JOURNAL OF CUINICALMICROBIOLOGY, Nov.1981, p. 0095-1137/81/110501-09$02.00/0
Production of
Antiserum to Respiratory Syncytial
Virus
Polypeptides: Application
in
Enzyme-Linked Immunosorbent
Assay
ALLAN
HORNSLETH,I*
PER CHR.GRAUBALLE,'
BIRGITTE FRIIS7 J0RGEN GENNER,3 ANDIB R. PEDERSEN'
Department ofClinicalVirology,Institute ofMedical Microbiology, University of Copenhagen,
Copenhagen';PediatricDepartment,CopenhagenCounty Hospital,Gentofte2;andDermatology
Department, FinsenInstitute, Copenhagen,3 Denmark
Received 18 December1980/Accepted1June 1981
By
use of crossed immunoelectrophoresis techniques, respiratorysyncytial
(RS)
virus-specific precipitates
wereproduced
between RS virus cellular antigen
[solubilized
intris(hydroxymethyl)aminomethane-glycine
buffer,
pH 9]
andan-tiserum
raised in rabbits
against semipurified RS virus. When these precipitates
were
employed
asantigens for further immunizations
inrabbits,
antibodies
(anti-RSV-precip.I)
wereproduced
which reacted withonly
oneRS virusantigen whentested
by the crossed immunoelectrophoresis technique. Precipitates obtained
between
RS
virus
cellular
antigen
(labeled
with
L-[tS]methionine)
and
anti-RSV-precip.I
wereexamined
by polyacrylamide gel electrophoresis,
which showedthat
anti-RSV-precip.I precipitated RS virus polypeptides of molecular
weights 28,000to84,000.
Anti-RSV-precip.I
wasemployed
ascaptureantibodies in theenzyme-linked immunosorbent
assay,in which
RS virus cellular antigen
wasused
asthe
second
layer. Determination of human
RS virus immunoglobulin G antibodies by
this
enzyme-linked
immunosorbent
assaytechnique showed
ahigh degree of
sensitivity,
specificity,
and
reproducibility.
The
introduction of
newsensitive
enzymeim-munoassays
(2,
17) into
diagnostic
virology has
created
a demand for more potent and morespecific
immunesera.Thepurpose of thepresent reportis
todescribe the
production
of antiserum
to
respiratory
syncytial
(RS)
virus
polypeptides
(molecular weight
[MW],
28,000
to84,000)
by
the
useof crossed
immunoelectrophoresis
(CIEP)
techniques.
Further
characterization of
this antiserum and
abrief
summary of results
obtained with it
inthe
enzyme-linked
immuno-sorbent
assay(ELISA)
will also bepresented.
RS virus is
asingle-stranded
ribonucleic acid
virus
belonging
tothe
family
Paramyxoviridae
(14).
Multiplication
occursin the
cytoplasm
of
the host
cell, independent
of
cellular
deoxyribo-nucleic acid
(11),
with maturation
taking place
by
budding
from altered
plasma
membranes
(16).
Previous reports have shown that six toeight RS
viruspolypeptides
(MW
ranging
from20,000 to
80,000)
can be demonstrated inRS
virions
orinRS virus-infected cells
by
polyacryl-amide
gel
electrophoresis (PAGE)
(4, 5, 13, 18,
24,
32).
According
to a recent World HealthOrganization
report,RS
virusis themostimpor-tant
respiratory
diseasepathogen
of infants andyoung
children (31).
It is difficult
toproduce
RS
virions orRS
viralantigens
free of host cell material. CIEP
tech-niques (30) facilitate the purification of viral
antigens,
asshown
by previous
reportsfrom this
laboratory
(8, 9, 12,25).
Theimmunization of
rabbits with precipitates (consisting
of rabbit
antisera and viral antigens) obtained by CIEP
techniques has made it possible
toproduce
spe-cific antisera
toseveral
herpessimplex virus
(HSV) antigens (27)
aswell
as torotavirus
(8)and
influenza virus antigens
(12).MATERLALS
ANDMETHODS
Cell culture. Vero and HEp-2 cells were grown in
Roux bottles with Eagle minimal
essential
medium(Earles salt solution) and 10% fetal bovineserumand
maintained in the samemedium with 2%fetalbovine
serum. Forisolation of RS virus from specimens of
nasopharyngeal secretions from patients with acute
respiratorydisease, HEp-2cells were grown in
plastic
tubes (12 by100mm; Nunc,Denmark).
RS virus strain. The Long strain of RS virus,
passedapproximately5times inKBcells and
approx-imately 55 timesin HEp-2 cells, was used in these
studies.
Titration
of RSvirus. Virustitrationwasdonebyplaque
titration
in Verocellmonolayersin 2.5-cmpetri501
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dishes.RS virus waspassed twiceinVerocellcultures
(Roux bottles), and tissue culture maintenance me-dium was harvested from cultures showing 3+ cyto-pathic effect. The maintenance mediumwasclarified
by low-speedcentrifugation and supplemented with
bovine serum albumin (finalconcentration,0.1%)
be-fore being stored in small volumes at -70°C. Virus
diluted in tissue culture maintenance medium was
absorbedfor 2 h (at35°C inaC02incubator). After
removal of the rest of the inoculum from the petri
dishes, Leibovitz L-15 medium containing 10% fetal bovine serum and 0.8% agarose(19)wasoverlaid. After 5days of incubationat35°CwithoutC02, the mono-layers were fixed with 100%methylalcohol for 10min,
stained with methylene blue(1%, wt/vol, indistilled
water) for 30 min after removal of the overlay, and
destained for5min with 96%ethylalcohol.
Titration of RS virusneutralizing antibodies.
Antibody titrationwasdoneby plaque reductiontests.
Verocellculture maintenance mediumcontaining
ap-proximately 100plaque-forming unitsper 0.3ml was
incubatedwith equalvolumes of theserumdilutions
atroom temperature. After1 h,0.6 mlof these mix-tures was addedtoeach Vero cellmonolayer (in
2.5-cm petri dishes) and incubated for 2 h in a C02
incubatorat37°Cbefore therestofthe inoculumwas
removed andtheLeibovitz-agaroseoverlay described
above wasadded. The titerwas read after 5daysof
incubationat35°CwithoutC02andcalculatedasthe
highestserumdilutiongivingmorethan 50% reduction
oftheplaque-formingunitcountwhencomparedwith
controldisheswithoutserum.
Production of antiserumto gradient-purified
RSvirus. One-liter volumes of tissue culture main-tenance medium from RS virus-infected HEp-2 cell
monolayers in Roux bottles (showing 3+ cytopathic
effect)weretreatedaccordingtothemethod described
by Senterfit and Baldridge (22). After precipitation
withpolyethyleneglycol (PEG 6,000), the resuspended
sedimentwasultracentrifuged(26,000 rpm) for16h at
4°C (Beckman SW27) on a continuous 30 to 70%
(wt/vol) sucrose gradient made in
tris(hydroxy-methyl)aminomethane
(Tris)-ethylenediaminetetra-acetate buffer as described (22). Virus-containing
bands(1.20to1.22g/ml)wereharvested,dilutedwith
phosphate-buffered saline (PBS),andsedimentedby
furtherultracentrifugationat25,000rpmfor3h
(Beck-manSW27) at4°C. This final sediment, dilutedin a
small volumeofPBS,containedapproximately300x
106 virions per ml (checkedby electron microscopy;
seebelow) and constituted theantigen.
Rabbits weregivenfour intracutaneous injections (at intervals of10 days) ofapproximately 30 x 106
virions in PBS, which had been mixed with equal
volumesofcomplete Freundadjuvant and sonicated
for30 s(BransonB12sonifier,18kilocyclesper s)just
before the immunizations. Serum harvested 8 days
after the lastimmunizationisreferredto as
anti-RSV-prim.
Electronmicroscopy. Estimationofthe number of RS virions was made according to the method describedbyPyrhonen (20).Volumes(10,l)ofserial virusdilutions werelayeredonto200-squaregrids,and virus wasallowed to absorb for 30 s. Excess fluid was
drained by filter paper, and the grids were flooded
twice withcontrast (2% solution ofammonium molyb-date inwater). After drying, the grids were examined in aPhilips Transmission EM 301 at a primary mag-nification of 45,000. At least10 squares were examined. Thus a titer could be read as the highest dilution still containing intact virus particles.
RS virus cellular antigen employed in CIEP
techniques and in ELISA. RS virus HEp-2 cell
monolayers showing 3+ cytopathic effect were gently washed twice in PBS and harvested in Tris-glycine buffer (0.08 M Tris, 0.02 M glycine; pH 9), 1 ml per
Roux bottle. The harvested cells were frozen and
thawed three times and sonicated twice (30 s each
time at 18 kilocycles per s on ice in a Branson B12
sonifier). After further centrifugationat5000 x g for
30 min (Sorvall S-3Automatic) at 4°C, the
superna-tantfluidconstituted the antigen. Control antigen was
made inexactly the same way from uninfected HEp-2
cells.
CIEP. The CIEP technique wasperformedas
de-scribed previously (8, 25, 30). Details regarding the exactexperimentalconditions are given in thefigure legends.
Immunization ofrabbits with agarose gel
pre-cipitate produced by CIEP. RSV prepre-cipitate I
(shown in Fig. 1A) was used as the antigen. Each
rabbit was immunized intracutaneously three times
(withthreeprecipitates each time) at intervals of 10
days and bled10days after the lastimmunization,as
described earlier(12, 26).
Humanserumsamples examined. Pairedserum
samples examinedwerefrom7children (1 to 6 years
old) admitted tothe pediatric departmentof
Copen-hagen County Hospital (Gentofte) for acute lower
respiratory disease. The firstserumsample wastaken
onadmission, andthe secondwas takenapproximately
4weekslater.
Titration of RS virus antibodies by indirect
immunofluorescence (IF). RS virus-infected HEp-2
cell monolayerson coverslipsin Laytontubes,infected
2days earlierat amultiplicity of infectionof 0.1, were
washed twice with PBS and fixed inacetone for 10
min at room temperature. Dilutions of human sera
were added for 1 hat 37'C. An optimal dilution of
fluorescein isothiocyanate-conjugated rabbit
anti-hu-manimmunoglobulin G (IgG) (DAKO
Immunoglob-ulins A/S,Copenhagen;code no.F1090)wasadded for
45min at37°C. The titer wasdefinedasthehighest
dilution of patient'sserumgiving definitebright
fluo-rescence ofgranular inclusions in thecytoplasm.
Labeling of RS virus-infected HEp-2cells with
L-[35S]methionine. One Roux bottle infected 48 h
earlierat amultiplicityofinfectionof 0.05 was
incu-bated (at35°C) with 25 ml of maintenance medium
containing 25% of the normal concentration of methi-onine.L-[35S]methionine(0.5mCi;(NewEngland Nu-clear Corp., lot no. 1271-172; specificactivity, 1,010.6
Ci/mmol, 1.0mCi in 0.1 ml) was added to thisbottle,
and after further incubation for 24 h, the cells and
mediumwereharvested. The cells showed advanced cytopathic effect and werebeginningto detachfrom the bottle when they were harvested. The labeled
HEp-2cells were mixed withRSvirus-infected
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503
2cellsfrom another Roux bottle cultured
simultane-ously, but withoutisotopeand withnormalmethionine
concentrationin the maintenance medium(25 ml).RS
viruscellularantigenwasproducedfrom this mixture
of labeled andunlabeled cellsasdescribedabove. The
media from the labeled and unlabeled bottles were
mixed, and RS viruswaspurifiedfrom thismixtureas
described below.
Purification of L-[5S]methionine-labeled RS
virus. The mixture of tissue culture maintenance
media referred to abovewasclarified (3,000 x g, 30
min,4°C), and 25mlwas centrifugedfor90 min at
26,000Xg(SW28.1rotor, BeckmanultracentrifugeL
2-65B) at4°Con adouble cushion ofsucrose. This
consisted of5mlof 65%(wt/vol)sucroseand5mlof
20% (wt/vol) sucrose made up in0.01MHEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid),
0.5 MNaCl, and0.01M
ethylenediaminetetraacetate
(pH 7.4). Thevirus-containingbandwasisolated,
di-luted to 10 ml with 0.01 M HEPES and 0.002 M
ethylenediaminetetraacetate (pH 7.4), layeredon a20 to 50% (wt/vol) continuous potassium tartrate
gra-dient, andcentrifugedfor18h(4°C) at22,000xg in
the rotorindicated above. Thisgradientwasmade up
in 0.01 MHEPES and0.02M
ethylenediaminetetra-acetate (pH 7.4). Twenty-eight fractions were
har-vested from thegradient.A
200-pd
sampleof thepeakfraction(showingmaximumcountsper minute anda
specific gravityof1.21g/ml)wasdiluted upto4ml in PBScontaining1% of theproteininhibitoraprotinin
(Midran, Novo Industries, Copenhagen, Denmark)
andcentrifugedfor1hat55,000 x g(SW60rotor)at
4°C. The sedimentwassuspendedin50
pl
ofelectro-phoresissample buffer (12) containing 2% aprotinin
beforebeingtestedby analyticalsodiumdodecyl
sul-fate(SDS)-PAGEasdescribedbelow.
SDS-PAGE.AnalyticalSDS-PAGE ofsolubilized
samples ofpurified RS virus and of solubilized gel
precipitates was carried out in vertical slab gels as
describedpreviously (12),with thefollowing
modifi-cations: the thickness of the gel was 1.5 mm, the
stackinggel contained 4% (wt/vol) acrylamide, and
theseparationgelcontained 10%(wt/vol) acrylamide.
The sample bufferwas supplementedwith 2% (wt/
vol) aprotinin; theelectrophoresiswascarriedoutfor
260 min with aconstant current of40mA.
Autora-diographywascarriedout at-70°Cfor2weekswith
KodakXRY-1film.
Preparation of the immunoglobulin fraction
from rabbit antisera and from humansera.
Im-munoglobulinfractionswereobtainedbythe method
describedbyHarboe andIngild (10).
Determination of concentrations of
immuno-globulinsolutions. Immunoglobulinconcentrations
weredeterminedbythemethoddescribedby
Bram-halletal.(3).
Examination of nasopharyngeal secretions
from children for cellscontaining RS virus
anti-gen.Nasopharyngealsecretionswereexaminedbythe
methods outlined by Gardner and McQuillin (6).
Washedcells fixedonordinary microscopeslideswere
incubated with anti-RS serum (bovineserum;
Well-come Research Laboratories, Beckenham, England)
at37°Cfor1h, followedby incubation (alsoat37°C)
for 45 min with fluorescein isothiocyanate-conjugated rabbit anti-bovine serum (Wellcome).
ELISA employed. Determination of IgG
anti-bodies in human sera was performed on polystyrene microtest plates (A/S Nunc, Denmark; code no. 2-62162) by employing the following reagents: (i) rabbit anti-RS virus precipitate I serum (anti-RSV-precip.I;
seebelow), immunoglobulinfraction containing 35 mg
ofimmunoglobulin per ml;(ii) RS virus cellular
anti-gen (or control antianti-gen) harvested in Tris-glycine buffer (pH 9) as described above; (iii) rabbit anti-human IgG, peroxidase conjugated (DAKO Immuno-globulins A/S; code no. P1090); (iv) bicarbonate buffer
(pH 9.6) (15 mM Na2CO3, 35 mM NaHCO3, 3 mM
NaN3); (v) washing solution (pH 7.2) (0.5 MNaCl, 2.7
mMKCl, 1.5 mM KH2PO4, 6.5 mM.JU2HPO4.2H20,
and 0.5 ml of Tween-20 added per 1,000 ml); (vi)
dilution buffer (pH 7.2) (same as washing solution,
with 5 gof bovine albumin
[Sigma
A8022, fractionV]
and10 ml of phenol red [0.1%, wt/vol] added per 1,000
ml); (vii) staining solution (pH 5) (34.7 mM citric acid,
66.7mMNa2HPO4.2H20, 500 mg of
1,2-phenylenedi-amine-dihydrochloride[Fluka], and 500
pl
of 30%H202added per 1,000 ml).
Serum dilutions of 1:100, 1:400, 1:1,600, and 1:6,400
were examined by the following procedure utiizing
the reagents described above. (i) The wells were coated
for 1 h with100,ul each of rabbit anti-RS serum diluted
1:5,000 in bicarbonate buffer.(üi)The plate was washed
four times for 1 min (with intervals of 1 min) in
washing solution. Between fillings the plate was tapped, upside down, against soft tissue. (iii) RSV antigen or control antigen diluted 1:100 in dilution
bufferwasadded to thewells (100,l per well)
over-night at 4°C. (iv) The plate was washed four times
again(see step ii). (v) Human serum samplesdiluted
inwashing solution were added to the plate for 1 > at
roomtemperature. (vi) The washing procedure a
repeated. (vii)Anti-humanIgG (reagent 3), diluted 1:
1,000 in dilution buffer, was added for 1 h at room
temperature(100
pl
perwell).(viii)Thewashingpro-cedure was repeated. (ix) Staining solution without
H202 and phenylenediamine was added to the plate
for 1 to 2 min. (x) Complete staining solution was
addedtotheplate (100
pl
perwell) for4min.(xi) Thereaction wasstopped by adding 150 yl of 2 N H2SO4
perwell. The resultswereread inaVitatronUniversal
photometer at472nm, butpositive or negative results
and increase or no increase of optical density (OD)
values between serum samples could usually be read by the naked eye. Definite increase of IgG antibody
concentrations between two serumsamples as
deter-mined by the ELISA described represents more than
adoubling of the OD values. The same positive human
standardserum wasrunonallplates. The small
(ap-proximately 5 to 10%) day-to-day variationoftheOD
values was corrected by comparing the day-to-day
results obtained with this positive serum.AUl serum
dilutions were tested with the control antigen also.
The final OD values of the positivesera were obtained
after subtraction of the values found with control
antigen. More than 90% ofthe OD values obtained
with serum dilutions of 1:400 and with control antigen
were less than 0.1, and background values for the
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plates (without antigen or serumadded) ranged from 0.005 to 0.015.
Titration of CF RS virus antibodies.
Comple-ment-fixing (CF) antibodytitration wasdone atthe
State Serum Institute in Copenhagen (by C.
Mor-dhorst) accordingto amethod described earlier(15).
TheantigenemployedwasmadefromRS virus (Long
strain)-infected GMK cell cultures.
HSVtype 1 reference antigen and antiserum
against HSVantigenno.11.Referenceantigenwas
prepared fromTritonX-100-solubiizedrabbit cornea
cells asdescribed earlier (25, 27). Antiserumagainst
theglycoprotein,antigenno. 11,wasraised inrabbits
asdescribed earlier(27, 29).
RESULTS
Production
ofimmunoprecipitates by
CIEP between antibodies
tosemipurified
RS
virus(anti-RSV-prim)
andRS
virus
an-tigen. Figure
1(A
andB)
shows the results ofCIEP in which
anti-RSV-prim
wasemployed
inboth
plates
in the second-dimensiongel.
RSvirus antigen was
employed
in the first-dimen-siongel
inFig.
1A, and controlantigen
wasemployed
inFig.
lB.The
antibodies
used in theseplates
weread-sorbed
withHEp-2 cells
beforeuse(6
x 107cells
perml ofserum for18hat4°C). The
antigens
used were cellular
antigens
sonicated in Tris-glycinebuffer
as outlined above. In thisfigure
and in
Fig.
2 anemptygel (without antigen
orserum) was interposed between the first- and
second-dimension
gels
toimprove
theprecipi-tatepatterns.
The
precipitate
markedRSV-precip.I,
whichcould be found
only
in theRSantigen
plate,
wasused for further immunization. This
precipitate
was cut outof thewet
gel,
and severalimmuni-zations with
precipitates
weregiven
intracuta-neously
torabbits asdescribed
above. Thean-tiserumobtained
by
thisprocedure
iscalled
anti-RSV-precip.I.
Characterization of rabbit
anti-RSV-pre-cip.I.
(i)
Lackof reaction
withRS virus
glycoproteins
inCIEP.
Figure
2 shows theprecipitate
obtainedby CIEP,
with the sameantigenasinFig. 1in thefirst-dimension
gel
andwith
anti-RSV-precip.I
antibodies in thesecond-dimensiongel; the
immunoglobulin
fractionwasadjusted
to aconcentration ofapproximately
30g/liter. Figure2BisanalogoustoFig. 2A, except that thelectin concanavalin A
(ConA)
wasin-corporated
inthefirst-dimensiongel.
ConAwasemployed
at a concentration known to absorbvirus antigens of glycoprotein nature (12, 28). Thesimilarity of the precipitates in Fig.2Aand
B demonstrates that the antibodies
employed
(anti-RSV-precip.I)
did notprecipitate outanyglycoproteins fromthe
antigen
when aconcen-FIG. 1. (A) CIEP of RS virusHEp-2cellular
anti-gen.First-dimension gel:60ilofthe antigen
prepa-ration was electrophoresed in 1.5-mm-thick 1%(wt/
vol) agarose (Litex Type HSA; Glostrup, Denmark)
in Tris-barbital buffer (pH8.6; ionicstrength 0.05): 36mMTris, 12 mM barbital, 3 mM NaN3, and 0.4
mMcalciumlactate.Currentwas 8V/cmfor90 min.
Second-dimensiongel: 1.3-mm-thick agarose gel as
describedabove, containingrabbit antibodies
(anti-RSV-prim) toRS virus. Anti-RSV-prim wasused as
theimmunoglobulin fraction at 15
jul/cm2
ofgel, as asolution(in 0.9oNaCl) containing20 gof
immuno-globulinperliter. Currentwas 2 V/cmofgelfor 18 h.
Electrophoreticbufferwasthe same as the gelbuffer.
StainingwaswithCoomassiebrilliantblue. This and
thefollowing CIEPexperiments shown were all
per-formed on 7-by-10-cm glass plates.An intermediate
gelsection (30 by 70by 1.4 mm) was interposed be-tweenthefirst- and second-dimensiongels. (B)This
gelisanalogoustothat in(A), except that the control
HEp-2cellular antigen was electrophoresed in the
first-dimensiongel.
tration ofConA of 60
jUg/cm2
wasemployed in thefirst-dimension gel. Whenantiserum
against the glycoprotein antigen, HSV antigen no. 11,wasaddedtothe second-dimensiongelandHSV
type1
reference
antigenwasaddedtothefirst-dimension gel, antigen no. 11 was precipitated
out (to the left in the intermediate gel in Fig. 2A). This precipitate was removed in Fig. 2B by the ConA andconsequently constituted a
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POLYPEPTIDES
ANTI-RSV-PRECiPi
ANTI-HSV-AG-1-(5jLCM2)
A
EMtPTY GEL
| 75
LJ
RSV-AG
Io
o7p
HSV-AG^'
.ANTI-RSV-PRECIPI
J(O
Oy/CM2)
ANTI-HSV-AG-11
(5pyICM2)
*375pL
RSV-AG
7pl
HSV-AG'oON-A
6OuG/i
A
ANTI-RSV-PREC-P
I(1OpLICM2,
I
~~~~~A
EMPTY GEs
>75DU
7)
R;V <zANTI-RSV-PRECTP I
(lCpl
/CM
B
e..
.
FIG. 2. (A)CIEPof RSvirusHEp-2cellular
anti-genand HSVtype1 reference antigen. First-dimen-siongel: 75IL oftheRS virusantigenand7,l ofthe
HSV antigen were electrophoresed.
Second-dimen-siongel: (a)rabbitanti-RSV-precip.Iata
concentra-tionof10
jl
of immunoglobulin fraction (adjustedto30g/liter in 0.15 M NaCl)per cm2 of gel and (b)
rabbit antibodies against HSV antigenno. 11 ata
concentrationof5pl of immunoglobulinfraction
(ad-justed to20g/liter in 0.15 MNaCl)per cm2. The
anionic detergent TritonX-100 was added at0.5%
(vol/vol)toboth thegel bufferand theelectrophoresis
buffertofacilitatetheprecipitation oftheHSV
anti-gen.Experimentalconditionswereotherwiseas
de-scribedfor Fig.1.(B)Thisgelisanalogoustothat in
(A)exceptthat ConA (60 pg/cm2) wasincorporated
in thefirst-dimension gel.
tivecontrolinthe
experiment.
(ii)
Theimmunogenic
potency inhumanRS virus infection of the
precipitated
anti-gen. The anti-RSV-precip.I immunoglobulins
were produced for diagnostic purposes. It was
thereforeimportanttoknowwhetherantibodies
to the antigen precipitated out by CIEP with
rabbit antibodies (as inFig. 2A)were also
pro-duced in human infections. This question was
evaluated by the CIEP-intermediate gel
tech-nique (1).
InFig. 3 thesameantibodies andantigenwere
employedasin Fig.2A. InFig.3A the
interme-diategelisempty; in Fig.3B and Cthe
immu-n
A,/V
j IGRSV-NEG d15pl/CMt2)
A~~~~~
-7F,L... RZ.,
-4-'I'j -'f
ANTi-R5-PRECiPIT
c
HUMAN IG
RS5 - nS
05vP /CM2J
s5.le - V-
-X
FIG. 3. CIEP ofRS virus HEp-2cellular antigen.
The RS virus antigenemployed in the first-dimension
gel and the RS virus antibodies employed in the
second-dimensiongelare thesameasdescribedfor
Fig. 2. Intermediate gel section: (A) empty; (B)
im-munoglobulin fraction of humanserum(without RS
virus antibodies) added at 15 ,ul ofa solution of
immunoglobulinin 0.15 MNaCl(10g/liter)per cm2;
(C) immunoglobulin fraction ofhuman serum
(con-taining RS virus antibodies) added at 15 1il qfa
solutionof immunoglobulin in 0.15M NaCl (10g/
liter)per cm2. Experimental conditionswere
other-wiseasdescribed for Fig. 1.
noglobulin fractions of human sera (15
pl/cm2)
were incorporated into this gel section. In Fig.
3B theimmunoglobulin fraction employed was
preparedfroma pool of10 humanserum
sam-k
.J
4-...
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14,1981
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pleswithoutantibodiestoRS virus (checked by CF, IF, and ELISAtechniques). In Fig. 3C the immunoglobulin fraction added tothe interme-diate
gel
wasprepared
fromapool
of10human serumsamples
with intermediate to highanti-body
titers to virus. Theimmunoglobulin
con-centrations of these pools were
adjusted
to ap-proximately 10 g/liter. All 20 sera were from children (1to3yearsold)
withlowerrespiratory
disease
(pneumonitis
orbronchitis).
A compari-son ofFig.
3A andFig.
3C reveals that the antigen migration wasinhibited
intheinterme-diate gel
inFig. 3C (containing
humananti-bodiesto
RS
virus),
whereas no inhibition was seeninFig.
3B.Thesefindings strongly
indicate that the antigensreacting
inprecipitation
with rabbitanti-RSV-precip.I antibodies
arealso im-munogenicduring
RS virusinfections
in chil-dren.(iii)
Antibody
titers measuredby
plaque
neutralization
and IFtechnique.
When theimmunoglobulin
fraction(30
g/liter)
fromanti-RSV-precip.I
serum wastestedby plaque
reduc-tion in Vero
cell
monolayers,
the titerwasfoundtobeless than1:50.For
comparison,
thepositive
and
negative
human serumpools (described
above) showed
plaque
reduction titers ofap-proximately
1:3,200and
less than 1:50,respec-tively.
The titer of the
immunoglobulin fraction
ofanti-RSV-precip.I
serum(30g/liter)
determinedby the IF
technique
was 1:320, whereas theimmunoglobulin
fractions of the positive andnegative
humanserumpools (10 g/liter)
were 1:160andless than 1:10,
respectively.
Anti-RSV-precip.I
antibodies gave afine
granular
cyto-plasmic
fluorescence
in theHEp-2 cells
used in the IF assay.(iv) Determination of
MW ofRS virus
polypeptides
precipitated by
anti-RSV-pre-cip.I antibodies.
Polypeptide
MWsweredeter-mined
by
PAGE in vertical slabgels
withL-[35S]
methionine-labeled RS virus and
precipitates
formed between
L-[35S]methionine-labeled
RS viruscellular
antigen
andanti-RS-precip.I
PartA andpart Bof theprecipitate shown inFig. 4
were cut out of the wet
gel
and solubilized inSDS-containing
Tris buffer beforebeing
tested.Anti-RSV-precip.I
didnotproduce precipitates
with cellular
antigen
produced from uninfected controlHEp-2
cell cultures. Thereforeprecipi-tates
produced
withcontrolantigen
fromL-[35S]
methionine-labeled control cultures couldnotbe tested. The
gels
werecalibrated withamixture of sixproteins (MW
14,400to94,000)
run atthesametime (12).Figure5shows
autoradiography
ofsolubilized RS virus
(track
c). Thesamples,
whichwere
layered
onthegel
corresponding toFIG. 4. CIEP of L-[35S]methionine-labeled RS vi-rus cellular antigen. First-dimension gel: 75 ,ul of the antigenicpreparation waselectrophoresed. Second-dimension gel: anti-RSV-precip.I employed in the same way as described for Fig. 2 and 3. Experimental conditions were otherwise as described for Fig. 1.
<;e 4;
am. im
FIG. 5. SDS-PAGE performed with
L-[35S]methi-onine-labeled RS virus and gel precipitates
consist-ing of
L-[35S]methionine-labeled
RS virus cellularantigen andanti-RSV-precip.L (a) solubilized
puri-fied RS virus; (b) solubilizedprecipitate,part A of
precipitate shown in Fig. 4;(c) solubilizedprecipitate,
part BofprecipitateshowninFig.4.
tracks a,
b,
and c, contained approximately 100,000, 40,000, and 40,000 cpm,respectively.
Several
of the polypeptides shown in track awerealso foundintracks b andc. Apolypeptide ofMW 28,000 was seen in trackcand in tracka
butwasscarcely visible in track b.
J. CLIN. MICROBIOL.
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Determination of IgG antibodies in
hu-man RS virus infections
by
use ofanti-RSV-precip.I
as capture antibodiesin
ELISA. Serumpairsfrom 147 infants and chil-dren admittedto
hospital
for lowerrespiratorytractdiseaseweretested forRSvirus IgG
anti-bodies by ELISA. The technique employed is
described indetail above. The majority ofthe
patientswereless than 2yearsold. In 46 of the
patients the ELISA showed increases of RS antibodyconcentration. In Table 1 the
results
ofvarious diagnostictestsperfonned on seraand
samples of nasopharyngealsecretions from these
46patientsaresummarized.In 26 of the patients
RS
virus could be found innasopharyngeal se-cretions, either bydirect immunofluorescent ex-aminationofepithelialcells (present inthese-cretions)orbyinoculationofasmall amount of
secretioninto each of twoHEp-2tube cultures.
Thirty-twoofthepairedserumsamplesshowed
titer increasesofRSvirusIgGantibodies when
examinedbythe indirect IFtechnique.Ineight patients, only ELISAgave adefinite diagnosis
of RS virus infection. No patients with titer
TABLE 1. Forty-sixserum pairs showingincrease in
OD when tested for RS virus IgG antibodies by
ELISA, compared with otherdiagnostic tests
No. of serum pairs showing:
Notier
RessTit;eri Serocon-
increase
Results
Ttri-version'
orsero-creaseby b CF
conver-CF(24.9)b
(3Y3.5)b
sion byCF (36.3)b
ODincrease by 28d 8e lof
ELISA
RS virus in secre- 17 5 4
tion(by IFor
isolation)
Titer increase by 28 3 1
IF
No RSV in secre- 0 3 5
tion, notiter
in-creaseby IF
'Range of OD values for first sera (dilution 1:400
examined) in 46 serum pairs, 0.000 to 0.799; mean,
0.174. Range ofOD values for second sera (dilution 1:400 examined) in 46 serum pairs, 0.261 to 1.968; mean, 0.848.
bMean numberofdaysbetween blood samples.
C<4
to4.d Mean ageof children inthe group, 18.9months.
'Meanage, 13.9 months.
fMeanage, 13.1 months.
increases
byother
tests (CF or IF techniques)werenegative by
ELISA.
DISCUSSION
Vertical slab
gel PAGE of precipitates
pro-duced
betweenRS virus HEp-2 cellular antigen
and rabbit
anti-RSV-precip.I (raised against
gel
precipitates
designated
RSV-precip.I) showed
that
anti-RSV-precip.I reacted
inthe CIEP
as-saywith six
polypeptides
whichhad
MWs rang-ing from approximately 28,000 toapproximately
84,000.
Wunner and
Pringle (32) and Cash
etal.
(4,
5)have
described
sevenRS virus
polypep-tides (MW
25,000 to48,000)prepared
from
RS
virus grown
in
BSC-1cells.
These authorselai
that the three predominant polypeptides
were aglycoprotein of
MW 48,000(VGP
48), anucleo-capside-polypeptide of
MW 41,000(VP 41),and
a
polypeptide of
MW 27,000(VP 27).Levine (13)
examined
RS virus produced
inHeLa
cells,
found (by
PAGE)
sevenRS virus
polypeptides
of
MW 22,000 to 79,000,and described three of
these
polypeptides (MW
22,000, 56,000,79,000)
as
glycoproteins. However, in
a recentpublica-tion, Peeples and Levine (18) modified their
PAGE
techniqueand obtained
MWsfor
the twolarge glycoproteins of
49,000and
90,000.These
authors furthermore reported that the dominant
nucleocapsid polypeptide has
aMW
of
44,000(18). Ueba
(24)advocated that RS virus
precip-itated
byPEG
6,000should be filtered through
a
Bio-Rad
Biogel
A 15-mcolumn before further
purification
on sucrosegradients. This author
(24) found (by
PAGE)
sevenRS virus
polypep-tides
of MW25,000 to 75,000.In
the present paper, Tris-glycine bufferwith-out
detergent
wasemployed for the
preparation
of the antigen used in CIEP
techniques. When
ConA
wasadded
tothe
first-dimension
gel
inCIEP,
noinhibiting effect
wasobserved
onthe
antigens precipitated
by anti-RSV-precip.I but
ConA did inhibit the
precipitation
of
HSV
anti-gen no. 11,
which
wasincluded in the
sameplate
as a
positive control. This antigen is
aglycopro-tein, according
toearlier
findings (27).
Further-more,
anti-RSV-precip.I
did
notneutralize
RS
virus when assayed in plaque-reduction
tests.These
findings do
not supportthe
possibility
that
anti-RSV-precip.I is able
to reactwith
RS
virus surfaceglycoproteins.
Levine (13) and
Peeples
and Levine (18)ex-amined
RS
viruspolypeptides by different
PAGE techniques and showed that the migra-tion of the
glycoproteins
in thediscontinuous
buffer
system ofLaemmli
wasdifferent
from themigration
of theglycoproteins observed
when aneutral buffer
system wasemployed.
Thebuffer
system of Laemmli was also
employed
in theVOL.14,1981
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present investigation, but with some modifica-tions asdescribed above.
Tyrrell
etal. (23)ex-amined measles virus
polypeptides by
SDS-PAGE and found that theaddition
of the pro-teaseinhibitoraprotinin reduced the breakdown ofsomeof thepolypeptides
during processing
of the material. In the present report,aprotinin
was added both to the
gel
where labeled RS virusprecipitateswereproduced
andtothesam-ple buffer used for the
preparation
of thesam-ples for
SDS-PAGE.
Proteaseinhibitors
havenot been
employed
inpreviously published
in-vestigations
concerning
RS viruspolypeptides.
Differences in PAGE
techniques
employed
(as mentionedabove)
and otherdifferences
inmeth-ods
(e.g.,
differentcell cultures
and the use ofactinomycin
Dincell
culture mediumby
someinvestigators) can
probably
explain
thedifferent
MWs for RS virus
polypeptides published
by
different
groups(13, 18, 24,
32).
The MWfor
RSvirus
polypeptides
determined withgel
precipi-tates, as
reported
in the present paper, alsodiffers
slightly
from the MWpreviously
pub-lished. MWs
for
thesepolypeptides (shown
inFig.
5,tracks b and c, in thisreport)
fall into the
samerangeasthe MWsreported by
Peeples
and Levine (18) andby
Ueba(24).
Thelimited
num-ber of PAGE
experiments
presented
inthisar-ticle
isnotsufficient
for anyidentification
of theRS virus
polypeptides
found. Theseexperiments
nevertheless show that the
antiprecipitate
hy-perimmuneserum
produced
(anti-RSV-precip.I)
precipitates
only polypeptides
fromRS
virus-infected
HEp-2
cells which are also present inpurified
RS virus.CIEP
techniques
using
humanIgG (with
andwithout
RS
virusantibodies)
addedto interme-diategel
sections have shown that thepolypep-tides,
oratleast
some ofthem,
precipitated by
anti-RSV-precip.I
areimmunogenic
during RS
virus infections in children. This statement is
supported
by
the greatsensitivity
andspecificity
of ELISA when these
polypeptides
areemployed
as
antigens
formeasuring
RS virusIgG
anti-bodies in infants and small
children.
Gerna et al. (7) measuredRS virus
IgG
anti-body
by
animmunoperoxidase technique
inwhich fixed HEp-2 cells or human
embryonic
lung fibroblasts in tissue culture
microplates
were
employed
asantigen.
They
found that theimmunoperoxidase technique was more sensi-tive than theCF
technique
for thedetection
ofantibody
rises, especially
in 2- to 4-month-oldpatients.
Richardsonetal.
(21)
measuredRS antibodiesby
ELISAusing
asuspension
offreeze-thawed RS virus-infectedHEp-2
orGMKcells(in tissue culture maintenancemedium)
asantigen
ab-sorbeddirectly to the plate in carbonate buffer (pH 9.8). They diluted the serum samples in PBS-Tween, supplemented with 1% fetal bovine serum and19% of an uninfectedcell suspension, toreduce nonspecific binding of the test serum (21). They found that the ELISA technique was capable ofdetectingserological responses, espe-cially in younginfants 1to 3 monthsold where these responses could bedemonstrated only with difficulty by use of either CF or plaque-reduction techniques (21). These authors nevertheless could not demonstrate increased sensitivity of ELISA (as compared with the other techniques mentioned) in children more than 7months old (21). In the present report, RS virus IgG anti-bodies have been determined by ELISA with morewell-defined RSV virus antibodies and an-tigens employed as first and second layers on microtest plates. When this methodology was employed, ELISA also showed greater sensitiv-ity than other diagnostic tests (serology by CF or IFtechniques,
nasopharyngeal
secretions ex-amined by directimmunofluorescence
orby vi-rus culture) in children more than 1 year old. Furthermore, this technique showed good repro-ducibility, low background OD values, and low OD values with control antigen. The use of specific antisera produced against viral antigens purified by CIEP techniques as capture anti-bodies inELISA
offers great possibilities of the attainment of more accurate information by di-agnosticvirological serology.
ACKNOWLEDGMENTS
This work was supported by a grant from the Danish Medical Research Council.
WethankC.Mordhorst,theState SerumInstitute, Copen-hagen,forperformingCF tests for RSvirusantibodies, and MereteEskildsen forexcellent technical assistance.
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