Specific Serum Immunoglobulin G (IgG), IgA, and IgM Antibodies

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 28, No. 12 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, Centers for Disease Control, 1600 Clifton Road, N.E., Atlanta, Georgia Received 17 May 1990/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, 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. 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 did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive 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 and infants <1 year of age, F MAb assays detected specific IgG 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 recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection. Respiratory syncytial (RS) virus is the single most common cause of lower respiratory tract infection in infants and 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 dependence on the complement fixation test (21) for serologic diagnosis has been superseded by more sensitive and convenient enzyme immunoassays (4, 12, 13, 20, 22). The recent availability of monoclonal antibodies (MAbs) to RS virus offers an opportunity for further improvements in diagnostic assays. Because MAbs of the desired specificity are such a convenient source of high quality reagents, we chose to test our previously developed MAbs as detectors in capture immunoassays for specific immunoglobulin G (IgG), IgA, and IgM antibodies to RS virus. To evaluate the impact of the epitope and protein specificities of MAbs on these assays, we compared individual and combinations of MAbs to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of RS virus with a standard polyclonal antibody (PAb). We also compared the results of individual MAbs with those obtained by Western immunoblot. MATERIALS AND METHODS 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 adults with unrelated respiratory tract infections were used to compare sensitivities of MAb and PAb assays. * Corresponding author Specificity studies were conducted with the following: (i) 20 paired acute- and convalescent-phase serum specimens from persons with diagnostic rises in specific antibodies to 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 viruses A and B; three with adenovirus), (ii) 60 specimens from healthy adult blood donors, (iii) 22 specimens from healthy infants <2 years of age, and (iv) 10 serum samples positive for rheumatoid factor. Antigen. Antigen was prepared from RS virus reference strains Long (5), (6), and A2 (17). Virus was absorbed for 1 h at 37 C onto monolayers of HEp-2 cells. Eagle minimal essential medium supplemented with fetal calf serum (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 then freeze-thawed three times, and the cellular debris was pelleted by low-speed centrifugation (500 x g) for 15 min. Supernatants from the respective strains were pooled and stored at -70 C. Uninfected cells were processed 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 broad strain reactivity were selected from a large panel of previously characterized MAbs (1-3) for evaluation (Table 1). All MAbs were derived from the same immunizing strain, A2. MAbs were purified by precipitation with equal volumes of saturated ammonium sulfate and by separation on DEAE- Sephacel (Pharmacia, Uppsala, Sweden). Biotinylation was performed by procedures previously described (2).

2 VOL. 28, 1990 MONOCLONAL ANTIBODIES IN RS VIRUS SEROLOGY 2745 TABLE 1. MAbs to RS virus MAb Subclass Epitopea inhibition Neutralization h IgG2A(K) N NDb ND 131-4g IgG2A(K) N ND ND 131-2a IgGlK Fla <20 < lh IgG2A(K) F2 <20 2, c IgGlK F3 >500 20, g IgGlK G1,3 ND < f IgG2B(K) G4 ND <20 a Protein specificity of MAbs was previously determined by immunoprecipitation of radiolabeled or biotinylated proteins. The uppercase letter designates the protein, the first numeral designates the antigenic site determined by blocking 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 designates an epitope distinguished from others at an antigenic 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 Disease Control (anti-human IgG and IgM MAbs are available from Dimension Laboratories, Inc., Mississauga, Ontario, Canada). Capture MAbs were purified by precipitation with equal volumes of saturated ammonium sulfate followed by two washes with 40% saturated ammonium sulfate. All MAbs were dialyzed against 0.01 M phosphate-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 volume of radioimmunoprecipitation assay buffer containing 0.01 M Tris hydrochloride (ph 7.4), 0.15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100, and 0.5% sodium dodecyl sulfate. Viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose paper as previously described (10). Strips of the nitrocellulose paper were reacted with serum specimens diluted 1:100 in PBS with 0.15% Tween 20 (PBS/T) for 2 h at room temperature. The strips were then washed three times with PBS/T and reacted for 1 h at room temperature with the peroxidaseconjugated goat anti-human IgG (heavy and light chain) antibody (Kirkegaard & Perry, Gaithersburg, Md.) diluted 1:3,000 in PBS/T. After three washes, a solution containing 0.5 mg of 3,3'-diamino-benzidine per ml of PBS with 1,ul of 3% H202 per ml was added and incubated for 10 min. Color development was stopped by washing four times with deionized water. The appearance of a band with the convalescentphase specimen 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 proteins. MAb capture 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 microtiter 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 to four wells each and incubated for 1 h at 37 C. Plates were washed three times, and positive antigen and negative control cells diluted 1:300 were added to duplicate wells for each specimen and incubated overnight at room temperature. The next day, plates were 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, Amersham, United Kingdom) was added, and the plates were incubated for 20 min at 37 C. After five washes, a solution containing 0.1 mg of 3,3',5,5'-tetramethyl-benzidine (Sigma Chemical Co., St. Louis, Mo.) per ml and 1.6,ul of 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 temperature. Color development was stopped by the addition of 2 M H2PO4, and the A450 was read by using a MR580 Micro-Elisa Autoreader (Dynatech Laboratories). PAb capture immunoassays. PAb immunoassays were performed 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/T with 2% normal goat serum served as detector antibody, and (ii) peroxidase-labeled goat anti-bovine antibody (Kirkegaard 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 checkerboard titrations and were standardized for all assays. The screening dilution of the 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 of RS virus strains were determined separately and mixed proportionately to serve as assay antigen. Later, assay incubation times were shortened for added convenience; capture antibody and antigen incubations were reduced from overnight to 1 and 3 h at 37 C, respectively. To further shorten assay time, detector MAbs were mixed with antigen and incubated in a single step with no loss of sensitivity. Total assay time was 6 h. Expression of results. Results were expressed as P - N Antibodya 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 (%) 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 detected in the acute (A)- or convalescent (C)-phase serum; Increase, significant increase (ie., from absence to presence ofthe respective band or a subjective doubling in intensity of the band) in antibody detected between serum pairs.

3 2746 ERDMAN AND ANDERSON J. CLIN. MICROBIOL. LO r- ra a... AC(: AC; a N m AC AC AC r-v-._._. CD Oi AC m 2 a.w É. 5.. _h amf. - -N.-~ P G[P) à. 22K FIG. 1. Western blot of acute (A)- and convalescent (C)-phase serum specimens from six adults recently infected with RS virus. A hyperimmune anti-rs virus horse serum (HaRSV) was run as positive control. The nitrocellulose was cut into strips and aligned for 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 the RS virus proteins. values, defined as the average differences in A450 measured between duplicate wells of positive (P) and negative (N) antigen. To account for the residual nonspecific sticking of viral proteins that could not be eliminated, results were standardized to permit comparisons between assays by subtracting the P - N value of a diluent blank without serum (PB - NB) from each serum specimen (Ps - NS). A specimen was considered positive if (Ps NS) (PB NB) (PB NB)INs] was was A450 and if the PIN value [Ps >2; PIN values were included to account for specimens with high background. This cutoff was approximately equivalent to the mean plus 3 standard deviations of 60 normal serum specimens. The same cutoff was used for all three assays. A significant rise in specific antibodies to RS virus was defined as a 250% increase in P - N values between acute- and convalescent-phase specimens, a factor exceeding 3 standard deviations above the mean difference between 20 paired specimens collected from persons with other virus infections. RESULTS Western blot. Of 23 acute- and convalescent-phase serum pairs from persons with recent RS virus infection, 14 had detectable rises in specific IgG antibody to one or more of the virus structural proteins, as determined by Western blotting. Antibody prevalence and response rates to the specific proteins are presented in Table 2, and the patterns of five representative patients are illustrated in Fig. 1. Only 2 of 10 (20%) persons with strong IgG responses to the G protein by Western blot had a detectable response with the corresponding capture immunoassay, whereas 4 of 6 (67%) and 10 of 10 (100%) individuals with responses to F and N by Western blot were detected by the respective capture immunoassays. Specificity of MAbs. The ability of biotinylated MAbs to detect RS virus proteins bound by serum antibody in capture immunoassays was evaluated by capturing RS virus antigens with unlabeled MAbs and then reacting them with biotinylated detector MAbs. As seen in Table 3, 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 at different antigenic sites and by F MAbs but were not reactive when captured by themselves or by N MAbs. Comparison of MAb and PAb assays. Of the two N MAbs and three F MAbs, h (N) and 131-2a (F) gave the best signal-to-noise ratios for their respective proteins. MAb h (N) assays correlated well with PAb assays for all immunoglobulin classes (Fig. 2). MAb 131-2a (F) assays had generally lower values but identified specific IgA in eight serum specimens that were negative by h (N). MAb 131-2g and 130-Sf (G) assays were insensitive for specific IgG and IgM antibodies, with activity confined to the IgA antibody class. Assays based on a pool of MAbs h (N) and 131-2a (F) detected specific IgG in 54 of 55 (98%), IgA in 51 of 52 (98%), and IgM in 16 of 17 (94%) serum specimens positive by the PAb assay; inclusion of MAbs to G protein did not improve assay sensitivity for any immunoglobulin class (data not shown). Ten additional serum specimens from seven persons with IgG or IgA responses to RS virus by both assays had detectable IgM antibodies by MAb but not by PAb assay. N MAb assays detected specific antibodies in both infants <1 year of age and adults, F MAb assays detected IgA and IgG in adults and IgA in infants, and G MAb assays only detected IgA in adults (Table 4). The MAb pool gave the best results for both age groups and for all Capture TABLE 3. Relative reactivity of MAb pairs to RS virus Reactivity of detector MAbb MAb Epitopea h (N) 131-4g (N) 131-2a (Fla) 133-lh (F2) 131-2g (Gi, 3) 130-5f (G4) h N g N a Fla lh F g G1, f G a See Table 1, footnote a. b Reactivity of MAb pairs is represented as a range from maximum (+ + +) to no detectable (-) reactivity.

4 VOL. 28, 1990 MONOCLONAL ANTIBODIES IN RS VIRUS SEROLOGY H(N) 131-2A(F) 131-2G(G) F(G) H(N) 131-2A(F) IgG '..0 4.z.Z3.T...:: IgA IgM Absorbance (P-N) 0.5 Absorbance (P-N) 0.5 FIG. 2. Comparison of P - N values of PAb and MAb assays for RS virus IgG, IgA, and IgM antibodies. Spearman rank correlation coefficients for the monoclonal pool h and 131-2a versus polyclonal antibody were r = (IgG), r = (IgA), and r = (IgM). immunoglobulin classes and was further evaluated in parallel with PAb assays for specificity. Specificity of MAb assays. The specificities of the optimized MAb assays were evaluated with serum specimens from 6 normal infants <2 months of age, 16 infants aged 2 to <24 months, and 60 adult blood donors. Specific IgG was detected in all infants <2 months old and all adults, IgA was 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, suggesting recent infection. Furthermore, specific IgM was not detected in the acute- or convalescent-phase serum specimens from 20 patients with diagnostic antibody rises to other 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 not be confirmed by PAb assay). Ten specimens positive for rheumatoid factor were negative for specific IgM antibodies. DISCUSSION The advantages of MAbs are apparent, but identifying those suitable as detector reagents for antibody immunoassays can :P. Il -J q., 1iizZZ prove problematic. Some have low affinity (26) or high nonspecific binding (personal observation). Others exhibit restricted strain 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 solid phase (7). Furthermore, MAbs must complement the proteinspecific antibody response of the host, which can vary with immunoglobulin 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 compatible with the host antibody response. Because RS virus is enveloped and disassociates into individual protein or protein complexes when exposed to the detergent Tween 20 present in the assay diluent, a single detector MAb may not identify all proteins captured by host antibody, and MAbs with different protein specificities may give different results with the same specimens. Our results validate this concern (detector MAbs to the F and G proteins failed to react with antigen captured by N MAbs and vice versa) and underscore the need to carefully evaluate detector MAbs during assay development.

5 2748 ERDMAN AND ANDERSON J. CLIN. MICROBIOL. 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 assays and response F G N Infants Adults Infants Adults Infants Adults Infants Adults IgG il Increase Decrease IgA Increase Decrease IgM Increase Decrease a PAb and MAbs 131-2A (F), 130-2G and 130-5F (G), and H (N) were used. For each test, sixteen infants were tested; five had acute- and convalescent-phase serum specimens. Twenty-seven adults were tested; fourteen had acute- and convalescent-phase serum specimens. As expected, the sensitivities of detector MAbs varied with immunoglobulin class and patient age. F MAbs were sensitive in assays for specific IgG in adults and IgA in adults and infants but were insensitive for IgM. G MAbs were moderately sensitive in assays for specific IgA in adults 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 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 specimens from healthy infants <2 months of age in which IgG of probable maternal origin was present, although IgM and IgA 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 from adults that contained IgA or IgG antibodies. Specific IgM was also absent in persons with other virus infections. A rise in specific IgG antibodies to RS virus in a child with herpes simplex virus type 1 infection may have been due to a mixed infection; paired sera from other patients with herpes infection did not show a similar rise in IgG or IgA antibodies to RS virus. The human antibody response to RS virus has been shown to include antibodies against all the RS virus structural proteins. In Western blot (8, 11) and indirect enzyme immunoassay (16, 23) responses to G are usually detected. Surprisingly, in our capture immunoassays, G MAbs detected very little antibody to RS virus demonstrable by Western blot; essentially no IgG or IgM but some IgA antibodies were identified. We suspect that a number of conditions conspired to limit the effectiveness of G MAbs in these 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 directly to the microtiter plates; (ii) multiple epitopes on the G protein could have been blocked or altered after binding to host antibody; and (iii) since the signal in capture immunoassays depends on the proportion of captured antibody with the desired specificity, it is possible that antibodies to the G protein represent only a small proportion of the total antibody response to RS virus. On the other hand, assays using N and F MAbs readily detected IgG antibodies to RS virus and, when combined, gave results as good as or better than those with PAbs for all immunoglobulin classes evaluated. 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