JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1979, p. 471-475 0095-1137/79/04-0471/05$02.00/0
Assay
of Human Interferon in Vero Cells
by
Several Methods
PAULO C. P. FERREIRA, MARIAL. P. PEIXOTO,MARIA A. V. SILVA,AND ROMAIN R.GOLGHERt*
Departamento de Microbiologia,Instituto de CienciasBiologicasda Universidade Federal de Minas
Gerais, Belo Horizonte, Minas Gerais,Brasil Received forpublication22January1978
Fourmethods for theassayof human interferon in Verocells werecompared basedontheinhibition of viralcytopathic effect(CPE)intubes, the inhibitionof CPE inmicroplates, the reduction of plaques, and theinhibition ofquantitative hemadsorption. For inhibition ofCPE,Sindbis virus, vesicular stomatitis virus, poliovirus type 2, and vaccinia virus were used for challenge. In the plaque
reduction method, Sindbis virus, vesicular stomatitis virus, and poliovirus were employed, and Newcastle disease viruswasused in thequantitative hemadsorp-tion assay. Sindbis viruswas mostsusceptibleto interferoninthose tests mea-suring inhibition of CPE, but vesicular stomatitis virus was as sensitive in the plaque reduction method. Highest titers of interferon were recorded in micro-plates, especially with Sindbis virus as the challenge agent, followed by the quantitative inhibitionassay.TheCPEinhibition methodwasthesimplest, and the quantitative hemadsorption assay was the most rapid to perform. Repro-ducibilities, as shown by the coefficient of variation, were 15, 39, and 59% for plaque reduction, CPE inhibition in tubes, and CPE inhibition in microplates, respectively.
Theassayof human interferon has been per-formedby usingsemiquantitative and
quantita-tive methods(7).Inthe last fewyears,agrowing interest inhuman interferon has ledtothe de-velopment of newprocedures (1, 2, 12, 14, 17). However, the relative merits ofnewlyreported techniques aredifficulttojudge since,with the
exception ofa few reports (3, 10), descriptions
are not usually accompanied by comparative studieswithcommonlyusedprocedures.
There-fore,themany different types of assays and the
diversity of cells and ofchallenge viruses that canbeemployedmake it difficult to decidewhat
method and cell-virus combinationtoadopt for
aparticularpurpose.
In this report, one cell line was used (Vero cells) to ensure a better comparison between different procedures, and several of the more
widely used methods forassayof interferon (7)
werestudied in order toprovidea moreuniform
assessmentof themethodology for titrating hu-maninterferon and tofacilitate the choice of an assay foraspecificneed.
MATERIALS AND METHODS
Cells.Verocellsweregrownin medium 199 supple-mented with 5%sheep serum,penicillin,streptomycin, and amphotericin B. For experiments cells were
tPresent address:NationalInstituteforMedicalResearch, Mill Hill, LondonNW71AA, United Kingdom.
seeded asfollows: in tubes (16 by 150mm), 100,000 cells perml;in 60-mlbottles,106cellsper5ml; and in
microplates,50,000cells per0.15ml per well. Viruses. Newcastle disease virus (NDV), Victoria strain, received from KurtPaucker,waspropagated in
embryonatedeggs. The viruswastitratedby plaque assayin Vero cells.
Vaccinia virus, vesicular stomatitis virus (VSV), Indiana strain (donated by Kurt Paucker), poliovirus type2,Lansing strain,andSindbis virus (kindly
sup-plied by Norman B. Finter) were grown in Vero or HeLacells(poliovirus).Titrationof these viruses was carriedoutin Verocellsbydeterminingthe 50%tissue culture infective dose in tubesorwellsin microtiter
platesby the method of Reed and Muench (16),
em-ploying fourtofivetubesorsixwellsperdilution. Interferon. Interferonwasprepared bythe method ofGresser (8) inprimary human amnioncellsexposed
toliveorultraviolet-irradiated NDV. The fluids were collected after24 or72h of incubation andheldatpH
2 for 3 days before neutralization and storage at -20°C. One unit of interferon,asdetermined by using the inhibition ofcytopathic effect (CPE) in tubes with VSVaschallenge,wasequivalent to 3.3international
reference units.
Interferon titrations. (i) Inhibition of CPE in
tube cultures. The interferonpreparationwasdiluted
intwofold stepsinmaintenance medium(medium199
supplemented with 1% sheep serum and antibiotics [MM1]).A1-mlamountof each dilutionwasaddedto
fourtubesof Verocellswith an in vitroage of3to 4
days. After24 hof incubation at 37°C, the medium
wasremoved, and 1.0ml ofvirussuspension,diluted in maintenance medium without serum (MMO) to 471
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472 FERREIRA ET AL.
containapproximately 100 50% tissue culture infective doses, was added to the cultures. The tubes were
reincubated, and, when viral CPE had destroyed75to 100% of control cultures, the final reading was
re-corded. The interferon titerwasexpressedasthe
re-ciprocal of the highest dilution which causedan
inhi-bition of 50% of the CPE. In each test, a parallel
titration of the challenge viruswasincluded.
(ii) Inhibition of CPE in microplate cultures. At 1 day after seeding, the mediumwasreplaced with
0.075 ml of MM1 perwell. Interferonwas diluted in
fourfoldstepsinMM1, and 0.075 mlof each dilution
wastransferred from transfer plates(Cooke
Engineer-ing Co., Alexandria, Va.) to four or five wells by
capillarity. After 24 h of incubationat37°C, the fluids
were removed, and the cells were infectedwith 0.15
mlof challenge virusperwellanddiluted in MMOto
containapproximately 100 50% tissue culture infective doses.Titrations ofchallenge virusaccompanied each
test and showed that 30to 1,000 50% tissue culture infective doseswereusedasinocula.
(iii) Plaque reduction test. Interferon prepara-tionswerediluted in twofoldsteps,and0.5 ml of each
dilution was added to a 60-ml bottle of Vero cells
containing 5mlofmaintenance medium with2%
se-rum. Two bottles were employed per dilution. The cells were incubated at37°C for 24 h. Themedium
wasthen discarded andreplaced with 0.4 ml of Hanks
saline containing about 100plaque-forming units of virus. After virusadsorption, the cellswereoverlayed
withmedium and 1%agarose.Plaqueswereread after
staining with crystal violet-ethanol solution. (iv) Quantitative hemadsorption. The technique describedby Finter(4),adapted for microtiter plates,
was used. Interferon was diluted and addedto Vero
cellsasforthemicroplateassay.After24 h at37°C, thecellswereinfectedwith ND inMMO, withaninput multiplicity of 200. Excess viruswasremoved by
wash-ing the monolayerswith Hankssalinesolutionafter1
h ofadsorption; each well then received 0.15 ml of
MM1,and theplateswerereincubated at37°C for 24 h. At that time the medium was removed, and the
monolayerswerewashed withphosphate-buffered
sa-line(0.15MNaCl,0.1 Msodiumpotassium phosphate, pH 7.2) at4°C. Toeachwell,0.1 mlof 1%guinea pig erythrocyteswasadded,andafter 25minat4°C, the
cellswerewashed withphosphate-buffered saline,and the attached erythrocyteswerelysed with 0.1 ml of
distilledwaterper well.The contents ofeightwells
weremixed, and theoptical density was determined
at 410nmwithaBeckman GTspectrophotometer. Resultsobtained,lessvalues fromcellcontrols,were
plotted as optical density against the logio of the
interferon dilution. The 50% reductionpointwas
cal-culatedfromthecurve,and thecorresponding inter-feron titer could be determined.
RESULTS
Inhibition of CPEintubes.Interferon prep-aration A was employed in the experiments
showninTable 1. Sindbis virus and VSVwere inhibitedtoapproximately thesamedegree,
re-quiring2.3and3.3reference units,respectively, toreduce viral CPEby50%. Poliovirusexhibited
intermediate sensitivity, and vaccinia viruswas insensitive to interferon. Titers varied from 80 to 160, 20to 160, and30 to 60forSindbisvirus, VSV, and poliovirus, respectively. Despite the highvariation when VSVwasusedaschallenge virus, repeated titrations (n = 22) in different
batches ofcells of thesameinterferon
prepara-tionshowedacoefficient of variation (standard deviation/mean) of 0.39.
Inhibition of CPE microplates. Another
interferonpreparation (preparation B) was em-ployed in studies for which the resultsareshown in Table 2. Sindbis virus required the least
amount of interferon (0.07 reference units) to
give a 50% reduction in CPE. Poliovirus and VSV needed, respectively, 4and 16timesmore
interferon to achieve the same effect, whereas vacciniaviruswasagainnotsusceptibleto inhi-bitionby interferon.
Acoefficient of variation of0.59wasfoundin
25assayswithSindbis virus.
Plaque reductiontest.Titers obtainedwith
preparation Bwere800, 715, and407when Sind-bis virus, VSV, and poliovirus were used as
TABLE 1. Assayof human interferon in Vero cells: inhibitionof viral CPE in tube cultures
Interferonti- Reference Relative Virus ter (perM)a UnitS/milat sensitivityC
endpoint'
Sindbis 120(4)d 2.3 1
VSV 83(6) 3.3 0.7
Polio 37(4) 7.4 0.3
Vaccinia <10 (3) >27.7 <0.08
aReciprocal of interferon dilution inhibiting 50% of
theCPE.
b Number of reference unitsrequiredtoinhibitviral CPE by 50%.
'Ratio of reference unitsat end-point dilution of Sindbis virusinrelationtodifferentchallenge viruses.
dThenumberofexperiments is given in
parenthe-ses.
TABLE 2. Assayof human interferon inVerocells: inhibitionof viral CPE in microplates
Interferon Reference
Vu
Interfpero
units/0.15 Relativesen-Virus titer (per mlatend sitivity'
0.15ml)a pointpontb
Sindbis 1,280d 0.07 1
VSV 80 1.0 0.07
Polio 320 0.3 0.25
Vaccinia <10 >8.5 <0.008 aReciprocalof interferon dilutioninhibiting50%
of
theCPE.bNumberof reference unitsrequiredtoinhibit viral CPEby50%.
c Ratio of reference units atend-point dilution of
Sindbisvirus in relationtodifferentchallengeviruses.
dMeantiterof at least threeexperiments.
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ASSAY OF HUMAN INTERFERON IN VERO CELLS 473
challenge, respectively. Graphs of the
experi-mental results(Fig. 1) showed that there was a
goodlinear response inthe50%range,butatthe
extremes of the curves the numbers ofplaques
varied considerably. Nosignificantdifference in
sensitivitywasfoundbetween VSV and Sindbis
virus, and polioviruswas only slightly more re-sistant to interferon.When thesameinterferon preparation was assayedeight times with VSV
orSindbis virus, thecoefficient of variationwas 0.15.Plaque formationby vaccinia viruswas not
influencedbythegreatest amountof interferon used in thesetests(datanotshown).
Quantitativehemadsorption.Sendaivirus,
asoriginally described byFinter (4), could not
be used because the amount ofhemagglutinin produced inVerocellswas toolow.Butinfection ofcells with NDV gave stronghemadsorption, and the curves obtained with this virus are
shown inFig.2.Therewas agood linearresponse
inthe25 to75%range,andthecalculated titers ofpreparation Bwere430,800, and1,280, with
a meanof 836,displayinghigh sensitivity.
How-ever, thevariabilityinassays over aperiodof1
weekwaslarge (Fig. 2,curvesAandB). Comparisonof methods. Because titers de-pendon the 50%endpoint andonthevolumes ofinterferonappliedtovaryingamountsof
me-100.
75'
I-z 0
I-to
a.
50-
25-VIRUS
POLIO O--o
SINDBIS h---4 VSV *
),a 1,1 1,4
INTERFE RON INTERNATIONAL UN!TS log.,,)
FIG. 1. Plaque reduction curves of different vi-rusesininterferon-treatedVero cells.Barsrepresent
range of findings; the points represent the mean.
PFU, Plaque-formingunits.
0,50-0
c 0,40
2
z 0,30
-a
F 0,20,
0,10
EXP
A 0--0
B v---O
N . _.
I/
/
C ,---!
I i
./
f0~~~~~~
/
I/ //
/!/
*<~~~~~A
t . ,~~I,
-I -2 -3 -4
INTERFERON DILUTIONS (log10)
FIG. 2. Curves from quantitative hemadsorption
assaysof interferon in Vero cells.
dium in thedifferent cell culturesystems,a more precisemannerofcomparingthesensitivities is
to calculate the amountofinterferon thatwas needed in each method to reduce the effect of viral infectionby half. Thus, interferon
prepa-ration B wastitrated with the CPE method in tubes, and itspotencyininternationalreference units was ascertained. These data, the data in Tables 1 and 2, and the amounts in reference units for theplaqueassayandquantitative hem-adsorption were calculated (Table 3). The mi-crotechniques (inhibition of CPE and quantita-tive hemadsorptioninmicroplates) showed the highest sensitivities. When Sindbis virus was used forchallenge,33and110timesmore inter-feron had to be available to reach a 50% end point in the CPE tube method and plaque re-duction test, respectively, compared withCPE inhibition in microplates. VSV and poliovirus alsobehavedsimilarly, althoughthedifferences
wereless marked. NDVwashighlysensitiveto human interferon, since as few as 0.2 interna-tional reference unitsgavea50%endpoint,five times less than theamountneededtoinhibitthe CPE ofVSV (a virus commonly usedfor chal-lenge in interferon assays) in microplates. The small volumes and number of cells needed (six times lessthan in the tubemethod and60times
less than in the plaque reduction procedure) mightexplainthese differences (5, 15).
DISCUSSION
Sindbis virusisvery sensitivetohuman inter-feron (Tables 1and2),ashas beenpointedout
byothers (1, 18).However, thetime ofreading VOL. 9,1979
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TABLE 3. Assayof humaninterferon in Vero cells: comparison of interferon reference units needed for50% inhibition inseveral methods
Totalinterferon reference units needed for50%inhibitionof:.
Assay measuring inhibitionof:
Sindbisvirus VSV Poliovirus NDV
CPE (tubes) 2.3(33)b 3.3 (47) 7.4 (106)
CPE (microplates) 0.07 (1) 1.0 (14) 0.3 (4)
Plaques 7.7(110) 8.7(124) 15.4(220)
Quantitative hemadsorption 0.2 (3)
aRecorded per
milliliter
for the tube assay, per 5.5 ml for the plaque assay, and per 0.75 ml for the assay in microplates and quantitative hemadsorption.bFigures in parentheses denote relative sensitivity of method in relation to the microplate assay with Sindbis
virus.
the CPE in Vero cells infectedwith Sindbis virus or VSV is critical, because viral inhibition by interferon could be overcome rapidly by
pro-longed incubation. The coefficient of variation of0.39for thistypeofassayis in thesamerange asshownby Siewersetal. (18).
Moreover,thecomparativesensitivitiesto
in-terferon of twoviruses in one system was not
necessarily paralleled in another system. For instance, 16 timesmore interferon was needed
toreduce theCPE of VSV than that of Sindbis virus in microplates (Table 2), whereas in the macrotechnique the ratiowasonly1.4(Table 1). Polioviruswas moreinhibitedby interferon than
wasVSV in the microtechnique, and the
oppo-site occurred in the tube method. Thesefindings
can best toexplained by differences in the
ex-perimental conditions under which these tests are carried out (2, 12). However, multiplicity does not appear to be a factor since 30-fold differences in the dosage ofVSV did notalter the interferon titer in the microplate assay, as
hasalso been reported for the tube method (7). VSV and Sindbis virus were about equally susceptibletointerferonintheplaquereduction
assay. VSVplaques appearedearlier(36instead of48h forSindbisvirus), andtheywereeasier
toread. Thecoefficients of variationintheassay
of human interferon of0.25 (13),0.26 (19),and
0.15(seeabove)showahighdegreeof
reproduc-ibility.
In the quantitative hemadsorption method, high titers of interferon were revealed when NDV wasusedaschallengevirus. NDV is con-sideredtobeavirusresistenttointerferon(11), but, under the experimental conditions em-ployed, the growth of NDV was probably re-strictedto onecycle,andit has been shown(9) that the inhibition ofmultiplicationofNDV and
VSVin a single cycle were in the same range.
Curves similar to those shown in Fig. 2 were obtainedin themacrosystembyFinter(4).
From the data obtained and following the criteria described by Finter (7),acomparisonof the characteristics of the various methodscan
be made. Amethod is considered simple when
a minimum ofpreparation, technical expertise, andequipmentareneeded, and CPE inhibition
measured in tubes or microplates, as well asthe
plaque reduction method, were the simplest. TheCPE inhibition method in microplateswas very convenient, taking little time to perform and to read and allowing a larger number of samplestobe handled compared with the other methodsstudied. Because thepresenceof NDV hemadsorption could be demonstrated after24 hofinfection of Vero cells and thehemoglobin content measurement took about 1 h, this methodwasthemostrapid.
The coefficient of variation demonstratedthat
the plaquereduction method wasthe most
re-producible procedure, withacoefficient of vari-ationofonly0.15,compared with0.39and0.59
for theCPE method in tubes and microplates, respectively. The poor reproducibility of
semi-quantitative methods when visual reading of CPE isused iswell documented(7).
When many samples are tested and a high sensitivity is required, the CPE inhibition in
microplateswith Sindbis virusasthechallenge should be the method of choice. Ifrelative
po-tencies of standard preparations of interferon
are to be compared, or when there are other
conditionswhensmall differencesintitermust beascertained,theplaquereduction assay with VSV should bepreferred.
Themajor disadvantageofCPE inhibitionin
microplates,ascompared withothermethods,is its poorreproducibility. The inhibitionofgrowth ofSindbisvirus in Verocellscanbequantitated by the dye uptake method (6, 12) or by the
hemagglutinin yield-reductionassay (14), and a
modificationof the latter method to a
quantita-tive assaymightlead toimproved results.
Com-474 FERREIRA ET AL. J. CLIN. MICROBIOL.
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parative studies including these modifications will be required to determine whether this is in fact the case.
ACKNOWLEDGMENTS
We thank Kurt Paucker, Ian M. Kerr, and Bryan R. G. Williams for reviewing the manuscript. Excellent technical assistance was provided by Joao R. dos Santos, Maria L. Trindade, and Bernadete M. Jesus.
This work wassupported by aConselho Nacional de De-senvolvimento Cientifico eTecnol6gicogrant and by the Fin-anciadora de Estudos eProgramas 132 program.
LITERATURE CITED
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11-27. In N. B. Finter (ed.), Interferons and interferon inducers. NorthHolland Publishing Co.,Amsterdam. 12. McManus, N. H. 1976. Microtiter assay for interferon: microscpectrophotometric quantitation of cytopathic effect.Appl.Environ.Microbiol.31:35-38.
13.Merigan,T.C.,D. F.Gregory,and J. K.Petralli.1966. Physical propertiesof human interferonprepared"in vitro"and "in vivo".Virology29:515-522.
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