith

'25I-labeled

goat antisera tohumanimmunoglobulins. Primary infection withVZV was associated with early production of IgM and IgG antibodies and rapid development of lymphocyte transformationtoVZVantigen. Among

eight

subjects with varicella tested 1 to 4daysafter onset, sevenpatients hadIgG andsixpatientshad IgM antibodies; all patientshadbothIgGandIgM antibodieswithin 7 days.AnIgM

response wasdocumented byradioimmunoassayin 18 of 26patientswithherpes

zoster.VZV antibodiescould beassayedby

radioimmunoassay

inunfractionated

serumwith commercialgoatantiseratohumanimmunoglobulin and commercial VZVantigen. VZV-specific IgG bindingwaspresent inallsera from42 subjects withaVZVantibody titer of-1:8 asdeterminedby indirectimmunofluorescence andcellular

immunity

toVZVasdeterminedby

lymphocyte

transformation and who had had varicellaatleast20yearsbefore testing. The geometricmeantiter

was 1:6,309, and titers were 21:16,384 in 20 subjects.

Antibody

was present as

determinedbyradioimmunoassay in14samples negative by complement fixation and in five samples negative by complement fixation and immune adherence hemagglutination.No

specific

bindingwasobserved in21 serafromsubjects who

were not immune to VZV as determined by indirect immunofluorescence or

lymphocyte transformation despitethe presence ofherpes simplexor

cytomega-lovirusantibody indicated by

complement

fixation in15 sera.High titers ofVZV

IgM antibody weredetectedinunfractionatedsera despite thepresenceofhigh titers ofVZV

IgG antibody.

The VZV

radioimmunoassay

provided a sensitive

and

practical

method formeasuring VZV

IgG

andIgM antibodies.

Primary infection with varicella-zoster virus (VZV) is common in

early

childhood. Varicella

is

usually

mild in

healthy

children but

life-threatening disseminationofthe viruscan occur

in children with

congenital immunodeficiency

disorders or

malignant

diseases (7). A reliable method for

determining

susceptibility

to

vari-cella is

important

because the

severity

of the

infection can be reduced

by

the

prophylactic

administration ofzosterimmune

globulin

to

im-munocompromised

children (13). The indirect

immunofluorescence methods forVZV

antibody

aresensitive, but these

techniques

require

labo-ratoryfacilitiesfor

handling

varicella virus,and their

reliabiity

canbeaffected

by

the

subjective

interpretation involved (26, 27). The standard

serological

methods,

such as the complement

fixation technique, have notbeen useful in

de-tectingVZVantibodyforprolonged periodsafter theprimaryinfection (25).None of these meth-ods has been

easily adaptable

to detect VZV

immunoglobulin M (IgM) antibody in

unfrac-tionatedserum.

The reactivation of latent VZV results in

herpes zoster, which is a frequent occurrence among patients with malignancy and organ

transplant

recipients

(18, 19). Inadditionto its

clinical application, a very sensitive assay for

VZVIgG and IgM antibodiesmight detect

defi-cienciesinantibody production that contribute tothe pathogenesis ofsevereprimary varicella and to the reactivation oflatent VZV virus in immunocompromised patients.

The solid-phase radioimmunoassay(RIA) has been asensitivemethod forthe

detection

of IgG

antibody to herpesviruses, including VZV,

herpes

simplex,

andcytomegalovirus (9, 10, 16), and has also been used to measure IgMantibody tocytomegalovirus (16). In this study, a

simpli-fied solid-phase RIA technique with

commer-ciallyavailable reagents wasemployed to mea-sure both IgG and IgM antibodies to VZV in

367

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368 ARVIN AND KOROPCHAK

unfractionatedserumsamples.TheRIAmethod wascompared with other assaysforVZV

anti-body, including complement fixation, indirect immunofluorescence assay (IFA), and immune

adherancehemagglutinationassay(IAHA).The

VZV IgM and IgGresponsesofnormal subjects

wereevaluated with the RIAtechnique during

primaryvaricella and acuteherpeszoster. MATERIALS AND METHODS Subjects.Serawereobtainedfromhealthy subjects whoseimmunitytoVZVwasestablished by IFA and

bytheVZV-specific lymphocyte transformationassay

(seebelow).The VZV-immune adults had hadprimary varicella 20yearsor morebefore testing. Fiveofthe subjectswhowerenotimmuneby thesecriteria sub-sequently developed varicella.Serawerealso obtained

from otherwisehealthysubjects with acute varicella

orherpeszoster.Thediagnosiswasconfirmedby

VZV-specificdirect fluorescence ofepithelial cellsfromthe skin lesions (24). Serawere tested forantibodies to

herpes simplex and cytomegalovirus by the

comple-mentfixationtechnique (17).

Antigenpreparation.Oneroller bottle(490 cm2) ofconfluenthuman foreskin fibroblastcellswas inoc-ulated with a VZVstrain (passage 44) originally iso-lated from a patient with herpes zoster and kindly provided by LucyRasmussen. When 75% of the cells showedcytopathic effect, theywereremovedand used

toinfect five roller bottlescontainingconfluent fibro-blast monolayers. After 72 h, when 75% cytopathic effectwas observed,theinfectedcellswereremoved with glass beads, washed once with phosphate-bufferedsaline(PBS), pH 7.2,suspendedtoa

concen-tration of 5 X 10" cellsper ml and sonicated. After

centrifugationat400 xgtoremovelargercelldebris, the antigen was dispensed in working volumes and stored at-70°C. Control antigen waspreparedfrom uninfectedcells bythesameprocedure.

Thecommercial antigenwaspreparedfor use asa

VZVcomplementfixationantigen byFlow Laborato-ries, Inc., Rockville,Md.

Varicella-zoster antibody testing by comple-mentfixation,IAHA,andIFAand VZV lympho-cytetransformation.TheVZVcomplementfixation

assay wasperformed byastandard methodusingtwo

units ofantigenandtwounits ofcomplement (17).For the IAHAassay(12, 19),serial dilutions ofsera(0.025

nml) were incubatedwith 0.025 ml ofVZVantigenin

the wells ofamicrotiter"V"plate(Flow Laboratories) for1 hat37°C.The usualoptimaldilution ofantigen

was 1:8 to 1:16.Complementwasaddedatadilution of 1:80to 1:100. Afterincubationat37°C for 40min, dithiothreitolinVeronal-bufferedsaline and ethylene-diaminetetraaceticacid(0.025mlperwell)anda 1.5%

suspension of human "O" cells (0.025 ml per well)

which had been screened forsensitivityinthisassay were added. The plateswere agitated for1 min and left atroom temperature for 1 h. AUl sera werealso tested with control antigen. The antibody titer was

readasthereciprocalof the maximum serial dilution

resultingin 3+hemagglutination.

Ourmodification of the IFA method has been

de-scribed (1). Monolayers of human foreskin fibroblast cells grown in chamberslides (Lab-Tek Division, Miles Laboratories,Inc., Naperville, Ill.)wereinfected with VZV. Whenseveral foci of infectedcellswere visible in each well, but before the cytopathic effect was extensive, the slides were fixed in cold acetone for 5 to 10min. The fixedslides were stored at -70°C. Begin-ning at 1:8, serialdilutions of the serum samples to be tested and known negative and positive sera were coded and added in triplicate to the slide wells. After incubation for 1 h at 37°C, the slides were washed three times with PBS, and fluorescein-conjugated guineapig antihumanIgG (Microbiological Research Co.,Bountiful, Utah) diluted 1:20 was added. After 1 h ofincubation at 37°C, theslideswere washedthree times with PBS and examined under afluorescence microscope. The titer was read as the highest dilution of serum which showedfluorescence ofinfected cells in at least two of the three wells.

VZV cell-mediated immunity was assessed by in vitro lymphocyte transformation to VZV antigen as described previously (1). Ficoll-Hypaque-separated peripheral blood mononuclearcells in30% heat-inac-tivated human serum wereincubated with VZV anti-gen for 5 days. Lymphocyte transformation was de-tected by comparing tritiated thymidineincorporation inantigen-stimulatedcultures(;3H-antigen) with con-trol antigen cultures

(;3H-control).

The results were expressed as astimulation index ;H-antigen/3H-con-trol. Astimulation index of at least threefold to VZV antigen was seen in normalseropositivesubjects (1).

RIA for varicella-zoster IgG and IgM anti-bodies. TheRIA for VZVIgG antibodywasa modi-fication of the method described by Friedman et al. (10). Commercial VZV antigen (Flow Laboratories) was diluted to 1:5 with PBS, pH 7.2, and antigens preparedin thislaboratoryweredilutedto 1:8 to 1:10 for use in the assay. Three lots of the commercial antigen were used with equivalentresults. VZV anti-gen (0.025ml)oruninfected controlantigen (0.025 ml) was added to each well of a polyvinyl "U" plate (Dynatech Co., Alexandria, Va.), and the plate was allowedto dry overnight at roomtemperature. After onewashing withPBS,thewells were filled with PBS containing 20% fetal calf serum andincubated for 1 h at37°C. Thewells were then rinsed twice with PBS andallowedto dry. Each serumsample tobe tested wasadded to twowellscontainingantigen and to two control wells. The sera were diluted in PBScontaining 10%fetalcalfserumand0.05%Tween 20(J. T. Baker Chemical Co., Phillipsburg, N.J.) using a microtiter platedilutor(Cooke Co.,Alexandria, Va.). Theplates wereincubated at 37°C for 1 h and washed three times with PBS containing 0.05% Tween 20 before

'2'l-la-beledantihumanglobulinwasadded.

The specificbinding of VZV IgG antibodywas as-sessed using goat antihuman IgG, specific for the Fc fragment (Cappel Laboratories, Downingtown, Pa.) thathad been labeled with '2'Iby amodified chlora-mine T method (20). '25I-labeled monospecific goat antihuman IgM antibody (Tago Co., Burlingame, Calif.) purifiedby affinity chromatography wasused to detect IgM antibody bound to the VZV antigen-coatedwells. Afterlabeling,67to76%of thecountsin J. CLIN. MICROB10L.

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RIA FOR IgM AND IgG ANTIBODIES TOVZV 369 thesepreparationswereprecipitable with 10%

trichlo-roacetic acid. Thespecific activity of the labeled prep-arationsranged from 8to10,iCiof

"25I

permicrogram

ofprotein.

For use in the RIA, the iodinated globulin was dilutedtocontain2X 10" cpm/ml. Afterpreparation of theplatesasdescribed, '251-labeled antihumanIgG

orIgM (0.025 ml perwell)wasaddedtoeachwell, and theplateswereincubated for 1 hat37°C. Theplates were thenrinsed five times with cold PBScontaining 0.05% Tween20and allowedto dry. The wellswere separated bypassinga"hot wire" below the surface of theplate, transferredto countingvials, and counted for1 mininagammascintillationcounter(Beckman

Instruments,Inc., Fullerton,Calif.). Controls used in each assayincluded knownpositive and negativesera anddiluent alone.Specific antibodybindingwas con-sideredtobepresentateachserumdilution for which theratioof themeancountsper minute (cpm)of the twowells containingserum and VZV antigento the mean cpm of the two wells containing serum and control antigen was -2.5. The maximum cpm seen after the incubation of positive sera with antigen

ranged from 6,000to 15,000 cpm per well;the maxi-mum cpm after the incubation of antibody-positive

serumwithcontrol antigenrangedfrom200to 1,000

cpm per well. The incubation ofnegative serum or diluent with VZV antigenorwith controlantigenalso producedcountsin this range.

Separation ofIgG and IgM serum fractions. The methods used for the removal or separation of IgG andIgM antibody from thetestseraincluded the

adsorptionofIgG by using staphylococcal proteinA (3), the separation of IgG and IgM by usinga gel

filtrationtechnique,andincubation with 2-mercapto-ethanol (2). The gel purification procedure, kindly performed by Gregory Filice, used Bio-Gel A-5M,

which isa6% agarosegel (Bio-Rad Laboratories, Rich-mond,Calif.)toseparate7S and19Sglobulin.

Rheumatoidfactor. Sera thatwerepositivein the VZVIgM assayweretested for rheumatoid factor with a commercial latex agglutination method

(Calbi-ochem,LaJolla,Calif.) (21). VZVIgGandIgM anti-body titerswereassayedinserafrom six adults with hightitersofrheumatoidfactor, rangingfrom1:1,280 to 1:5,120, who had had varicella at least 20 years beforetesting.

RESULTS

BothlaboratoryandcommercialVZVantigen

preparations

were.

sufficiently

adherent to the

wells of the

polyvinyl

microtiter plates to

pro-videan

adequate

antigen-coated

surface for the

detection of

specific antibody binding.

The op-timalconcentration of VZV antigen was deter-minedbyevaluatingthespecific binding detect-able in sevensera with VZVantibody titers of 1:8byIFA andnodemonstrable VZVantibody by the complement fixation or IAHA assays.

Using a 1:5 dilution of the commercialantigen

and 1:8to 1:10dilutions ofourlaboratory anti-gen, antibodywas detectable byRIA at serum dilutions of 1:256 or higher in sera with low

positive IFA titers. Parallel testing of these sera showed equivalent VZV antibody titers with

commercialantigen diluted 1:5 and our labora-toryantigen diluted 1:8 in PBS.

With the commercial antigen, specific IgG bindingwas detected in all 42 sera from subjects remote from infection who had VZV antibody as determined by IFA (Fig. 1). No specific binding wasobserved in 21 sera from subjects who were not immune to varicella by IFA or in vitro

lymphocyte transformation (Fig. 1). When

25I-labeled goat antihuman IgM was used to detect bound IgM,

ail

8 patients with acute varicella

and 18 of 26patients with acute herpes zoster had VZV IgMantibody (Fig. 1 and 2). None of the 15 subjects remote from infection who had VZV IgG antibody had detectable VZV IgM antibody (Fig. 1). No specific binding occurred when sera from nine subjects who were not immune tovaricellawere tested.Binding curves

for

'251-labeled

antihuman IgG and IgM in

un-fractionated serum from a subject with acute varicellaareshown in Fig. 3.

Twelve serum samples fromseropositive

sub-jectsweretestedtodetermine the reproducibil-ity of theassay.Of the 12 sera, 10 had IgG titers within a twofold dilution, and two samples showed a greater thanfourfoldvariation intiter.

As is the case with other methods, it was nec-essary to test paired sera in the same assay to

obtain the most accurate comparison of VZV

IgG and IgM antibody titers early and later in

thecourse ofacuteinfection (Fig. 2).

Someassaysforthe

detection

of viralantibody

bysolid-phase RIA have been based on a com-parison of the

'25I-labeled

antihuman IgG bound

afterincubating the test serum with viral antigen

andthebindingmeasured when a known

nega-tiveserum was incubated with theantigen (15).

SpecificVZVIgGbindingcouldbeassessed with

reference

to astandard negative serum without

significantly altering the antibody titer in 20

seropositive

subjects. However, the

elimination

of the incubation of each serum with control

antigen produced false-positive results among

subjects whohad negative VZV IFA titers and nolymphocyte transformation to VZVantigen.

Of 10 nonimmune subjects, 2 showed binding ratios greater than 2.5 at serum dilutions of 1:16 whenbinding was assessed in relation to a stand-ard negative serum. The binding ratios were consistently less than 2.5 when the calculation

wasbased onbinding of the subject's serum to control antigen.

Thesensitivity of the VZV RIA for measuring

IgG antibody wasevaluated in 14 subjects who

had no antibody to

varicella

as determined by

complement

fixation (Fig. 4). The VZV RIA

VOL.12,1980

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370 ARVIN AND KOROPCHAK

AJ

4.2x106

in,

1.x10

zo

2.6x105

CL CL

- Q 65,536 _

. 16,384

-z 4096

-o

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C:_z <16 k

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-m -_

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VZV IgGANTIBODYTITER 4 VZV IgMANTIBODYTITER

FIG. 1. IgG and IgM antibody titers to VZVmeasured by RIA in nonimmune and immune subjects identified by VZVIFA and VZVlymphocyte transformation. The antibody titers are indicated along the

vertical axis. Theantibody titeris thehighest dilutionoftheserumsample forwhichthe specific binding

ratio(definedasthemean cpmafterincubation intwowellscontaining VZVantigen dividedby themean cpmafterincubationin twowellswithcontrolantigen)is-2.5. Theleftpanelshowsthe VZVIgG antibody titers in nonimmune subjects, immune subjects who had varicellamore than 20years before testing, and subjectswithacuteherpeszostertested 2to14and21 to35daysaftertheonset. The right panelshowsthe VZVIgM antibodytitersinimmunesubjectswhohadvaricellamorethan 20yearsbefore testingandsubjects

withacuteherpeszoster.

titerswerelowerinthisgroup (geometricmean

titer[GMT], 1:380)thanintheunselected

pop-ulation ofseropositive subjects (GMT, 1:6,309),

but the GMTby VZVRIA wasfourfoldhigher

than the GMT measuredbyIFA.TheRIAand IFA were equivalent in detecting seropositive

subjects in this group and in the unselected

population,butantibodywasdetectablebyRIA in more dilute serum samples from the same

individual. TheIAHAfailed todetectantibody

infivesera thatwere positive byRIA and the GMTbyRIAwas12-foldhigherthantheGMT

byIAHA(Fig. 4).

Cross-reactivitywithherpes simplexvirusand

cytomegalovirus antibodies did not appear to affect the VZVantibodyresults byRIA.Despite

thepresenceofantibodytoherpessimplexvirus in sixsubjectsandantibodytocytomegalovirus

in ninesubjects, VZVRIA titerswerenegative

in those individualswhowereVZV-susceptible asdeterminedbyIFAandlymphocyte

transfor-mation. When the assay was used to measure

VZV IgG antibodyinimmunesubjects withno

recent VZV infection, the VZV GMTs were equivalentbetweengroupsthathad antibodyto

herpes simplex virus or cytomegalovirus and

thosethatdid not. The VZVGMT in 17 herpes simplex virus-seropositive subjects was 1:2,454

compared with 1:5,623 in 18herpes simplex vi-rus-seronegative subjects;theVZVGMTs in 15

cytomegalovirus-seropositive and 17

cytomega-lovirus-seronegative subjectswere1:3,238and 1:

5,254,respectively.

Sera from six immunesubjectswithout recent VZVinfectionwhohadVZV IgG antibody titers ranging from 1:1,024to 1:2.6x 105and who also

hadhightitersofrheumatoid factorwere

eval-uated todetermine whetherrheumatoid factor producedafalse-positiveresult in the VZVIgM assay. Rheumatoid factor, which is usually an

IgM antibody against IgG, couldattach to the VZV IgG bound to the VZV antigen. The 125j_

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RIA FOR IgM AND IgG ANTIBODIES TO VZV

DAY DAY16 DAY DAY16

25

20

x

w

0 15

z

o

0 -J

m 10

ci>

5

DAY DAY16 VZV liG ANTIBDDY TITER VZV IlMANTIBDDYTITER VZVLYMPHOCYTE

TRANSFORMATION

FIG. 2. IgG and IgM antibody titers to VZV measured byRIA and VZV antigen-stimulated in vitro lymphocytestimulation inpatientswith acutevaricella.Theleftand the centerpanelsshowthe VZVIgGand IgM antibody titers, respectively, inpairedseraobtainedfromeight patientstestedI to4and 7 to 16days

aftertheonsetofacute varicella. Theantibodytiter isthehighestdilutionoftheserumsample forwhichthe specificbindingratio(definedasthemeancpmafterincubation in two wellscontainingVZVantigendivided bythemeancpmafterincubationin twowellswith controlantigen)is22.5. Theright panelshowstheVZV antigen-stimulatedlymphocyte transformationresponsesofthesepatients. Thestimulation indexisaratioof cpm measured after addingtritiatedthymidineto VZVantigen-stimulated lymphocyte cultures tocpm in control cultures incubated with uninfectedcell antigen. A stimulation index ofgreater than threefold is positive;the shadedareaindicatesastimulation indexless thanthreefold.

labeled antihumanIgM mightthen bind to the

rheumatoidfactor, resulting in apositive assay forIgM in serumthat did not contain specific VZV IgM. However, five of the six sera tested werenegativeintheVZVIgMassaydespite the presenceof rheumatoid factor. One serumhad

an IgM titerof 1:16. This titerwaslower than

theVZVIgMtiters of mostpatientswithrecent

varicellaorherpeszoster(Fig. 1and2). The IgM antibody detected in the patients with acute infection was considered VZVspecific because

none of thesera from thesepatients contained

rheumatoidfactor.

Both IgG and IgM antibodies to VZV were

presentveryearlyinthecourseofprimary var-icella infection in normal subjects (Fig. 2).

Amongeight patients tested within4days after

theonsetofexanthem, sevenhadIgGantibody

and six had IgM antibody. VZV IgG and IgM antibody titers continued to increase 7 to 16

days after the appearance of the rash in most

patients. The production of VZV IgG and IgM antibody within a few days after the onset of clinicalvaricellawasaccompanied byapositive

invitrolymphocytetransformationresponseto

VZVantigen in fourpatients.Antibody produc-tion precededlymphocyte transformationin the other four patients. The only patient with no

detectable VZV IgG, IgM,orlymphocyte

trans-formationwastestedjust 12 h afterappearance ofthe exanthem.

VZVIgM antibodywasdetectable by RIA in

18of 26patients with herpeszoster,including13 patients tested 3 to 5 weeks after the onsetof the infection (Fig. 1). All of the patients with herpes zosterhad VZV. IgG antibody and posi-tive lymphocyte transformation responses to

VZVantigen.

Thepresenceofhigh titers of VZV IgG

anti-body in patients withacutevaricella and herpes

zoster did not interfere with the detection of VZV IgM antibody in unfractionated serum. >4.2x16

<q

4.2x10

Z 0

1.0x106

a CC

_ 2.6x10 <ci

211

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372 ARVIN AND KOROPCHAK

4096 6.5x104

64 1024 16,384 2.6xl0O

SERUM DILUTION

FIG. 3. Binding of '25I-labeled goat anti-human IgG and IgMantiseraafter the incubation of

unsep-aratedserumandserumfractions fromasubjectwith

acutevaricella with VZVantigen. Thefigureshows thespecific bindingincpmof

"'M-labeled

goat

anti-serumtohumanIgGdetectedafterthe incubationof unseparatedserumandserumtreated with 2-mercap-toethanol(ME)with VZVantigen.Thespecific bind-ingin cpm of '251-labeledgoatantiserum to human

IgM after the incubation of unseparated serum, se-rum adsorbed with staphylococcal proteinA (staph A), andserumtreatedwith ME is shown. The shaded areasindicate therangeofcpmmeasured when 1251.

labeledanti-human IgG (S)orIgM (O) wasadded afterthe incubationoftheserawith controlantigen. The horizontal axis indicates the dilutionsatwhich eachserumpreparationwastested.

Testing done after adsorption of serum with

staphylococcalproteinAand aftergelfiltration to remove IgG showed that the antibody de-tected with the VZVIgM procedurewaspresent

in theIgMfractionand thatIgMtiterswerenot increased by the removal ofIgG (Fig. 3). VZV

IgG antibodyremained detectable after

adsorp-tion withstaphylococcal proteinAorgel

filtra-tion in some cases, reflecting thesensitivity of the RIA method.VZVIgGwasmeasurable when

4096 20481

w

1-o

O

z

r.,

N

1024f 5121 256 128 641 321 16 8 <8

CF IAHA IFA RIA

VZV IgG ANTIBODY ASSAY

FIG. 4. Comparison of IgG antibodytiters to VZV measured by IAHA, IFA, and RIA in seropositive

subjects, tested more than 20years after varicella

infection, who had no detectable VZV antibody, as

determined by complement fixation. Twofold serial dilutions are indicated along the vertical axis. The

horizontalaxisshowsthe VZVantibodyassaysthat werecompared.

the totalIgG had been reduced toless than0.9 to 1.8 mg/ml. IgM antibody was not detected

after the treatment of VZV IgM-positive sera

with2-mercaptoethanol, but IgG antibody

per-sisted (Fig. 3).

DISCUSSION

The solid-phase RIA technique provedtobe

asimpleand sensitive method for the

measure-ment of VZV IgG and IgM antibodies. Using commercially available reagents, this method

detected VZV antibody in very dilute serum

samples in which no VZV antibody had been

detectedbyIAHAorIFA. The RIA methodwas

not subject to the false-negative results found with theIAHAtechniqueinolderpersons with-out recentinfection notedby Forghanietal. (8)

and observed in thepresentstudy.The RIAwas as reliable as the IFA in detecting antibody

amongsubjectsremotefrom infection and

mea-suredhigher titersofantibodyin thesesubjects. Among21VZVseronegative subjectswhowere

susceptible by lymphocyte transformation, the IFAmethodgaveone false-positiveresult

com-paredwithnonebyRIA. Thefalse-positiveIFA

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RIA FOR IgM AND IgG ANTIBODIES TO VZV 373

titerwasduetothedifficulty of accurately judg-ing differences influorescence between

negative

andlow-positive sera.Theendpoint in 'he RIA methodwasclearandeliminatedtheproblem of subjectiveinterpretation. Although theRIA was notcompared withthe VZV enzyme

immunoas-saydescribedby

Forghani

etal.(8), thehighest IgGtiterobserved with thatassay was1:2,048 in

subjects remote from infection, whereas the

mean titer forasimilar

population

was 1:6,309

by RIA and 20 subjects had titers 1:16,384. Sinceserafrompatientswho weresusceptibleto varicella by lymphocytetransformationand IFA wereconsistently negative for VZV by RIA, and

fiveof thesesera wereobtained fromindividuals

who subsequently developed varicella, we be-lieve that the method can be usedclinically to

identifyVZV-susceptible patients.

Animportant advantage of the RIAtechnique

was itsapplication tothemeasurement ofVZV IgMantibody. Althoughthe IFAprocedurehas beenused tomeasure VZVIgMantibody,

com-petitionbetween IgG andIgM forbindingsites can cause

difficulty

with this method (6). The capacitytomeasureIgMantibody without frac-tionatingserumand thecommercialavailabiity of purified

monospecific

goat antihuman IgM antiserum made the RIA a practical assay for

VZV IgM

antibody.

Withthe

monospecific

an-tiserum,

specific binding

ratioswere ashigh for

IgMasfor

IgG

antibody

withoutanyalteration

ofthe

procedure.

Although

rheumatoid factor

caninterferewith thedetectionofspecificVZV IgM antibody in a few instances, this problem shouldarise

only

withseracontaining veryhigh

titersofrheumatoid factor. Even in these sera,

false-positive IgM titers can be

expected

tobe lowerthantiters observedduringVZV infection. Thephenomenon is not

likely

tobe important

in mostclinicalcircumstances,sincerheumatoid factorwas not detected in patientswith either

varicella or herpes zoster. Preliminary studies

show thatthe RIA technique can also be used to measureVZV-specific IgA antibody.

Sixty-ninepercentofpatientswithherpes

zos-ter had VZV

IgM antibody

as determined by

RIA. These data substantiated theobservations

of Ross and McDaid (22) andBrunelletal.

(4),

who reported that VZV IgM antibodycould be detected by IFAinpatientswithherpes zoster.

Fifty percent ofpatients had IgM antibody as

determined by IFA according to Ross and McDaid(22). Schmidt and Lennette foundIgM

antibodyinonly 1 of22 patientstested by VZV neutralization with theIgMserumfraction(23). The VZVIgMdatabyRIA contrast withresults obtained when the IAHA method wasmodified to measureVZVIgM.Usingthe IAHAmethod,

Gernaetal.have suggested thataspecific VZV IgMresponse occurswithvaricella butnotwith herpes zoster (11). The VZV IgMtiters we ob-tained in serumfromsubjects withherpes zoster were as high as those in patients with primary varicella in some cases. The presence of VZV IgM antibody does notdifferentiate primary var-icellafrom generalized herpes zoster.

The studies of kinetics of the immune re-sponse to primary varicella showed that both

IgM and IgG antibody and VZV-specific lym-phocyte transformation develop very early in the course of clinical infection in otherwise

healthysubjects. The simultaneousappearances ofIgG and IgM antibodies within 4 daysafter theonset of varicella demonstrated by RIA con-trastwith the apparent delay in VZV IgM

pro-duction assessed by the IFA method (5). Cra-dock-Watson et al. detected IgM antibody within7days in 31%of patients, when 100% of patientshadhigh titers of IgM antibody byRIA within 7 days. In addition to comparing the

antibodyresponse tovaricella and herpeszoster

innormal subjects with the response in immu-nocompromised patients, the VZV RIA for IgG

and IgM antibody will be useful to determine whether the antibody response tothe varicella vaccine now being evaluated in high-risk

pa-tientsissimilarto that seenafternatural infec-tion (14). The sensitivity of the method will allow further investigation of therole of mater-nally acquired antibody in modifying varicella,

the effect ofreexposure of immune subjects to the virus, and other aspects of the natural his-toryofVZVinfection.

ACKNOWLEDGMENT

We gratefully acknowledge the assistance of Catherine

Albin, Gregory Filice, HalstedHolman, andJanet Kahle. LITERATURE CITED

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