Vol. 28, No. 12JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1990, p. 2744-27490095-1137/90/122744-06$02.00/0Copyright © 1990, American Society for Microbiology

Monoclonal Antibody-Based Capture Enzyme Immunoassays forSpecific Serum Immunoglobulin G (IgG), IgA, and IgM Antibodies

to Respiratory Syncytial VirusDEAN D. ERDMAN* AND LARRY J. ANDERSON

Respiratory and Enterovirus Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases,Centers for Disease Control, 1600 Clifton Road, N.E., Atlanta, Georgia 30333

Received 17 May 1990/Accepted 24 September 1990

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) ofrespiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA,and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies(PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assaysdeveloped with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgMantibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgMantibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays wereinsensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAbassay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens werepositive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age:N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults andinfants <1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adultsand infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs wascomplementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positiveby PAb assay. These MAb assays were also specific with specimens tested from persons without a history ofrecent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunityto have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.

Respiratory syncytial (RS) virus is the single most com-mon cause of lower respiratory tract infection in infants andyoung children (9). Whereas antigen detection remains themethod of choice for rapid diagnosis of RS virus infection,antibody detection serves as an important adjunct in bothclinical and epidemiological studies (14). Historical depen-dence on the complement fixation test (21) for serologicdiagnosis has been superseded by more sensitive and con-venient enzyme immunoassays (4, 12, 13, 20, 22). The recentavailability of monoclonal antibodies (MAbs) to RS virusoffers an opportunity for further improvements in diagnosticassays. Because MAbs of the desired specificity are such aconvenient source of high quality reagents, we chose to testour previously developed MAbs as detectors in captureimmunoassays for specific immunoglobulin G (IgG), IgA,and IgM antibodies to RS virus. To evaluate the impact ofthe epitope and protein specificities of MAbs on theseassays, we compared individual and combinations of MAbsto the fusion protein (F), attachment protein (G), and nu-cleoprotein (N) of RS virus with a standard polyclonalantibody (PAb). We also compared the results of individualMAbs with those obtained by Western immunoblot.

MATERIALS AND METHODS

Serum specimens. Sixty-three serum specimens from 27adults and 16 infants <1 year of age selected from fourdifferent outbreaks of RS virus and six serum specimensfrom adults with unrelated respiratory tract infections wereused to compare sensitivities of MAb and PAb assays.

* Corresponding author.

Specificity studies were conducted with the following: (i) 20paired acute- and convalescent-phase serum specimens frompersons with diagnostic rises in specific antibodies to otherviruses (three with parainfluenza virus 1, 3; three withherpes simplex virus type 1; one with mumps; two withparvovirus; five with enterovirus; three with influenza vi-ruses A and B; three with adenovirus), (ii) 60 specimensfrom healthy adult blood donors, (iii) 22 specimens fromhealthy infants <2 years of age, and (iv) 10 serum samplespositive for rheumatoid factor.

Antigen. Antigen was prepared from RS virus referencestrains Long (5), 18537 (6), and A2 (17). Virus was absorbedfor 1 h at 37°C onto monolayers of HEp-2 cells. Eagleminimal 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 untilmonolayers developed cytopathic effect of 3 to 4+. Thecultures were then freeze-thawed three times, and the cellu-lar debris was pelleted by low-speed centrifugation (500 x g)for 15 min. Supernatants from the respective strains werepooled and stored at -70°C. Uninfected cells were proc-essed similarly for negative control antigen.MAbs. Detector MAbs to different epitopes of the N, F,

and G proteins of RS virus with strong affinity and broadstrain reactivity were selected from a large panel of previ-ously characterized MAbs (1-3) for evaluation (Table 1). AllMAbs were derived from the same immunizing strain, A2.MAbs were purified by precipitation with equal volumes ofsaturated ammonium sulfate and by separation on DEAE-Sephacel (Pharmacia, Uppsala, Sweden). Biotinylation wasperformed by procedures previously described (2).

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TABLE 1. MAbs to RS virus

MAb Subclass Epitopea inhibition Neutralization

130-12h IgG2A(K) N NDb ND131-4g IgG2A(K) N ND ND131-2a IgGlK Fla <20 <20133-lh IgG2A(K) F2 <20 2,560143-6c IgGlK F3 >500 20,480131-2g IgGlK G1,3 ND <20130-5f IgG2B(K) G4 ND <20

a Protein specificity of MAbs was previously determined by immunopre-cipitation of radiolabeled or biotinylated proteins. The uppercase letterdesignates the protein, the first numeral designates the antigenic site deter-mined by blocking antibody assays, the lowercase letter designates an epitopedistinguished from other epitopes at an antigenic site by reaction patternsagainst strains of RS virus, and the second numeral designates an epitopedistinguished from others at an antigenic site by different patterns of antibodyblocking.

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 Disease Control (anti-human IgG and IgM MAbsare available from Dimension Laboratories, Inc., Missis-sauga, Ontario, Canada). Capture MAbs were purified byprecipitation with equal volumes of saturated ammoniumsulfate followed by two washes with 40% saturated ammo-nium sulfate. All MAbs were dialyzed against 0.01 M phos-phate-buffered saline (PBS), pH 7.2, and stored at -70°C.Western blot. The Long strain of RS virus grown in HEp-2

cells as described above was solubilized in a minimal volumeof radioimmunoprecipitation assay buffer containing 0.01 MTris hydrochloride (pH 7.4), 0.15 M NaCl, 1% sodiumdeoxycholate, 1% Triton X-100, and 0.5% sodium dodecylsulfate. Viral proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreduc-ing conditions and transferred to nitrocellulose paper aspreviously described (10). Strips of the nitrocellulose paperwere reacted with serum specimens diluted 1:100 in PBSwith 0.15% Tween 20 (PBS/T) for 2 h at room temperature.The strips were then washed three times with PBS/T andreacted for 1 h at room temperature with the peroxidase-conjugated goat anti-human IgG (heavy and light chain)antibody (Kirkegaard & Perry, Gaithersburg, Md.) diluted1:3,000 in PBS/T. After three washes, a solution containing0.5 mg of 3,3'-diamino-benzidine per ml of PBS with 1 ,ul of3% H202 per ml was added and incubated for 10 min. Colordevelopment was stopped by washing four times with deion-ized water. The appearance of a band with the convalescent-phase specimen or a subjective doubling in the intensity ofbands between paired specimens was considered to be asignificant increase in antibodies to RS virus-specific pro-teins.

MAb capture immunoassays. Anti-human IgG, a mixtureof anti-human IgAl and IgA2, and anti-human IgM MAbsdiluted 1:1,000 in PBS were each added to Immulon Ilmicrotiter plates (Dynatech Laboratories, Alexandria, Va.)and incubated overnight at 4°C; all reagent volumes were 75,ul per well, 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%Tween 20, and serum specimens diluted 1:100 were added tofour wells each and incubated for 1 h at 37°C. Plates were

washed three times, and positive antigen and negative con-

trol cells diluted 1:300 were added to duplicate wells for eachspecimen and incubated overnight at room temperature. Thenext day, plates were washed three times and biotinylatedanti-RS virus MAbs were added and incubated for 1 h at37°C. Plates were washed three times, a 1:3,000 dilution ofstreptavidin peroxidase (Amersham International, Amer-sham, United Kingdom) was added, and the plates were

incubated for 20 min at 37°C. After five washes, a solutioncontaining 0.1 mg of 3,3',5,5'-tetramethyl-benzidine (SigmaChemical Co., St. Louis, Mo.) per ml and 1.6 ,ul of 3% H202per 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. Color development was stopped by the addition of 2 MH2PO4, and the A450 was read by using a MR580 Micro-ElisaAutoreader (Dynatech Laboratories).PAb capture immunoassays. PAb immunoassays were per-

formed as described above with the following modifications:(i) bovine anti-RS virus PAb (Burroughs Welcome Co.,Research Triangle Park, N.C.) diluted 1:3,000 in PBS/G/Twith 2% normal goat serum served as detector antibody, and(ii) peroxidase-labeled goat anti-bovine antibody (Kirke-gaard and Perry) diluted 1:10,000 in PBS/G/T with 2%normal goat serum served as conjugate. Both reagents were

incubated for 1 h at 37°C.Immunoassay optimization. Reagent dilutions as well as

incubation times and temperatures were evaluated by check-erboard titrations and were standardized for all assays. Thescreening dilution of the test sera was selected to saturateavailable capture sites with minimal nonspecific bindingbetween immunoglobulin classes (as determined by assaywith 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 of RS virus strains were determined sep-arately and mixed proportionately to serve as assay antigen.Later, assay incubation times were shortened for addedconvenience; capture antibody and antigen incubations werereduced from overnight to 1 and 3 h at 37°C, respectively. Tofurther shorten assay time, detector MAbs were mixed withantigen and incubated in a single step with no loss ofsensitivity. Total assay time was 6 h.

Expression of results. Results were expressed as P - N

TABLE 2. Detection of serum antibody and antibody responses to RS virus proteins by Western blot

No. of samples with positive detection of or antibody response to RS virus protein (%)Antibodya

G F N P M 22K

DetectedA 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 detected in the acute (A)- or convalescent (C)-phase serum; Increase, significant increase (ie., from absence to presence ofthe respectiveband or a subjective doubling in intensity of the band) in antibody detected between serum pairs.

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2746 ERDMAN AND ANDERSON

LO r-

rA

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FIG. 1. Western blot of acute (A)- and convalescent (C)-phaseserum specimens from six adults recently infected with RS virus. Ahyperimmune anti-RS virus horse serum (HaRSV) was run aspositive control. The nitrocellulose was cut into strips and alignedfor display. The G, F, N, P, precursor G protein product G(P), M,and 22K proteins are indicated. Serum pairs 1985, 1991, 1992, 1993,and 1995 show rises in specific IgG antibodies to one or more of theRS virus proteins.

values, defined as the average differences in A450 measuredbetween duplicate wells of positive (P) and negative (N)antigen. To account for the residual nonspecific sticking ofviral proteins that could not be eliminated, results werestandardized to permit comparisons between assays bysubtracting the P - N value of a diluent blank without serum

(PB - NB) from each serum specimen (Ps - NS). Aspecimen was considered positive if (Ps - NS) - (PB - NB)was 20.05 A450 and if the PIN value [Ps - (PB NB)INs] was

>2; PIN values were included to account for specimens withhigh background. This cutoff was approximately equivalentto the mean plus 3 standard deviations of 60 normal serumspecimens. The same cutoff was used for all three assays. Asignificant rise in specific antibodies to RS virus was definedas a 250% increase in P - N values between acute- andconvalescent-phase specimens, a factor exceeding 3 stan-dard deviations above the mean difference between 20 pairedspecimens collected from persons with other virus infec-tions.

RESULTS

Western blot. Of 23 acute- and convalescent-phase serumpairs from persons with recent RS virus infection, 14 haddetectable rises in specific IgG antibody to one or more ofthe virus structural proteins, as determined by Westernblotting. Antibody prevalence and response rates to thespecific proteins are presented in Table 2, and the patterns offive representative patients are illustrated in Fig. 1. Only 2 of10 (20%) persons with strong IgG responses to the G proteinby Western blot had a detectable response with the corre-sponding capture immunoassay, whereas 4 of 6 (67%) and 10of 10 (100%) individuals with responses to F and N byWestern blot were detected by the respective capture immu-noassays.

Specificity of MAbs. The ability of biotinylated MAbs todetect RS virus proteins bound by serum antibody in captureimmunoassays was evaluated by capturing RS virus antigenswith unlabeled MAbs and then reacting them with biotiny-lated detector MAbs. As seen in Table 3, no single detectorMAb could detect all RS virus proteins. N MAbs reactedstrongly with antigens captured by themselves and by NMAbs at different antigenic sites but reacted weakly whencaptured by F or G MAbs; F MAbs reacted strongly withantigens captured by themselves, by F MAbs at differentantigenic sites, and by G MAbs but reacted weakly whencaptured by N MAbs; and G MAbs reacted with antigenscaptured by G MAbs at different antigenic sites and by FMAbs but were not reactive when captured by themselves orby N MAbs.Comparison of MAb and PAb assays. Of the two N MAbs

and three F MAbs, 130-12 h (N) and 131-2a (F) gave the bestsignal-to-noise ratios for their respective proteins. MAb130-12h (N) assays correlated well with PAb assays for allimmunoglobulin classes (Fig. 2). MAb 131-2a (F) assays hadgenerally lower values but identified specific IgA in eightserum specimens that were negative by 130-12h (N). MAb131-2g and 130-Sf (G) assays were insensitive for specific IgGand IgM antibodies, with activity confined to the IgA anti-body class. Assays based on a pool of MAbs 130-12h (N) and131-2a (F) detected specific IgG in 54 of 55 (98%), IgA in 51of 52 (98%), and IgM in 16 of 17 (94%) serum specimenspositive by the PAb assay; inclusion of MAbs to G proteindid not improve assay sensitivity for any immunoglobulinclass (data not shown). Ten additional serum specimensfrom seven persons with IgG or IgA responses to RS virusby both assays had detectable IgM antibodies by MAb butnot by PAb assay. N MAb assays detected specific antibod-ies in both infants <1 year of age and adults, F MAb assaysdetected IgA and IgG in adults and IgA in infants, and GMAb assays only detected IgA in adults (Table 4). The MAbpool gave the best results for both age groups and for all

TABLE 3. Relative reactivity of MAb pairs to RS virus

Capture Reactivity of detector MAbbMAb 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 See Table 1, footnote a.b Reactivity of MAb pairs is represented as a range from maximum (+ + +) to no detectable (-) reactivity.

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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)

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FIG. 2. Comparison of P - N values of PAb and MAb assays for RS virus IgG, IgA, and IgM antibodies. Spearman rank correlationcoefficients for the monoclonal pool 130-12h and 131-2a versus polyclonal antibody were r = 0.836 (IgG), r = 0.903 (IgA), and r = 0.782 (IgM).

immunoglobulin classes and was further evaluated in parallelwith PAb assays for specificity.

Specificity of MAb assays. The specificities of the opti-mized MAb assays were evaluated with serum specimensfrom 6 normal infants <2 months of age, 16 infants aged 2 to<24 months, and 60 adult blood donors. Specific IgG wasdetected in all infants <2 months old and all adults, IgA wasnot detected in infants <2 months old but was detected inmore than 50% of the adults, and IgM was not detected ininfants <2 months old or adults. Specific IgA and IgMantibodies were detected in one 18-month-old infant, sug-

gesting recent infection. Furthermore, specific IgM was notdetected in the acute- or convalescent-phase serum speci-mens from 20 patients with diagnostic antibody rises to otherviruses, although a significant rise in IgG antibodies was

detected in one person with herpes simplex type 1 virusinfection (results were consistent on repetition, and couldnot be confirmed by PAb assay). Ten specimens positive forrheumatoid factor were negative for specific IgM antibodies.

DISCUSSIONThe advantages of MAbs are apparent, but identifying those

suitable as detector reagents for antibody immunoassays can

prove problematic. Some have low affinity (26) or high nonspe-cific binding (personal observation). Others exhibit restrictedstrain reactivity; for example, some MAbs to RS virus are

group or strain specific (2). The activities of MAbs can vary,depending on whether antigen is presented free in solution,immobilized by antibody, or directly absorbed to the solidphase (7). Furthermore, MAbs must complement the protein-specific antibody response of the host, which can vary withimmunoglobulin class (25) and subclass (23, 24), patient age(15), and prior exposure to the agent (24).Our first concern in using MAbs as detector reagents in

capture immunoassays was identifying those compatiblewith the host antibody response. Because RS virus is envel-oped and disassociates into individual protein or proteincomplexes when exposed to the detergent Tween 20 presentin the assay diluent, a single detector MAb may not identifyall proteins captured by host antibody, and MAbs withdifferent protein specificities may give different results withthe same specimens. Our results validate this concern (de-tector MAbs to the F and G proteins failed to react withantigen captured by N MAbs and vice versa) and underscorethe need to carefully evaluate detector MAbs during assaydevelopment.

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2748 ERDMAN AND ANDERSON

TABLE 4. Detection of serum antibody and antibody responses to RS virus in infants and adults by PAb and MAb assays

No. of samples with antibody or antibody response to RS virus

Antibody detected MAb assaysand response F G N

Infants Adults Infants Adults Infants Adults Infants Adults

IgG il 27 3 26 1 2 12 27Increase 3 6 1 5 0 2 2 6Decrease 0 1 0 0 0 0 0 1

IgA 7 27 10 27 1 18 9 26Increase 2 6 2 6 0 3 3 5Decrease 1 7 1 4 0 3 1 6

IgM 7 8 1 2 0 0 13 14Increase 2 1 0 0 0 0 3 2Decrease 1 3 0 0 0 0 1 3

a PAb 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- andconvalescent-phase serum specimens. Twenty-seven adults were tested; fourteen had acute- and convalescent-phase serum specimens.

As expected, the sensitivities of detector MAbs variedwith immunoglobulin class and patient age. F MAbs weresensitive in assays for specific IgG in adults and IgA in adultsand infants but were insensitive for IgM. G MAbs weremoderately sensitive in assays for specific IgA in adults butwere insensitive for IgG and IgM. Only MAbs to N werebroadly reactive in all assays and, when combined with FMAbs, were comparable to PAbs in detecting specific IgG,IgA, and IgM responses to RS virus.Our optimized MAb assays were generally specific. IgM

and IgA antibodies were not detected in serum specimensfrom healthy infants <2 months of age in which IgG ofprobable maternal origin was present, although IgM and IgAantibodies were detected in one 18-month-old infant forwhom recent subclinical RS virus infection would not beunexpected, nor were they detected in commercial serumspecimens from adults that contained IgA or IgG antibodies.Specific IgM was also absent in persons with other virusinfections. A rise in specific IgG antibodies to RS virus in achild with herpes simplex virus type 1 infection may havebeen due to a mixed infection; paired sera from otherpatients with herpes infection did not show a similar rise inIgG or IgA antibodies to RS virus.The human antibody response to RS virus has been shown

to include antibodies against all the RS virus structuralproteins. In Western blot (8, 11) and indirect enzyme immu-noassay (16, 23) responses to G are usually detected. Sur-prisingly, in our capture immunoassays, G MAbs detectedvery little antibody to RS virus demonstrable by Westernblot; essentially no IgG or IgM but some IgA antibodies wereidentified. We suspect that a number of conditions conspiredto limit the effectiveness of G MAbs in these assays: (i) ourG detector MAbs were relatively less active than N and FMAbs with proteins bound by capture MAbs, whereasreactivity was comparable when the proteins were fixeddirectly to the microtiter plates; (ii) multiple epitopes on theG protein could have been blocked or altered after binding tohost antibody; and (iii) since the signal in capture immunoas-says depends on the proportion of captured antibody withthe desired specificity, it is possible that antibodies to the Gprotein represent only a small proportion of the total anti-body response to RS virus. On the other hand, assays usingN and F MAbs readily detected IgG antibodies to RS virusand, when combined, gave results as good as or better thanthose with PAbs for all immunoglobulin classes evaluated.These MAb assays offer laboratories a standardized meth-odology for serological studies of RS virus infection without

concern for long-term availability or variation in the qualityof reagents.

ACKNOWLEDGMENT

This study was partially supported by the Diagnostic Technologyfor Community Health (DIATECH) project funded by USAIDthrough cooperative agreement PPE-5935-A-00-5065-00 with theProgram for Appropriate Technology in Health (PATH).

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20. Richardson, L. S., R. H. Yolken, R. B. Belshe, E. Camargo,H. W. Kim, and R. M. Chanock. 1978. Enzyme-linked immuno-sorbent assay for measurement of serological response to res-piratory syncytial virus infection. Infect. Immun. 20:660-664.

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