Rapid, quantitative, semiautomated assay for virus induced and immune human interferons

0095-1137/80/09-0433/06$02.00/0

Rapid,

Quantitative, Semiautomated Assay for Virus-Induced

and Immune Human

Interferons

JON A. GREEN,`* TZE-JOU YEH,"3.ANDJAMES C. OVERALL, JR.23

Howard HughesMedical InstituteLaboratories2 and DivisionofInfectiousDiseases,Departmentsof

InternalMedicine' and Pediatrics,3 University of UtahCollege of Medicine, Salt Lake City, Utah 84132

Animproved human interferon (IF)assayisdescribed. This procedure is based ontheabilityofencephalomyocarditisvirustoreplicate in WISH cell

microcul-tureswith the production of discrete plaques in the presence ofa liquid tissue

culture medium. Performance of 50%plaque reduction endpointassays in

micro-culture required only 0.1 ml of specimen for determinations using duplicate dilutionsbeginningat1:3.Semiautomatedequipment facilitated simultaneous in situ dilution and distribution of multiple IF samples in cultures containing preformed WISH cell monolayers. An incubation period of5to6hwasadequate

fordevelopment of maximal antiviral activity by both virus- andimmune-induced IF. Sensitivity of the encephalomyocarditis microplaque reduction assay was

comparabletothat of othercommonly used techniques. The method is rapid,can

be completed within 30 h from the beginning of the IF assay, and is able to

accommodateasmanyas40to50samplesatasingle time.Encephalomyocarditis

microplaque reductionissuitable for thequantitation of IFas anantiviralagent oralymphokine.

Interestinhuman interferon (IF)has intensi-fied in recent years. Currently IF isbeing used

in the

prophylaxis

and therapy of human dis-eases (8,17). Inaddition,IF isincreasingly

rec-ognized as a lymphokine (9) with

immuno-modulatory

properties

(3, 15),andas a

potential

means of

increasing

an understanding of the

pathogenesis

ofviral and autoimmune diseases

(14). There is a

continuing

need, therefore, for further

development

of

improved methodologies

forrapidIFdetermination

(10).

Advances in human IF determination have producedassay systems that arerapid, sensitive, and

reproducible,

butrequire only small

quan-tities ofspecimen anda minimum oftime and

effort(10). Onesuch assay usesacontinuousline of human amnion

(WISH)

cells and endpoint determination basedon50%reductionin

vesic-ular stomatitis virus

(VSV)

plaques

which

de-velop in thepresence of aliquid culturemedium

overlay (12). This procedure is limited by for-mation of secondary microplaques and

coales-cenceofprimary microplaques withinthe 16- to

20-h incubation

period.

Inaddition,asoriginally described, only classical, virus-induced leuko-cyteorfibroblast IF wasassayedusing a 3- to 4-hincubation period. Subsequent investigations indicated that immune-induced IF required a

longerperiodof incubation to stimulatemaximal

antiviralactivity (5).

We have modified the previously described VSV-WISHcellmicroplaquereduction assay in

several ways. Encephalomyocarditis (EMC) vi-rus wassubstituted for VSV, witheliminationof secondary microplaque formation and a major

reductioninmicroplaque coalescence.

Simulta-neously, machine dilution and distribution of

multiple IF samples in culturescontaining

pre-formed WISH cell monolayers, as well as the use of a crystal violet stain for microplaque

enumeration were also incorporated into the

methodology.Finally,anabbreviatedincubation

period of5 to 6hwasexaminedandfoundto be sufficient for the development of maximal anti-viral activity by immune IF.

MATERIALS AND METHODS Cell cultures. Stationary WISH amnion cell

cul-tures, a continuous line established from a normal

human placenta (13), were originally obtained from

Flow Laboratories, Rockville, Md. Cultures were

maintained in Eagle minimal essential medium

(MEM) prepared with Earlesaltsolution and

supple-mented with 10%fetal bovine serumand antibiotics

(streptomycin,100

fug/ml,

andpenicillin,100ug/ml).

Virus. EMCvirus, obtained from Yvonne Bryson,

waspropagated in monolayercultures ofmouseLcells

and had atiter of

~107

plaque-forming units (PFU) per 0.1mlwhenassayedin humanWISH cell cultures

asdescribed below.

EMC plaque formationinliquid-overlay WISH

cell microcultures. Growth medium was removed,

and a 5x 10`' dilutionofEMC,calculatedtoproduce

approximately 20 to 30 PFU, was added in 0.02-ml

amountsof MEM with 2%fetalbovine serumbyusing

anEppendorftypeof transferpipette. One hourlater,

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an additional 0.1 ml of MEM containing 2%c fetal

bovineserumwasdispensed toeachmicroculture. At

22 to 24 h, the plates were fixed and stained, and plaques were counted as describedbelow.

IF preparations. Several IF preparations were

used. An IF-containingpoolwasinducedby Newcastle disease virus (NDV) in normal human peripheral bloodleukocyte cultures.Asecond IFpreparationwas stimulated byphytohemagglutinin(PHA) (Burroughs Wellcome, no. HA 16) in human peripheral blood

leukocytecultures fromhealthydonors.Virus-induced

IF (NI)V-IF),but not PHA-stimulated IF (PHA-IF),

wasresistant to exposure to pH 2 for 20 h and was neutralized by exposure tospecificantiserum directed against virus-inducedleukocyteIF. ANational

Insti-tute of Health (NIH) human leukocyte IFstandard,

G023-901-527, had a predicted titer of 20.0 x 10:

(range, 11.6 x 10" to 41.5 x 10') when assayed in

humanforeskin fibroblast cultures (data

accompany-ing NIH leukocyte standard). IF preparations were

stored in 0.5-mlvolumesat-70°Cat a 1:10dilutionof their original concentrations. Each sample was

de-frostedand used one timeonly.

IFdeterminationsby EMC plaque reductionin

WISH cells with liquid medium overlay. WISH

cellsat aconcentrationof7 x 104wereseeded in

0.1-ml volumes in 96-well microculture plates (Falcon

Plastics)20 to 24hbeforebeingused. Six IF-contain-ingspecimens at theappropriate dilutionwereadded

in duplicate 0.05-ml volumes to cultures in the first

rowof 12 micro-wells, each containing 0.1 ml ofthe

originalMEMgrowth medium. Six serial1:3(0.5log,()

dilutions were made withanautomated diluter

con-taining a row of 12 0.05-ml volume-calibrated wire

loops(Titertek,FlowLaboratories).Whenmorethan

one culture plate was used, two separate cassettes

eachcontaining12wireloopswereusedalternatelyso

that cooling between flame sterilization could occur

withoutappreciably delayingtheprocedure.

IF-containing WISH cell cultureswereplacedinan

unstacked fashionina5%C02,100% water-saturated

atmosphereat37°Cfor6 h.Supernatant mediawere

decanted into an autoclavable receptable containing

toweling to minimize splashing. Culture wells were

washedonetimeby addinganddecanting 0.1-ml

vol-umesof MEMcontaining2%fetal bovineserum.EMC

virus was then added as described above. Culture

plateswere returned to 37°C for 1 h to allow virus

adsorption,and0.1 mlof MEM with 2%fetalbovine

serumwasadded.

Readily identifiable EMC microplaques developed

within 22 to 24 h, at which time 20% Formalinwas

added for 30 min and theplateswere washed under

running tap water and stained with a 1% alcoholic

solution ofcrystalviolet(1).Plaque countingcould be done withthe unaided eyeorwithadissecting micro-scope or other magnifying device. The IF titer was

expressedas thereciprocal of the calculated dilution

thatreduced thenumber ofmicroplaques by50%(50%

plaquereduction) (6).

RESULTS

EMC plaque formation in WISH cell

monolayer cultures with liquid medium

overlay. EMC plaques, visible to the unaided eye in stained monolayers, formedwithin 24 h in confluent WISH cell cultures containing liq-uid medium without solidifying agents (Fig. 1). Secondary plaque formation and coalescence of plaques were not appreciable during the first 24 h of incubation at 37°C. Reproducibility of plaque formation was examined on eight

sepa-FIG. 1. EMC virus microplaque production in

WISH amnioncell culture monolayers. Rows A and B, Uninfected cells. Single defect in the two mono-layers in row A was produced by the diluting loop. Rows C and D, Cultures 24 h after additionof ap-proximately 20 PFU of EMC in an initial 0.02 mlof

inoculum. All cultures werefixed in Formalin and stained with 1% crystal violet. Finalmagnification,

5x.

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HUMAN INTERFERON ASSAY 435

rate occasions in whicha single EMC

prepara-tionwasaddedto

replicate

microculturewellsat a dilution estimated to contain 20to 30 PFU.

Data wereobtained foratotal of407individual cultures in which PFU were counted at 24 h (Table 1). In general, good correlation existed

between the estimated and actual number of PFUper 0.02mlof inoculum.

Effect of IF on EMC

plaque

formation. Responsiveness of EMC plaque formation to

inhibitionbyIF wasexamined under conditions

in which PFU

development

was related to 0.5

log1o dilution of NDV-IForPHA-IF (Fig. 2). For

purposesof

comparison,

the datawere "normal-ized" and actual

plaque

numbers at

specific

IF dilutions were converted to percentages of the

TABLE 1. Reproducibility ofEMC microplaque

formation in WISH cells in thepresenceof liquid

medium overlay

No. of inocu- No. ofplaquesper cul-Expt latedcultures" ture (avg±

SDb)

1 47 22.0±7.7

2 48 25.4±5.9

3 68 29.2±5.6

4 43 19.9±6.3

5 57 23.1±5.9

6 80 20.0±5.6

7 30 18.4±6.2

8 34 23.0± 3.4

Total 407 22.6±3.5'

"Each culturewas anindividual microculture well

inoculated witha0.02-mlvolume of a 5x10-6dilution

ofaseparatesample ofasingle EMC preparation.

hSD, Standard deviation.

'Mean±standarderror.

o

E

o il80

570

z60

E

50-o

Ô

20-10

J o

NDV-IF

Ir

2.4 29 3.4 3.9 2.0 2.5 LOGIOINTERFERON DILUTION

30 3.5

-90o

-80E3 oe

-70

5--60°

-50

-40E

-30F-₁

-202

10 w

h.l

,ï

a-FIG. 2. IF dose-response effect. Number of EMC microplaques developingin WISHcell cultures

ex-posed to0.5 logodilutions ofNDV-IForPHA-IF. Datahavebeen normalized such that eachpointisa

percentage representing the number of plaques in

duplicate,IF-treated culturesdividedbythenumber ofplaquesin virus control cultures.

number of plaques present innon-IF-containing

virus control cultures. An almost linear reduc-tioninEMCplaque number was noted to occur at four separate dilutions. The slopes of the curves for NDV-IF and PHA-IF were almost identical.

Durationof IF andWISH cellincubation and the development of antiviral

resist-ance. IFs produced by human leukocytes in response to NDV or PHA werecomparedwith

respect to the rate at which they induced the

antiviral state in WISH cell cultures (Fig. 3). In each of fourseparate experiments, NDV-IFand

PHA-IF wereadded to WISH cells for 0, 2, 4, 6, 8, 24, or 32 h. Attheselectedtimes,

IF-contain-ing dilutions were decanted, and monolayers

were washed once and challenged with EMC

virus. Both NDV-IF and PHA-IF induced a maximum antiviral state (synonymous with maximum IF titer) within 6 h of addition to

WISH cells.By 2h,NDV-IFand PHA-IFtiters

were 83 and66%of theirrespective maxima. At 4 h the titer of NDV-IF was 92% and that of

PHA-IFwas 85% ofpeak values. Basedon these

data, a 6-h incubation was adopted for routine

use in

subsequent

experiments.

Comparison ofthe modes ofsample di-lution and distribution on the titer of IF

preparations.

These studieswere donebefore thegeneraluseofthe semiautomated mechani-cal diluter forthein situ dilution and distribu-tion ofIF

preparations.

Twomethodswere

com-pared. Dilutions were prepared by sequential, manual pipetting of

individual

samples in glass

tubes and were manually transferredto WISH cell monolayercultures.Alternatively, machine-operateddilutingloopswereused for the

simul-taneousdilution and transferofsamplesin flat-bottom tissue culture wells containing pre-formedWISHcell monolayers. Developmentof antiviral activity wascompared at four dilutions

foreachmethod.Theresultsofmultiple exper-iments are presented in Fig. 4. As measured by

reduction

in EMC microplaque formation, no

appreciable difference

existedbetween the two

methodologies.

PHA-IF was used in these

ex-periments.

Similar

results(not shown) were ob-tained withNDV-IF.

Number of replicate cultures necessary for

accurate

and reproducible IF titer. If three or four replicate cultures were necessary for an accurate and reproducible IF titer of a

given specimen, fewer samples could be run in anassay (four and three samples per microcul-ture plate, respectively). Six samples could be assayed in eachmicroculture plate if only

dupli-catecultures were used. To examine this aspect oftheassaysystem, all eight rows with 12 wells

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24 32 O 2 4 6 8 HOURS OFINCUBATION

FIG. 3. Rate of onsetof antiviralstatein WISH cells exposed for various intervals to NDV-IF or PHA-IF.

EMCvirus wasadded after IF dilutions had been decanted and WISH cell monolayers had been washed

once with MEMplus 2%fetal bovine serum. The heavy, solid lineis the geometric mean of individual

experimentsrepresented bydashed lines. Vertical barsdepictthe standarderrorsof the geometricmean.Also

shownis the percentofmaximum IF titer.

each in a 96-well microcultureplate wereused to assay serial dilutions ofNDV-IF. The 50% plaque reductionendpointwascalculatedusing

a mean of the virusmicroplaquesfor: (i) all 12

cultures, and

(il) duplicate,

triplicate, or quad-ruplicate culturesfor the serial IFdilutions.The IF titers using two, three, and four

replicates

werethencomparedtothe titer based upon the mean plaque countsfor 12 cultures. Results of two experiments indicated that the IF titers

usingduplicates varieda meanof 24.8 ± 15.8% (range, 3 to47%) from the titer obtained when theplaquecountsfromall12cultureswereused. Titers using triplicate cultures varied 29.1 ± 19.8% (range, 12 to71%), and thoseusing quad-ruplicate culturesvaried 13.7 ± 10.4%(range, 3 to 25%). Similar results were obtained when

PHA-IFwasused. Whenall aspects of the assay system were considered (e.g., number of

speci-mensthatcould beruninagivenassay,amount ofspecimenrequiredforeach assay,technologist time, cost of materials), assays performed in

duplicate were considered sufficienttoprovide

an accurate andreproducible assessmentof the

IF titer. Using two rows per sample, repeated assayofour ownhuman IFstandard has resulted inIFtitersthat have varied38.7±27.8%(range, 4 to 108%) when the titers for 26 individual assayswerecomparedwith themeantiter forall assays.

Comparative sensitivity of EMC plaque reduction human IF determination. The

NIH-supplied Sendai-induced human leukocyte IF standard(G023-901-527)wasassayed on nine separateoccasions as the 50% plaque reduction endpoint of Sindbis virus plaques in human fore-skin fibroblast cultures by using the conven-tional agar overlay method. Results of these titrationswerecompared with thoseobtainedby

similarassay of the standard IFpreparation on

five separate occasions in theEMC WISH 50% plaque reduction liquid overlayprocedure (Ta-ble 2). Ahigh degree of accord existed between

thesetwotechniques and publishedresults

ob-tained when the NIH interferon standard was

assayed by the VSV 50% plaquereduction

tech-niquewith agaroverlay in human BUD-8cells. Comparison of titers for different techniques revealed a titerof 20.2 ± 12.1 x 103units with the Sindbis-human foreskin fibroblast assay,

23.8± 10.7 x 103unitsfor the EMC 50%plaque

reduction assay inWISH, and20.0 x

103

units,

previously reported, for VSV plaque reduction in 16 separate determinations in BUD-8 cells

(dataaccompanying the NIH-IFstandard).

DISCUSSION

The EMC-WISH microplaque reduction as-say for human IF satisfies many of the criteria for an improved, rapid IF bioassay (10) and

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HUMAN INTERFERON ASSAY 437

1.4 1.9 2.4 2.9

INTERFERONDLUTION(Logo)

FIG. 4. Comparison of manual and

semiauto-mated,mechanical methodsforthedilutionand

dis-tribution ofIF. Manualmethod (gray bars): Serial

threefold dilutions wereprepared with pipettes in

tubes.Samples ofthese dilutionsweretransferred by

useofmicropipettesto WISHcellcultures.

Semiau-tomated method(whitebars):12simultaneous,serial

threefolddilutionsweremadeinmicrocultures

con-taining preformed WISH monolayers. Transfer of

dilutions(distribution) occurredautomatically. Ver-tical lines representstandarderrorsof eightseparate experiments.

offers several advantages over previously

re-portedassaysystems (12, 16). EMC virus used in thisassayhad amuch moreuniform rate of

microplaque formation, less coalescenceof pri-maryplaques,andfewersecondary plaques dur-ing the 22-to 24-hincubationperiod under the liquid medium overlay than VSV used previ-ously forthispurpose (12). EMC isassensitive

to human IF as VSV in WISH cells, and the

EMC-WISH system isequivalent insensitivity totheoneused forthe NIHreference humanIF (G023-901-527).Minimalquantitiesofspecimen

are needed to perform an assay in duplicate, evenwhenbeginningatalowdilution. Theassay israpid and hasahigh sample capacity capable

ofaccommodatingasmanyas40to50specimens withresults attainableapproximately30 h from the beginning of the IF assay. The

semiauto-mated method of dilution saves considerable

time and isasaccurate asmanualdilution.

Re-quirements for media, plasticware, and other materials are reduced to a minimum with the

microculturesystem.Finally,apreviously

men-tionedadvantage arises from theuseofa

com-TABLE 2. Comparison of conventional and

semiautomated EMC- WISHmicroculture plaque

reduction assay methods in titrating NIH human IF standard'

Conventional Microculture liq-Expt agar overlay0 uidoverlay'

(Sindbis-HFF) (EMC-WISH)

1 36.5 32.8

2 17.9 13.2

3 43.0 28.0

4 19.5 33.6

5 6.3 11.6

6 10.0

7 26.0

8 7.6

9 15.0

Mean+SEd 20.2±12.1 23.8± 10.7

Range 6.343.0 11.6-33.6

aTiterinVSV/BUD-8 50% plaque

reduction

assay:

Mean,20.0x 10';range, 11.641.5 when assay N =6,

13.3-35.9 when N = 10(3).

b

0.5%

agaroseoverlay with conventional 18- to

22-hincubation period of IF on indicated human foreskin

fibroblast (HFF) cell monolayer.

eLiquid overlay technique with 6-h IF incubation

period.

dSE,Standard error.

merciallyavailable,continuouscell line(WISH)

with stable IFsensitivitycharacteristics.

Semiautomated mechanical equipment has been usedpreviouslytoprepareIF dilutions in

microcultureplates (2).However,inthissystem the IF dilutions were performed before the

ad-dition ofcellstoform the monolayer. With the

EMC-WISH assayprocedure, mechanical dilu-tion is carried out in microcultures with pre-formed cell monolayers. This offers the advan-tage of examination for quality of cell mono-layersbeforeuse, andeliminates potential inter-ference with growth of the monolayer by the

anti-divisional effectsof IF(17) orIF-containing leukocyte preparations(11). In the EMC-WISH

procedure, the integrity of the indicator cell

monolayer is unaffected by in situmechanical dilution, with the exception of an occasional round, central defect caused by the calibrated loop.

The current studies demonstrated efficient

mechanical dilution of IF in amedium contain-ing othermacromoleculesand somemicroscopic

cellular debris. The small dilution increments used may have reduced the risk of inadvertent sample "carry-over." No evidence of this phe-nomenon wasdetected when theplaque counts inserialdilutions ofhigh-titered specimens were examined. These findings support the earlier

demonstration of the efficiency of mechanical

dilutionofradiolabeled uridine (2).

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Activation of WISH cells to the maximal an-tiviral state was rapid, reaching a plateau by 6 to 8 hof incubation. No differencewasnoted in thekineticsof activationbyIF inducedby virus (NDV-IF) or by mitogen (PHA-IF). These data conflict with previous information which indi-cated that the onset of activation was longer

with immune IF thanwith virus-induced IF (4, 5, 7). The current findings are supported indi-rectly by the similar dose-response curves of NDV-IF and PHA-IF assayedafter6h of incu-bation in WISHcells (seeFig. 2). Delayedonset ofactivationwith immune IF would have been

anticipatedtoproducearelativelysteeper curve, i.e., less inhibition of plaque formation at the higherdilutions, particularlywith the short in-cubation period. Theexact reasons for the dif-ference in the observations reported here and thosereportedpreviously(4, 5,7)are notknown. Thisdifference may beafunction of the number oftimes IF-exposed monolayers are rinsed be-fore the addition of thechallengeinoculum(one

in thecurrent study, asopposed tothreetimes in previous studies) (4, 5, 7). Alternatively,

WISH cells were

challenged

with EMC at an

approximate multiplicity ofinfection of 0.0003, which is much lower than the

multiplicity

of infection of 10 used in studies which

demon-stratedprolongedinduction of the antiviralstate by immune IF(4,5,7). Preliminary

experiments

in our laboratory support both of these

hy-potheses. Whatever the finalexplanation,the 5-to6-hincubationperiodused in theEMC-WISH assay system is sufficient to provide a rapid, quantitative determination ofthe titer of both virus- and immune-induced human IF.

ACKNOWLEDGMENT

Thiswork wassupported,inpart,by Public Health Service grantAI-15074 from theNationalInstitutes of Health.

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