Copyright© 1990, AmericanSociety for Microbiology
Monoclonal Antibody-Based Capture Enzyme Immunoassays for
Specific
Serum Immunoglobulin G (IgG), IgA, and IgM Antibodies
to
Respiratory
Syncytial
Virus
DEAN D. ERDMAN*AND LARRY J. ANDERSON
Respiratory and Enterovirus Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, CentersforDiseaseControl, 1600Clifton Road, N.E.,Atlanta, Georgia 30333
Received 17 May1990/Accepted 24 September 1990
Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, andIgM capture enzymeimmunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serumspecimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but diddetect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low
sensitivity
of theG MAb assays is unclear, since many of these specimens were positive forIgGantibodies to Gby Western immunoblot. Thesensitivityof MAbassaysvaried with patient age: NMAb
assays detected specificantibodyresponses to RS virus in allimmunoglobulinclasses inbothadults
and infants <1year of age, F MAb assays detected specificIgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recentRS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity tohave long-term, standardized reagents and tests for serologicaldiagnosis of RS virus infection.Respiratory syncytial (RS) virus is the single most com-mon cause oflowerrespiratorytractinfectionin infantsand
young children (9). Whereas antigen detection remains the
method of choice for rapid diagnosis of RS virus infection, antibody detection serves as an important adjunct in both clinical and epidemiological studies (14). Historical depen-dence on the
complement
fixation test (21) for serologic diagnosis has been superseded by more sensitiveandcon-venientenzymeimmunoassays(4, 12, 13, 20,22). The recent
availability of monoclonal antibodies (MAbs) to RS virus
offersanopportunity forfurtherimprovements indiagnostic
assays. Because MAbsofthe desired
specificity
are such a convenientsourceof highqualityreagents, we chose to test our previously developed MAbs as detectors in captureimmunoassays for specific immunoglobulin G (IgG), IgA, andIgM antibodies to RS virus. Toevaluate the impact of
the epitope and protein specificities of MAbs on these assays, wecompared individualandcombinations of MAbs
to
the
fusion protein (F), attachment protein (G), and nu-cleoprotein (N) of RS virus with a standard polyclonal antibody(PAb). Wealso compared the results ofindividualMAbs with thoseobtained byWestern immunoblot. MATERIALSANDMETHODS
Serum specimens. Sixty-three serum specimens from 27 adults and 16 infants <1 year of age selected from four
different outbreaks of RS virus and six serum specimens
from adultswith unrelated respiratory tractinfections were used to compare sensitivities of MAb and PAb assays.
*Correspondingauthor.
Specificity studies wereconducted with thefollowing:(i)20
pairedacute- andconvalescent-phaseserumspecimensfrom persons withdiagnostic risesin specific antibodiesto other viruses (three with parainfluenza virus 1, 3; three with
herpes simplex virus type 1; one with mumps; two with parvovirus; five with enterovirus; three with influenza vi-ruses A and B; three with adenovirus), (ii) 60 specimens
from healthy adult blood donors, (iii) 22 specimens from
healthy infants <2 yearsofage, and (iv) 10 serum samples positivefor rheumatoid factor.
Antigen. Antigen was prepared from RS virus reference
strainsLong (5), 18537(6),and A2(17). Viruswasabsorbed
for 1 h at 37°C onto monolayers of HEp-2 cells. Eagle
minimal essential medium supplemented with fetal calf se-rum (2%), glutamine (292 ,ug/ml), penicillin (200 U/ml), streptomycin (200 ,ug/ml), and amphotericin B (10 ,ug/ml)
was then added, and the cultures were incubated until monolayers developed cytopathic effect of 3 to 4+. The cultures were thenfreeze-thawed three
times,
andthe cellu-lardebriswaspelleted bylow-speed
centrifugation
(500x g) for 15 min. Supernatants from therespective
strains were pooled and stored at -70°C. Uninfected cells were proc-essed similarlyfornegative control antigen.MAbs. Detector MAbs to different
epitopes
ofthe N, F,and G proteins of RS virus with strong affinity and broad strain reactivity were selected from a large panel of previ-ouslycharacterized MAbs(1-3) for evaluation
(Table
1).All MAbs were derived from the sameimmunizing
strain,
A2. MAbswere purified byprecipitation withequal
volumesof saturated ammonium sulfate and byseparation
onDEAE-Sephacel (Pharmacia, Uppsala, Sweden).
Biotinylation
wasperformedby procedures
previously
described (2).2744
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TABLE 1. MAbstoRSvirus
MAb Subclass Epitopea inhibition Neutralization
130-12h IgG2A(K) N NDb ND
131-4g IgG2A(K) N ND ND
131-2a IgGlK Fla <20 <20
133-lh IgG2A(K) F2 <20 2,560
143-6c IgGlK F3 >500 20,480
131-2g IgGlK G1,3 ND <20
130-5f IgG2B(K) G4 ND <20
aProtein specificity of MAbs was previously determined by
immunopre-cipitation of radiolabeled or biotinylated proteins. The uppercase letter designates the protein, the first numeral designates the antigenic site deter-minedbyblocking antibody assays, the lowercase letter designates an epitope distinguished from other epitopes at an antigenic site by reaction patterns against strains of RS virus, and the second numeral designatesan epitope distinguished from othersatanantigenic site by different patterns of antibody blocking.
b ND, Not done.
Anti-human immunoglobulin class capture MAbs IgG (HP6064), IgM (HP6083) (18), IgAl (HP6119), and IgA2
(HP6111) (19) were kindly provided by Charles Reimer, Centers for DiseaseControl (anti-human IgG and IgM MAbs
are available from Dimension Laboratories, Inc.,
Missis-sauga, Ontario, Canada). Capture MAbs were purified by precipitation with equal volumes of saturated ammonium sulfate followed bytwo washes with40% saturated
ammo-nium sulfate. All MAbsweredialyzed against0.01 M
phos-phate-buffered saline (PBS), pH7.2, and storedat -70°C.
Westernblot.TheLongstrain ofRS virusgrown in HEp-2
cellsasdescribed abovewassolubilizedin aminimalvolume ofradioimmunoprecipitation assaybuffercontaining0.01 M
Tris hydrochloride (pH 7.4), 0.15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100, and0.5% sodium dodecyl sulfate. Viral proteins were separated by sodium dodecyl
sulfate-polyacrylamide
gelelectrophoresis undernonreduc-ing conditions and transferred to nitrocellulose paper as
previously described(10). Stripsofthenitrocellulose paper were reacted with serum specimens diluted 1:100 in PBS
with
0.15%
Tween 20(PBS/T)
for2 h at roomtemperature.The strips were then washed three times with PBS/T and reacted for 1 h at room temperature with the
peroxidase-conjugated goat anti-human IgG (heavy and light chain)
antibody (Kirkegaard & Perry, Gaithersburg, Md.) diluted 1:3,000 inPBS/T. Afterthree washes, asolution containing
0.5 mgof
3,3'-diamino-benzidine
perml of PBSwith 1 ,ulof3% H202per ml wasadded andincubatedfor10 min. Color
developmentwasstopped bywashing four timeswith deion-izedwater.Theappearanceofabandwiththe
convalescent-phasespecimen or a subjective doubling in the intensity of bands between paired specimens was considered to be a
significant increase in antibodies to RS virus-specific pro-teins.
MAbcapture immunoassays. Anti-human IgG, a mixture of anti-human IgAl and IgA2, and anti-human IgM MAbs diluted 1:1,000 in PBS were each added to Immulon Il microtiterplates(Dynatech Laboratories, Alexandria, Va.) andincubated overnightat4°C; allreagentvolumeswere75 ,ulperwell, and PBS with 0.5%gelatin and 0.15% Tween 20
(PBS/G/T) was used as diluent for all subsequent steps. Plates were washed three times by using PBS with 0.05%
Tween20, andserumspecimens diluted 1:100wereaddedto four wells each and incubated for 1 h at 37°C. Plateswere washed three times, and positive antigen and negative con-trol cells diluted 1:300wereaddedtoduplicate wells for each specimen and incubated overnightatroomtemperature.The
next day, plateswere washed three times and biotinylated
anti-RS virus MAbs were added and incubated for 1 h at 37°C. Plates were washed three times, a 1:3,000 dilution of
streptavidin peroxidase (Amersham International, Amer-sham, United Kingdom) was added, and the plates were
incubated for 20 min at37°C. After five washes, a solution containing 0.1 mg of3,3',5,5'-tetramethyl-benzidine (Sigma ChemicalCo., St. Louis, Mo.)permland1.6,ulof 3%H202 per ml of 0.1 M citrate acetate buffer (pH 5.5) was added,
and the plates were incubated for 15 min at room tempera-ture.Colordevelopmentwasstopped by the addition of 2 M
H2PO4,and theA450wasreadby usingaMR580 Micro-Elisa
Autoreader(Dynatech Laboratories).
PAbcaptureimmunoassays. PAbimmunoassayswere per-formedasdescribed abovewith thefollowing modifications:
(i) bovine anti-RS virus PAb (Burroughs Welcome Co., ResearchTriangle Park, N.C.) diluted 1:3,000 in PBS/G/T with2%normalgoatserumservedasdetectorantibody, and
(ii) peroxidase-labeled goat anti-bovine antibody (Kirke-gaard and Perry) diluted 1:10,000 in PBS/G/T with 2% normalgoat serumservedasconjugate. Bothreagentswere incubated for 1 hat 37°C.
Immunoassay optimization. Reagent dilutions as well as
incubation times andtemperatureswereevaluatedby
check-erboard titrations andwere standardized for all assays.The screening dilution ofthe test sera was selected to saturate available capture sites with minimal nonspecific binding between immunoglobulin classes (as determined by assay with anti-human immunoglobulin class-specific conjugate). To ensure broad assay reactivity, a pooled antigen was
prepared from three RS virus strains, Long, 18537, and A2. Optimal dilutions ofRS virus strains weredetermined sep-arately and mixedproportionatelytoserve asassayantigen. Later, assay incubation times were shortened for added
convenience;captureantibody and antigen incubationswere reduced fromovernightto1 and3 hat37°C,respectively. To furthershortenassaytime, detector MAbsweremixed with
antigen and incubated in a single step with no loss of
sensitivity. Total assaytimewas 6h.
Expression of results. Results were expressed as P - N
TABLE 2. Detection ofserumantibody and antibodyresponses toRS virusproteins byWesternblot No.ofsampleswithpositivedetectionoforantibody responsetoRS virusprotein(%)
Antibodya
G F N P M 22K
Detected
A 21(91) 23(100) 19(83) 15 (65) 7(30) 7(30)
C 23(100) 23(100) 23(100) 22 (96) 13(57) 13 (57)
Increase 12(52) 6(26) 12(52) 11(48) 6 (26) 6(26)
a Detected, Antibody detectedin theacute(A)- or convalescent(C)-phaseserum; Increase, significant increase
(ie.,
fromabsence to presence ofthe respective bandor asubjective doublinginintensityof the band) in antibodydetected between serum pairs.on April 12, 2020 by guest
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LO
r-rA
AC(: AC;
a... a
N m
AC AC AC
r-v-._._.
Oi
CD
AC m 2
a
.W
205
É.
5..
_h amF .
à.
.-~ P
G[P)
22K 97.5 66
44
29
14.2
FIG. 1. Western blot ofacute(A)-and convalescent (C)-phase
serumspecimens fromsix adultsrecentlyinfected with RS virus. A
hyperimmune anti-RS virus horse serum (HaRSV) was run as
positivecontrol. The nitrocellulosewascutintostripsandaligned
fordisplay.TheG, F, N, P,precursorGprotein product G(P),M,
and 22Kproteinsareindicated. Serumpairs 1985, 1991, 1992,1993,
and1995 showrisesinspecific IgG antibodiestoone or moreof the
RS virusproteins.
values, defined astheaveragedifferences inA450measured between duplicate wells of positive (P) and negative (N) antigen. To accountfor the residual nonspecific sticking of viral proteins that could not be eliminated, results were
standardized to permit comparisons between assays by subtracting theP-Nvalueofadiluent blank withoutserum
(PB
-NB)
from each serumspecimen (Ps
-NS).
Aspecimenwasconsideredpositive if
(Ps
-NS)
-(PB
- NB)was20.05A450and ifthePINvalue
[Ps
- (PBNB)INs]
was>2; PIN valueswereincludedto accountforspecimens with high background. Thiscutoffwas approximately equivalent
to the meanplus 3 standard deviations of 60 normal serum
specimens. The samecutoffwasused forall threeassays.A significant rise in specific antibodiestoRS viruswasdefined as a 250% increase in P - N values between acute- and
convalescent-phase specimens, a factor exceeding 3 stan-darddeviations above themeandifference between 20paired specimens collected from persons with other virus infec-tions.
RESULTS
Western blot. Of 23 acute-andconvalescent-phase serum pairs from persons with recent RS virus infection, 14 had detectable rises in specific IgG antibody toone or more of the virus structural proteins, as determined by Western blotting. Antibody prevalence and response rates to the specific proteinsarepresented in Table 2, and thepatternsof five representative patientsareillustrated in Fig. 1. Only 2 of
10(20%)personswithstrongIgGresponsestothe Gprotein by Western blot had a detectable response with the corre-spondingcaptureimmunoassay, whereas 4 of 6 (67%) and 10 of 10 (100%) individuals with responses to F and N by Westernblotweredetected by the respectivecapture immu-noassays.
Specificity of MAbs. The ability of biotinylated MAbs to detect RS virusproteins bound byserumantibody incapture immunoassayswasevaluatedby capturing RS virus antigens with unlabeled MAbs and then reacting them with biotiny-lateddetector MAbs. AsseeninTable3,no single detector MAb could detect all RS virus proteins. N MAbs reacted strongly with antigens captured by themselves and by N MAbs at different antigenic sites but reacted weakly when captured by F or G MAbs; F MAbs reacted strongly with
antigens captured by themselves, by F MAbs at different antigenic sites, and by G MAbs but reacted weakly when captured by N MAbs; and G MAbs reacted with antigens captured by G MAbs atdifferent antigenic sites and by F MAbs butwerenotreactive whencaptured by themselvesor by N MAbs.
Comparison of MAb and PAbassays. OfthetwoN MAbs and three FMAbs, 130-12 h (N) and 131-2a (F)gavethe best signal-to-noise ratios for their respective proteins. MAb 130-12h (N) assays correlated well with PAb assays for all immunoglobulin classes (Fig. 2). MAb 131-2a (F)assayshad generally lower values but identified specific IgA in eight
serum specimens that were negative by 130-12h (N). MAb 131-2g and 130-Sf (G)assays wereinsensitive forspecific IgG and IgM antibodies, with activity confined to the IgA anti-body class. Assays basedon apoolof MAbs 130-12h(N)and 131-2a(F) detected specific IgGin 54 of 55(98%), IgAin 51 of52 (98%), and IgM in 16 of17 (94%) serum specimens positive by the PAbassay; inclusion ofMAbstoGprotein did not improve assay sensitivity for any immunoglobulin class (data not shown). Ten additional serum specimens from seven persons with IgG orIgAresponses to RSvirus by bothassays had detectable IgM antibodies byMAb but
notby PAbassay.N MAbassaysdetectedspecific antibod-ies inboth infants <1yearofageandadults,FMAbassays detected IgA and IgG in adults and IgA in infants, and G MAbassaysonly detected IgAinadults(Table 4).The MAb pool gave the best results for both age groups and for all
TABLE 3. Relative reactivityof MAbpairstoRS virus
Capture Reactivity of detectorMAbb
MAb
Epitopea
130-12h(N) 131-4g (N) 131-2a(Fla)
133-lh(F2) 131-2g(Gi,3) 130-5f(G4)130-12h N +++ + +
131-4g N +++ + + +
131-2a Fla + + ++ +++ ++ ++
133-lh F2 + ++ +++ ++ ++
131-2g G1,3 -+++ +++ - ++
130-5f G4 - -+++ +++ ++
a SeeTable 1,footnotea.
bReactivityof MAbpairsisrepresentedas arange from maximum(+++)to nodetectable(-)reactivity.
__ - -N
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131
-2A(F)
Absorbance(P-N) 0.5
131-2G(G)
1
30-5F(G)
Absorbance (P-N) 0.5
130-12H(N)
131 -2A(F)
. .
.::
FIG. 2. Comparison of P - Nvalues of PAb and MAbassaysfor RS virusIgG, IgA, and IgM antibodies. Spearman rank correlation
coefficients for themonoclonal pool 130-12h and 131-2aversuspolyclonal antibodywerer=0.836(IgG),r=0.903(IgA), andr=0.782(IgM).
immunoglobulinclassesandwasfurther evaluated inparallel with PAb assaysfor specificity.
Specificity of MAb assays. The specificities of the opti-mized MAb assays were evaluated with serum specimens from6normal infants<2months ofage, 16 infants aged2to <24 months, and 60 adult blood donors. Specific IgG was
detected inall infants <2months old andall adults, IgAwas
not detected in infants <2 months old but was detected in more than 50% of the adults, and IgM was not detected in infants <2 months old or adults. Specific IgA and IgM
antibodies were detected in one 18-month-old infant,
sug-gesting recent infection. Furthermore, specific IgM wasnot
detected in the acute- or convalescent-phase serum speci-mensfrom 20patientswithdiagnostic antibody risestoother viruses, although a significant rise in IgG antibodies was
detected in one person with herpes simplex type 1 virus infection (results were consistent on repetition, and could
notbeconfirmed byPAbassay). Tenspecimens positive for rheumatoid factorwerenegative for specific IgM antibodies.
DISCUSSION
Theadvantages ofMAbsareapparent,butidentifying those suitable asdetectorreagents forantibody immunoassays can
proveproblematic. Some have lowaffinity (26)orhigh nonspe-cificbinding (personal observation). Others exhibit restricted strain reactivity; for example, some MAbs to RS virus are
groupor strainspecific (2). The activities of MAbscan vary, depending on whether antigen is presented free in solution, immobilized by antibody, or directly absorbed to the solid phase (7). Furthermore,MAbs mustcomplementthe protein-specific antibody response of the host, which can vary with immunoglobulin class (25) and subclass (23, 24), patient age (15),andpriorexposure totheagent(24).
Our first concern in using MAbs as detector reagents in capture immunoassays was identifying those compatible
with thehostantibodyresponse. Because RS virus is envel-oped and disassociates into individual protein or protein
complexeswhenexposedtothedetergentTween 20present
intheassaydiluent, asingle detector MAbmay notidentify all proteins captured by host antibody, and MAbs with differentprotein specificities may givedifferentresults with the same specimens. Ourresults validate this concern
(de-tector MAbs to the F and G proteins failed to react with antigen captured byNMAbs and vice versa)and underscore the need to carefully evaluate detector MAbs during assay development.
130-12H(N)
.0
4
'.
IgG
IgA
IgM
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TABLE 4. Detection of serumantibody andantibody responses to RS virus in infants and adults by PAb and MAb assays No.of sampleswithantibodyorantibodyresponse to RS virus
Antibody detected MAb assays
andresponse F G N
Infants Adults Infants Adults Infants Adults Infants Adults
IgG il 27 3 26 1 2 12 27
Increase 3 6 1 5 0 2 2 6
Decrease 0 1 0 0 0 0 0 1
IgA 7 27 10 27 1 18 9 26
Increase 2 6 2 6 0 3 3 5
Decrease 1 7 1 4 0 3 1 6
IgM 7 8 1 2 0 0 13 14
Increase 2 1 0 0 0 0 3 2
Decrease 1 3 0 0 0 0 1 3
aPAb and MAbs 131-2A (F), 130-2G and 130-5F (G), and 130-12H (N) were used. For each test, sixteen infants were tested; five had acute- and
convalescent-phaseserumspecimens. Twenty-seven adultsweretested; fourteenhad acute- andconvalescent-phaseserumspecimens. As expected, the sensitivities of detector MAbs varied
with immunoglobulin class and patient age. F MAbs were sensitive in assays forspecific IgG in adults and IgA in adults and infants but were insensitive for IgM. G MAbs were
moderately sensitive in assaysforspecific IgA inadults but were insensitive for IgG and IgM. Only MAbs to N were
broadly reactive in all assays and, when combined with F
MAbs, were comparable to PAbsin detecting specificIgG,
IgA, and IgM responses toRS virus.
Our optimized MAb assays weregenerally specific. IgM and IgA antibodies were not detected in serum specimens
from healthy infants <2 months of age in which IgG of
probablematernaloriginwaspresent, althoughIgMandIgA
antibodies were detected in one 18-month-old infant for
whom recent subclinical RS virus infection would not be
unexpected, nor were they detected in commercial serum specimens fromadults thatcontainedIgAorIgGantibodies.
Specific IgM was also absent in persons with other virus
infections. Ariseinspecific IgG antibodiestoRSvirus in a
child with herpes simplex virus type 1 infection may have
been due to a mixed
infection; paired
sera from otherpatients with herpes infection did notshowa similarrise in
IgG orIgAantibodies toRS virus.
The humanantibodyresponse toRSvirushasbeenshown to include antibodies against all the RS virus structural proteins. In Westernblot (8, 11) and indirectenzyme
immu-noassay (16, 23) responses to Gare usually detected.
Sur-prisingly, in our capture immunoassays, G MAbs detected
very little antibody to RS virus demonstrable by Western
blot; essentiallynoIgG orIgMbut someIgA antibodieswere identified. We suspect that anumberof conditionsconspired
to limittheeffectiveness of G MAbs inthese assays: (i)our G detector MAbs were relatively less active than N and F MAbs with proteins bound by capture MAbs, whereas
reactivity was comparable when the proteins were fixed
directlytothemicrotiterplates; (ii)multiple epitopesonthe Gproteincould have been blockedoraltered afterbindingto hostantibody;and(iii) sincethesignalin capture immunoas-says depends on the proportion ofcaptured antibody with thedesiredspecificity, it ispossible thatantibodiestothe G protein represent only a small proportion ofthe total
anti-bodyresponse to RSvirus. On the otherhand,assays using
N and F MAbsreadilydetected IgG antibodies toRS virus
and,whencombined,gave resultsasgoodas orbetter than those with PAbsfor all immunoglobulin classes evaluated. These MAb assays offerlaboratories a standardized
meth-odologyforserological studies of RS virus infection without
concernforlong-term availability or variation in the quality of reagents.
ACKNOWLEDGMENT
Thisstudywaspartially supported bytheDiagnostic Technology for Community Health (DIATECH) project funded by USAID through cooperative agreement PPE-5935-A-00-5065-00 with the ProgramforAppropriate Technology in Health(PATH).
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