0095-1137/81/050957-06$02.00/0
Comparison
of
Fluorescent-Antibody, Neutralizing-Antibody,
and
Complement-Enhanced
Neutralizing-Antibody
Assays
for
Detection of
Serum
Antibody
to
Respiratory Syncytial Virus
TEJ N.KAUL,* ROBERT C. WELLIVER, AND PEARAYL. OGRA
Departments ofPediatrics andMicrobiology,State University ofNew YorkatBuffalo,andDivisionof
Infectious Diseases,
Children's
Hospital of Buffalo, Buffalo,
NewYork14222Received 15 October 1980/Accepted 26 January 1981
Acomparison of threeassaysfor the detection ofserumantibodytorespiratory syncytial virus (RSV)wascarriedouton47serumsamples obtainedsequentially
from infants andyoungchildren with RSV infection.Neutralizing-antibody(NA)
activitywasdeterminedbyasemimicromethod ofplaque reduction.
Complement-enhanced NAactivitywasdeterminedby the addition of guineapig complement
to NAassays. RSV antibodyresponses in immunoglobulin G, immunoglobulin
M, andimmunoglobulin A classesweredetermined byusing indirect immunoflu-orescencetechniques for thefluorescent-antibody(FAb) assay.AntibodytoRSV
wasdetectable by all three techniquesasearlyas4days after theonsetofillness. Atall phases of illness, titers obtained bycomplement-enhancedNAassays were
significantlygreaterthanthoseobtainedby NAorFAbassays (P<0.01). RSV-FAb titers determinedintheimmunoglobulin G class correlated well with those determined by complement-enhanced NAorNA assays. The data suggestthat the FAbassayfor detection ofRSVantibody inserumissomewhat less sensitive
but also less laborious andmorerapid than NAassays.
Serological
studies ofrespiratory syncytial
vi-rus(RSV) infectionarehinderedby the fact that antibody responsesafter RSV infection are di-minished in infants less than 6 months ofage (3), theagegroup inwhichsevereillness dueto
RSVoccurs most
commonly (17).
Theneutral-izing-antibody
(NA)
assayis the mostaccuratemethod currently used for detection of serum
antibodytoRSV but lacks
sensitivity
ininfantslessthan6monthsof age withRSV infection(3)
and is too laborious fortesting
large
numbers ofspecimens.
Recently,
wereported
ourexperi-ences with indirect immunofluorescence
tech-niquesfor the
fluorescent-antibody
(FAb)assay for the detection ofRSV antibody in immuno-globulin G(IgG),
IgM, and IgA classesin both serum (20) andnasopharyngeal
secretions(R. C. Welliver,Proceedings
ofthe 85thRossConfer-ence on Pediatric Research: Mucosal and Im-muneSystemin Health andDisease,inpress).
Other studies have demonstrated the appear-anceof NAactivitywhichcould be enhancedby
the addition offreshguineapigcomplement (C')
in the serum ofpatients recovering from infec-tion with severaldifferent viral agents (7, 8, 14, 18, 19,21). Potashetal. (18) reportedthat
neu-tralizing activity
infresh,
unheatedserumsam-plescollected fromsubjectsimmunized with
in-activated RSV vaccine could be markedly
re-ducedby heatinactivation of the samples. The neutralizing activity could be restored by
addi-tion offresh, nonimmune guinea pig or rabbit serum.
The current study was undertaken to deter-mine whether addition of C' enhances neutral-izing activity in the serum of patients with nat-ural RSV infection. Moreover, an attempt was made tolearniftheresults oftheFAb assay for
the detection of RSV antibody correlate well
with those of the NA orcomplement-enhanced
NA (CENA) assays.
MATERIALS AND METHODS
Studygroup. Forty-sevenserumspecimenswere
drawn,atvariousintervalsafterthe onsetofprimary
RSVinfection, from children who varied inagefrom
1to 12months.Thepatientswerediagnosedashaving
upperrespiratory illnessonly,pneumonia, bronchioli-tis,orasthma.Allpatientswereenrolled inaseparate ongoing study of respiratory illness in childhood. All
childrenwerecarefullymonitored and weretherefore
knowntobeundergoingprimaryinfectionwithRSV. At the time of initialenrollment,parents of the
partic-ipants were informed of the goals and risks of the study and of the fact that specimens obtainedmight be used laterforotherstudies of theimmuneresponse toRSV.Asigned statementofinformed consent was obtained at that time.RSV infection was confirmed
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by identification of viral antigen in nasopharyngeal
secretionsbyindirectimmunofluorescencetechniques
(11) and later by recovery of virusby tissue culture infectivity.
FAbtest.FAb titers weredetermined intheIgG,
IgM, and IgA classes as previously described (6).
Briefly, monolayersofHEp-2 cellswereinfected with astock strain of RSV and fixed withacetone toglass
slides. Cells wereincubated with serialtwofold dilu-tions ofserum for 30 min. Afterwashing, slideswere
labeled withfluorescein-conjugated goatanti-human IgG, IgM, orIgA globulin (Meloy, Springfield, Va.). Slides were washed and examined by fluorescence microscopy. Controls consisted of uninfected HEp-2
cellsand slides incubated with saline instead ofserum.
FAb assays under these conditions did notresult in the appearanceof fluorescenceinthe controls.
Neutralizationtests. NA assayswerecarriedout
with minor modification of a semimicromethod of plaquereduction describedpreviously (8, 13).Briefly,
2 x I05 HEp-2 cells were seeded into each well of plastic tissue culture plates (Linbro, New Haven,
Conn.) and incubated inahumidC02incubator over-night. Heat-inactivated serum wasdilutedtwofold in
growth medium in duplicate.Each0.1 mlof diluted
serum wasincubated with an equalvolume ofstock virus diluted to contain 100 to 150 plaque-forming
units per ml. C' (GIBCOLaboratories, GrandIsland, N.Y.), whichhad been titrated and dilutedtocontain 8 U/0.1 ml, wasadded to oneofthe pairs ofserum dilutions, and 0.1 ml of balanced salt solution was
addedtothe otherserumdilutionas acontrol.
Serum-virus mixtures with and without C'wereincubatedat roomtemperature for 1 h,after which 0.1 mlof each mixture was adsorbed onto duplicate wells oftissue culturecellmonolayers.Plateswerethen incubatedat 37°Cfor2hbefore removal of the inocula andrinsing
of cells with phosphate-buffered saline. Cell layers
were overlayedwith1 mlof1% agar in mediumwith 2.5%chickenserum(GIBCO).Platesweresealed and incubatedat37°C for72h,after whichanadditional overlaywasadded.Plateswereincubated4 moredays
and then fixed in 10% Formalin. After removal of the agar,thecellswerestained with 1%crystalviolet.NA and CENA titersweretakenastheserumdilutionsat
which a 60% reduction in plaque-forming units was
observed.
Controlsconsisted of virusdilutedinmedium
with-out serum, virus with C' without serum, and
serum-virus mixturestowhich heatedC'wasadded. Addition ofheated C' didnotresult inanincrease in titersas determinedbystandard NAassays. The incubation of C' and virus without serumdidnotresult ina reduc-tion inplaque-formingunits.
Statistical analysis.Regressionlinesweredrawn by the method of least squares, and the coefficients of correlation(rvalues)werecalculatedtodetermine the degree of statisticalsignificance.
RESULTS
Kinetics of FAb, NA, and CENA
re-sponses.AcomparisonofFAb, NA,andCENA responses is shown inFig. 1. Eachpointonthe individualcurvesrepresents thegeometricmean
2048r
1024 512 256 128 64 32 16 8 4
<4
0-4 5-10 11-28 35-56 60-90 o90
Days After Infection
FIG. 1. Kinetics of serum antibody response to RSV as determined by IgG-specific FAb (A A), IgA-specific FAb (A), IgM-,specific FAb (A----A), NA (-), andCENA(O) assays. Each point represents the geometric mean titer (indicated by cross bars) of atleast six samples.
(indicated by cross bars) of titers obtained from
at least six samples. RSV antibody, as deter-mined by the FAbassaywith IgG,was present at a geometric mean titer of 2.8 in the first 4 days ofillness. An increase in the IgG-specific FAb (IgG-FAb) titer was evident by 5 to 10 days after the onset of illness, and peak titers (111 to 138) wereattainedbetweenIland 56 daysafter the onset of illness. NA titers were two- to four-fold greaterthan IgG-FAb titers during the first
28daysofillness, after whichtitersobtained by
the NAassay orby the FAb assay for IgG were virtually identical. CENA titers were 2- to 6-fold and4- to30-fold greater than NA and IgG-FAb titers, respectively, at all phases after the onset ofillness.CENA titers reached a peak geometric
mean titer of 1,023 at 11 to 28 days postillness anddecreased to a geometric mean value of 99 by 90days postillness. Enhancement ofNA by C' was most prominent in the first 10 days of
ilness, atwhich time CENA titers were sixfold greaterthanNAtiters. From 35 days postillness until the end of the follow-up period, CENA titers exceeded NA and IgG-FAb titers by two
to fourfold. The differences between mean CENA titers and NAor FAbtiters were statis-ticallysignificantat all intervals, asdetermined bytheStudentttest (P<0.01).
ComparisonofCENA, NA,andFAb titers
at each serum dilution. Titers ofRSV anti-body obtained by eachneutralizationtechnique
areplotted against titers obtained byFAbassays in the three immunoglobulin classes at each serum dilution in Fig. 2and 3.Lines of identity
are superimposed to emphasize that titers
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2048r 1024
512 256 128 64 32 16 8 4 <4
00 0 a b O
- ~~~o.*
- & & * oe
-~& /
- oo / * e
- so oWithComplement
*WithoutComplement
<4 4 8 16 32 64 128 256 512 1024
RSV-IgGAntibodyTites(FAb)
FIG. 2. Comparison oftiters obtained with IgG-specific FAb assays for RSVantibody with those
obtained with NA(-)and CENA(O)assays.Lineof identity is superimposed.
tainedbyneutralizationassayswerealmost
uni-formly higherthan those obtained by FAb as-says for RSV-IgG, RSV-IgM, and RSV-IgA at
eachserumdilutiontested.
Correlationoftitersobtained by comple-ment-enhanced neutralization with IgG-, IgM-, and IgA-FAb titers. Regression lines
indicating the correlation of RSV-FAb titers in each immunoglobulin class tested with titers
obtained by complement-enhanced
neutraliza-tion are shown in Fig. 4. Since the plotting of serumdilutionsasshown inFig.2and 3 resulted
inanexponential scalewhichwasinappropriate
for the plotting ofregressionlines, the scale in Fig.4isalinearoneinwhich eachincrementon
the ordinateand abscissarepresents a
numeri-callyequal increasein antibodytiter.Slopesof theregressionlines indicate that theNAassay
results correlated best with FAb assay results
for RSV-IgG, less well forRSV-IgM, and least for RSV-IgA. However, calculation of
correla-tion coefficients for each regression line indi-cated a statistically significant (each r value,
>0.474; each P value, < 0.001) correlation of
FAb titers in each immunoglobulin class with titers obtained by complement-enhanced
neu-tralization.
Correlation ofCENA, NA, and IgG-FAb titers in seronegative to low-titer speci-mens.Inthe presentstudy,noserumspecimens wereseronegative forantibody when tested by
NA or CENA assays. The serum samples of
three patients with RSV infection (patients 1
through 3, Table 1) were seronegative by the
IgG-FAbassayandhad low titers ofNA.
Addi-RSV F-Ab IgATiters
00 83 Co
(% O O
a O
* .
co 00 »
"
0 00 Co
a/.
B
<4 4 8 16 32 64 128 256 512
RSV F Ab1gM Titers
FIG. 3. Comparison oftiters obtained with IgA-specific (A) andIgM-specific (B) FAbassaysforRSV
antibodywiththoseobtained with NA(-)and CENA
(O)assays.Linesofidentityaresuperimposed.
tionofC'increased the detectable NA titer2-to
16-fold. Incontrast, the serum samples of four
patientsknowntobeundergoingtheirfirst epi-sode of respiratory illness and known tobe in-fectedwith avirus other than RSV (patients4
through 7, Table 1) were also seronegative for RSV by the IgG-FAb assay and had low titers
of NA. Addition of C' did not result in an in-creasein theantibody titer in these patients.
DISCUSSION
The FAb assayhas been used inour
labora-tory (20; R.C. Welliver, in press) and by other researchers (15) for thedetection of RSV
anti-..2
qà-
ISI-.-N
1t1h 13,
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TABLE 1. NA and CENA titers in serum specimens seronegative for RSV IgG-FAbantibody
FAbtiterin Foldincrease
Patient no. IgG class NA titer CENA titer in NA titer by Nature of disease addition of C'
1 <4 4 8 2 Primary RSV infection
2 <4 4 16 4 Primary RSVinfection
3 <4 16 256 16 Primary RSVinfection
4 <4 4 4 0 Non-RSV infection
5 <4 4 4 0 Non-RSVinfection
6 <4 4 4 0 Non-RSVinfection
7 <4 4 4 0 Non-RSV infection
800_
z
'<- 700
600
500
400
o0 {
400 200 300 400 500 1200
RSV- FAb Titers
FIG. 4. Regression Unesdrawntoshow the
corre-lation of CENA titers with FAb titers in various
immunoglobulinclasses.Symbols:O,IgG-FAbtiters;
A, IgM-FAb titers; and A, IgA-FAb titers. Each r
value, :0.474,each Pvalue,<0.01. The ordinate and
abscissascaleislinearlyadjustedsothateach
incre-mentrepresents anumerically equalincrease in se-rumdilution.
body in serum and secretions. McIntosh and associates (15) studied 19serumspecimensand
foundanexcellent correlation betweenantibody
titers determinedbyNA assays (withoutadded
C')andby IgG-FAbassays.However,the results
of NAassaysforRSVantibodyin secretions did
not correlate well with the results ofIgA-FAb
assays for the detection of secretory antibody.
We undertook the current study to determine the correlation of titers of RSVantibodyinIgG, IgM, andIgA classes asmeasured by the FAb
assaywith the titers obtainedbyNAassayswith and without added C' atvarious intervals after natural infection. This study demonstrated a
goodlinear correlation(P<0.001)of theresults
ofIgG-FAbassayswith those of NA andCENA
assays.Thecorrelation of results of IgM-FAb or IgA-FAb assays with those of NA and CENA assays wasless direct, but still statistically sig-nificant. In fact, the use of IgG-FAb assays re-sulted indetection of titers which were identical
to those determined by NA assays in samples taken after the first month of illness. We believe that titers of NA observed in the first month of illness were greater than IgG-FAb titers because of the contribution of NA in the IgM class. Although we could not confirm it by separation ofthe IgM fraction of our samples on sucrose gradients because of limited sample volume, other studies (9) have shown that the 19S frac-tion of antibody developing in rabbits after vac-cination with herpes simplex virus contributes
tothe total amount of specific NA formed. The CENA assay appeared to be more sensitive than the IgG-FAb assay for the detection of RSV antibody. Nevertheless, the results of IgG-FAb assays correlated well with those of CENA as-says;also, the FAb assay was technicallysimpler toperform, and results of FAb assays were avail-able roughly 7 days earlier than those of NA or
CENAassays.Inaddition,the FAb assaycanbe usedfor thedetectionofRSV-IgM antibody and therefore may be useful in the early serological diagnosis of RSV infection (20). Finally, the ability to detect RSV antibody in the ilS IgA
class (15; R. C. Welliver, in press) makes this technique useful in the study of the secretory antibody response to RSV infection. The
appar-ent presence ofnon-antibody neutralizing sub-stances in secretions (15) may prohibit the use
of NA or CENA assays for the study of RSV secretoryantibody responses.
The current study also confirmsprevious
re-portsthat theaddition ofcomplementto stand-ardNAassaysmarkedly increases the sensitivity of such assays for the detectionof antibody to
RSV (1, 2, 18) aswellasmany other viruses (7, 8, 14, 19, 21). Potash and associates (18) found thatheating convalescentserumfromrecipients
ofRSV vaccine for 30 minat 56°C reduced the titer of NA and that the neutralizing activity could be restored by the addition of unheated
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rabbitorguinea pigserum. However, the
num-ber ofseratestedwasnotspecifiedinthe report. Buynak et al. (2) compared the results of NA
assayswithand withoutthe addition of fresh C'
in nine convalescent serum samples also
ob-tained from RSV vaccine recipients. The authors stated that the results of these nine selected specimens reflected the results of a more
ex-tended study. Enhancement of antibody titer after the additionofC'rangedfrom 2- to 32-fôld.
Finally, Baughman and associates (1) added C'
topooled ferret, guinea pig,and horsesera.NA
activity of ferret andguinea pig (butnothorse)
sera was enhanced by the addition of C'. The
currentstudy demonstrated that,atall intervals after natural infection, CENA titers were two-tosixfold greaterthan NA titers. Enhancement
of NAactivity by C'wasmostpronouncedin the
first 10 days after theonsetof illness. Inasimilar
fashion, Takabayashi and McIntosh (19) found that C' enhancementofNAactivity against
vac-cinia viruswasgreaterinseraobtained4weeks
after vaccination thaninseraobtained 6 months
after vaccination, whereas Hampar et al. (9) showed thatsera obtainedshortly after
immu-nization of rabbits with herpesvirus
demon-strated NAactivitywhich could beenhancedby the addition ofC',althoughNAactivityinsera
obtained later could be enhanced to a much
lesser degree by the addition of C'. Thereason
that addition of C' has suchavariableeffecton
NA activity in sera drawn at variousintervals
after infection is unknown. Hampar et al. (9) speculatedthat differentsubclassesofIgG anti-body, with differingpropensities for fixation of
C', werepresent atvarious intervals after
anti-genicexposure.
In thecurrentstudy,serumsamples obtained
fromfourpatients (4 through 7,Table1)known tohavenoprevious infectionwithRSV had NA
andCENA titersof 4.Since thesepatientswere younginfants,theobserved titersprobably
rep-resented maternal antibody acquired
transpla-centallyseveralmonthsearlier. Antibody titers
in these fourpatientswerenotenhancedbythe
additionof C'. The results obtained in thissmall number ofpatientssuggestthat theaddition of C'toNAassays maybeauseful test for
distin-guishing NA which is present as a result of
infection from that which isacquired
transpla-centally.
Dulbecco and associates (5) demonstrated that incubationof Westernequine encephalitis virusorpoliovirus with specific neutralizing
an-tisera did nottotally inactivate thevirus even
whenhigh concentrationsof antiserawereused.
Theresults of theirstudies indicatedthat virus remained active and infectious in the form of
virus-antibody complexes. Addition offresh
rab-bit serum to thevirus-antibody mixture
gener-allyresultedin a completereductioninthe titer ofinfectious virus. The authors suggested that the addition of fresh serum supplied certain
cofactorsnecessary for completeneutralization.
The results of the current study and others suggest thatthe factor supplied by fresh guinea pig serum was probably C'.
The persistence of infectious virus antibody complexes in vitro, as well as the kinetics of
formationand dissociation of these complexes, have been reviewed previously (14). In vivo,
infectious herpes simplex virus-antibody
com-plexes have been demonstratedin fluid obtained
fromvesicular lesions of humans with recurrent
herpes labialis (4). Poliovirus-antibody
com-plexes have been recovered from human feces fromwhich virus could beeluted by incubation of complexes at low pH (12). The recovery of
RSV from secretions containing specific
anti-body to the virus has been suggested as dem-onstrating the existence of virus-antibody
com-plexesinsecretionsobtainedafter RSV infection (15). It is well known thatinfection with RSV occurscommonly despite the presence of
trans-placentally-acquiredNA or serum andsecretory antibodyacquired by previous infection (10, 16).
Failure
to fixcomplementto suchvirus-antibodycomplexes, if theyoccur invivo, may be a
poten-tialmechanism by whichreinfections with RSV occur despite the presence of preexisting neu-tralizing serum or secretory antibody.
ACKNOWLEDGMENT
We are grateful to Frank J. Cerny for his help in the statistical analysis of the data.
This workwassupported by Public Health Servicegrants AI-15939 from the National Institute of Allergy and Infectious Diseasesand HL-21819 from theNational Heart,Lung,and Blood Institute.
LITERATURE CITED
1. Baughman,R.H.,J.D. Feneters,G. S. Marquis, and J. C. Holper. 1968. Effect ofcomplement andviral filtrationontheneutralization of respiratory syncytial virus. Apple.Microbiol. 16:1076-1080.
2. Buynak, E. B., R. E. Weibel, A.J. Carlson,A. A. Melean, and M. R. Hilleman.1979. Further investi-gation of live respiratory syncytialvirusvaccine admin-isteredparenterally. Proc. Soc. Exp. Biol. Med. 160: 272-277.
3. Chanock,R.M., H. W. Kim, and A. J.Vargosko.1961. Respiratory syncytial virus: virus recoveryand other observations during 1960 outbreak of bronchiolitis,
pneumonia,and minorrespiratorydiseases inchildren. J. Am. Med. Assoc. 176:647-653.
4. Daniels, C. A.,S.G. LeGoff,and A. L.Notkins.1975. Sheddingofinfectiousvirus/antibody complexesfrom vesicular lesions ofpatients with recurrentherpes labi-alis.Lancetii:524-528.
5. Dulbecco, R.,M.Vogt,and A. G.R.Strickland.1956. Astudyof the basic aspectsofneutralization of two
VOL.13,1981
on February 7, 2020 by guest
http://jcm.asm.org/
animal viruses, Western equine encephalitis virus and poliomyelitisvirus.Virology2:162-205.
6. Gardner, P. S., and J. MeQuillin (ed.). 1974. Rapid virus diagnosis. Application of immunofluorescence, p. 103-117.Butterworths,London.
7. Graham,B.J.,Y.Minamishima,G. R.Dressman,H. G.Haines,and M.Benyish-Melnick.1971. Comple-ment-requiring neutralization antibodies in hyperim-mune sera tohumancytomegalovirus.J.Immunol. 107: 1618-1630.
8. Grose, C.,J. B. Edmond, andP. A. Brunell. 1979. Complement enhancedneutralizing antibodyresponse tovaricella-zostervirus. J. Infect. Dis. 193:432-437. 9. Hampar, B., A. L. Notkins, M. Mage, and M. A.
Keehan. 1968. Heterogeneityinthe properties of 7S and 19S rabbitneutralizingantibodies toherpes simplex virus. J. Immunol. 100:586-593.
10. Johnson, K. M., H. H.Bloom,M. A.Mufson,and R. M. Chanock. 1962. Natural reinfection of adults by respiratory syncytial virus: possible relation to mild upperrespiratorydisease. N.Engl.J.Med. 267:68-72. 11. Kaul,A.,R.Scott,M.Gallagher,M.Scott,J.Clement,
andP. L.Ogra. 1978.Respiratory syncytialvirus in-fection:rapid diagnosis byuseofindirect immunofluo-rescence.Am. J. Dis. Child. 132:1088-1090.
12. Keller, R., and J. E. Dwyer. 1969. Neutralization of poliovirus by IgA coproantibodies. J. Immunol. 101: 192-202.
13. Kisch,A.L.,and K. M. Johnson. 1969. Aplaqueassay for respiratory syncytial virus. Proc. Soc. Exp. Biol. Med. 112:583-589.
14. Majer, M. 1972. Virus sensitization. Curr. Top. Microbiol. Immunol. 58:69-84.
15. McIntosh,K., H. B. Masters,I.Orr, R. Chao, and R. M.Baskin.1978.Theimmunologicresponse to infec-tionwith respiratory syncytial virus in infants. J. Infect. Dis. 138:24-32.
16. Mills, J., J. E. Vankirk, P. F. Weight, and R. M. Chanock.1971.Experimentalrespiratory syncytial vi-rus infection ofadults: possiblemechanisms of resist-ance toinfection andillness.J. Immunol. 107:123-130. 17. Parrott, R. H., H. W. Kim, J.O.Arrobio, D. S. Hodes, B. R. Murphy, C. D.Brandt, E. Camargo, and R. M. Chanock. 1973.Epidemiology ofrespiratory syn-cytialvirus infection in Washington, D.C. Am. J. Epi-demiol.98:289-300.
18. Potash, L., R. S. Lee, and B. M. Sweet. 1966. Increased sensitivity fordetection of respiratory syncytial virus neutralizing antibody by use of unheated sera.Bacteriol. Proc.,p. 122.
19. Takabayashi, K., and K. McIntosh. 1973. Effect of heat-labile factors on theneutralizationof vaccinia virus byhuman sera. Infect. Immun. 8:582-589.
20. Welliver,R.C.,T. N.Kaul,T. I. Putnam, M. Sun, K. Riddlesberger, andP.L.Ogra. 1980.Theantibody response toprimaryandsecondaryinfection with res-piratory syncytial virus: kinetics of class specific re-sponses.J. Pediatr. 96:808-813.
21. Yoshino,K.,and S. Taniguchi.1966.Evaluationof the demonstration of complement-requiring neutralizing antibodyas ameansforearly diagnosisofherpesvirus infection.J.Immunol. 96:196-203.
