Monoclonal capture antibody ELISA for respiratory syncytial virus: Detection of individual viral antigens and determination of monoclonal antibody specificities

Journal of Immunological Methods, 77 (1985) 247-258 Elsevier

247

JIM 03406

Monoclonal Capture Antibody ELISA for Respiratory Syncytial Virus: Detection of Individual Viral Antigens and Determination of Monoclonal Antibody Specificities * R. M i c h a e l H e n d r y 1,**, Bruce F. Fernie 2, Larry J. A n d e r s o n 3, Ellen Godfrey i and Kenneth McIntosh 1 I Division of Infectious Diseases, The Children's Hospital and the Department of Pediatrics, Harvard Medical School, Boston, MA 02115, : Department of Microbiology, Division of Molecular Virology and Immunology, Georgetown University Schools of Medicine and Dentistry, Rockville, M D 20852, and "~Respiratory and Enterovirus Branch, Division of Viral Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Heahh and Human Services, Atlanta, GA 30333, U.S.A. (Received 27 August 1984, accepted 15 November 1984)

An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus (RSV) that employs solid-phase monoclonal antibodies was developed. RSV antigens bound by these monoclonal capture antibodies were detected by addition of a polyclonal bovine antiserum, followed by anti-bovine enzyme conjugate and enzyme substrate. The sensitivity and specificity of the assay were determined by titrations of the solid-phase antibodies and by antigen titrations with both unpurified RSV-infected cell culture material and purified RSV nucleocapsids. The addition of a competitive binding step prior to the addition of antigen to the solid-phase antibody provides further evidence of the assay's specificity. Furthermore, the competitive binding assay enables the antigen specificity of monoclonal antibodies to be determined or compared without the use of purified antigens. Monoclonal capture ELISA is a convenient, rapid, and sensitive assay that can be used to measure specific RSV antigens in unpurified preparations as well as to determine anti-RSV antibody specificity and should prove useful in examining other complex antigen-antibody systems. Key words: ELISA - monoclonal antibody - respiratory syncytial virus proteins

Introduction One of the many uses of monoclonal antibodies in virology is for the identification of individual antigens in mixtures of proteins. The 2 major laboratory tech* Use of trade names and commercial sources is for identification only and does not imply endorsement by the Public Health Service or the U.S. Department of Health and H u m a n Services. ** To whom all correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

248 niques which are used for this function are immunoprecipitation and Western blotting. Both techniques have certain drawbacks. Immunoprecipitation methods generally require labeled antigens that must be either purified (Hunter, 1978) or subsequently analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Kessler, 1975). Some monoclonal antibodies fail to immunoprecipirate because of low affinity (Yolken, 1982), lack of binding to staphylococcal protein A (Richman et al., 1982), or lack of stable antigen binding under the conditions of the assay (Fernie et al., 1982). Western blotting, although not requiring labeled antigens, still requires electrophoretic separation and subsequent transfer to a solid phase prior to the detection of antigen-antibody binding (Burnette, 1981). Moreover, some monoclonal antibodies may be unable to bind to denatured antigens following their transfer to the solid phase (Erickson et al., 1982; DeBlas et al., 1983). We have sought methods for the immunochemical analysis of human respiratory syncytial virus (RSV) by using a panel of monoclonal antibodies (Cote et al., 1981; Kao et al., 1984) that would allow us to detect and quantitate individual viral antigens in unpurified preparations. In this report we describe an indirect enzymelinked immunosorbent assay (ELISA) that employs solid-phase monoclonal antibodies to capture specific RSV antigens. In addition, we have coupled this assay with a competitive binding step to define the specificity both of the antigens captured by the solid-phase antibody and of the competing antibody. The assay requires neither purified antigen nor labeled antigen or antibody.

Materials and Methods Cells and virus

HEp-2 cells, obtained from Flow Laboratories, were grown in 150 cm 2 flasks with Eagle's minimal essential medium (E-MEM) supplemented with 10% fetal bovine serum (FBS). The maintenance medium used for virus production was E-MEM with 2% FBS. RSV (long strain) was obtained from Robert Chanock (Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda. MD) and was plaque purified twice in our laboratory. After growth medium was removed, 2 ml of virus suspension in E-MEM was added to 150 cm 2 flasks of HEp-2 cells to give a multiplicity of infection equal to one. Virus was allowed to adsorb for 1 h at 37°C. Maintenance medium was then added, and the flasks were incubated at 37°C. When a 3 + to 4 + cytopathic effect was seen, the cells were scraped into the medium and disrupted with a probe sonicator for 1 rain at 4°C. Virus stocks and mock-infected HEp-2 cells were stored at - 7 0 ° C . Viral nucleocapsid was extracted from RSV infected HEp-2 cell lysates and purified by the method of Wunner and Pringle (1976). Antibodies

Horse anti-RSV neutralizing antiserum (HaRS) was obtained from Flow Laboratories. Bovine anti-RSV antiserum (lot M-117) raised in gnotobiotic calves (Cranage et al., 1981) was provided by E.J. Stott (Agricultural Research Council, Institute for Research on Animal Diseases, Compton nr. Newbury0 Berkshire). Monoclonal

249 a n t i b o d i e s were p r o d u c e d in the form of mouse ascitic fluids as d e s c r i b e d previously ( C o t e et al., 1981; C e p k o et al., 1983; K a o et al., 1984) (Table I). Mouse p l a s m a c y t o m a ( M O P C 21) p r o d u c e d in mouse ascites was o b t a i n e d from Bethesda Research L a b o r a t o r i e s . I m m u n o g l o b u l i n fractions were o b t a i n e d from mouse ascitic fluids by a m m o n i u m sulfate p r e c i p i t a t i o n at one third saturation, followed b y dialysis against p h o s p h a t e b u f f e r e d saline (PBS). T o t a l p r o t e i n was d e t e r m i n e d by the m e t h o d of B r a d f o r d (1976), m o d i f i e d for microtiter plates. A f f i n i t y - p u r i f i e d goat a n t i - b o v i n e and goat a n t i - m o u s e i m m u n o g l o b u l i n h o r s e r a d i s h p e r o x i d a s e conjugates were o b t a i n e d from K i r k e g a a r d and Perry L a b o r a t o r i e s .

Monoclonal capture E L I S A N i n e t y - s i x well flexible r o u n d - b o t t o m polyvinyl chloride microtiter plates (Dynatech L a b o r a t o r i e s ) were washed with distilled water before use. To d e t e r m i n e the b i n d i n g of m o n o c l o n a l a n t i b o d i e s to microtiter wells, i m m u n o g l o b u l i n fractions were diluted in PBS, and 0.1 ml was a d s o r b e d to microtiter plates overnight at 4°C. T h e contents of the wells were replaced with 0.1 ml of PBS plus 0.5% gelatin ( P B S - G ) a n d i n c u b a t e d 30 min at 37°C to block u n b o u n d sites on the plastic. The plates were washed 3 times (for 5 min each time) with PBS plus 0.05% Tween 20 (PBS-T) a n d shaken to remove excess fluid. G o a t a n t i - m o u s e i m m u n o g l o b u l i n c o n j u g a t e d to h o r s e r a d i s h p e r o x i d a s e was diluted 1 : 1 0 0 0 in P B S - G plus 0.15% T w e e n 20 ( P B S - T - G ) a n d 0.1 ml i n c u b a t e d in the wells for 1 h at 37°C. A f t e r a final wash with PBS-T, 0.125 ml of o r t h o p h e n y l e n e d i a m i n e ( O P D ) substrate (0.4 mg of O P D per ml in 0.15 M c i t r a t e - p h o s p h a t e buffer ( p H 5.5) c o n t a i n i n g 0.03% H 2 0 2 ) was a d d e d p e r well. C o l o r was allowed to develop in the d a r k for 30 60 rain at r o o m

TABLE I CHARACTERIZATION OF ANTI-RSV MONOCLONES Antibody designation

Protein Specificity (MW) a

lsotype b

13-1 16-2 18-3 25-2 26-2 6-3 10-F 1 446 2Hx-2 MOPC 21

F (66 K) NP (44 K) G (84 K) NP (44 K) NP (44 K) NP (44 K) G (84 K) P (33 K) Adenovirus hexon unknown

T1,K T2a,K T1,K T1,K y2a,K y2a,K T3,K 72a,K T2a,K

a As determined by immunoprecipitation (and/or immunofluorescence pattern). F = 66 kDa surface glycoprotein; G =84 kDa surface glycoprotein; NP =44 kDa nucleoprotein; P = 33 kDa phosphoprotein. b As determined by Ouchterlony immunodiffusion.

250

temperature, and the reaction was terminated by adding 0.025 ml of 3.5 M HCI per well. Absorbance at 490 nm was determined with an ELISA spectrophotometer (Model EL 307 IP Biotek Instruments, Burlington, VT). Outer wells of the microtiter plates received only substrate, and the spectrophotometer was adjusted to give a mean absorbance of zero on these wells. All assays were done in duplicate wells and reported as mean absorbance at 490 rim.

Monoclonal capture antibody ELISA was a modification of a method previously described (Hendry and McIntosh, 1982). Briefly, immunoglobulin fractions were diluted in PBS to a total protein concentration of 2 ~tg/ml, and 0.1 ml was added to each well. The plates were incubated at 4°C overnight or until needed. The wells were blocked with PBS-G and washed as described above. Unpurified RSV or purified nucleocapsid diluted in PBS-T-G plus 3 mM EDTA (PBS-T-G-E) was then added (0.1 ml per well) and incubated for 2 h at 37°C. Plates were washed 3 times with PBS-T, and 0.05 ml of bovine anti-RSV antiserum diluted 1 : 500 in PBS-T-G was added. The plates were then incubated for 1.5 h at 37°C. After another PBS-T wash, 0.1 ml of peroxidase-conjugated goat anti-bovine immunoglobulin diluted 1 : 1000 in PBS-T-G was added and incubated for 1 h at 37°C. Plates were washed with PBS-T and OPD substrate was added as described above.

Competitive binding EL1SA Capture antibody was adsorbed to plates as described above. Serial dilutions of competing antibodies diluted in PBS-T-G-E were added to equal volumes of RSV antigen diluted 1 : 25 in PBS-T-G-E and incubated overnight in glass tubes at room temperature. The subsaturating antigen dilution was determined from antigen titrations as described in the previous section. Capture antibody wells were blocked with PBS-G, washed with PBS-T, and 0.1 of the antigen-antibody mixture was added per well and incubated for 2 h at 37°C. The contents of each well were aspirated, and the plates were washed 3 times with PBS-T. RSV antigen bound was measured by the sequential addition of bovine anti-RSV, goat anti-bovine peroxidase, and OPD substrate, as above. Specific binding was determined by subtracting the mean absorbance obtained with control capture antibody (2Hx-2 or MOPC 21) from the mean absorbance of the anti-RSV capture antibodies for each dilution of competing antibody.

Results

Determination of monoclonal antibody binding to plastic' Ammonium sulfate-precipitated immunoglobulins were adsorbed to microtiter wells to determine their ability to bind to the solid phase (Fig. 1). In all cases, an input of 2 / ~ g / m l total protein saturated the available binding sites on the plastic, as evidenced by the lack of additional binding at the higher input concentration. Differences in the amount of anti-mouse conjugate binding to the individual

251 monoclones appeared to be independent of both the antigen specificity and the immunoglobulin subclass (Table I). The range of absorbances for conjugate bound at antibody excess (20/~g/ml) indicates that the amount of anti-mouse conjugate is not a limiting factor. Similar binding assays with a panel of 8 monoclones against G protein and 9 monoclones against F protein gave similar results (data not shown).

Titration of monoclonal capture antibodies The monoclonal capture antibodies were titrated against 1 : 5 0 dilutions of standard stock preparation of unpurified RSV and mock-infected HEp-2 cell supernatants, as well as a purified nucleocapsid preparation. Bound RSV antigens were detected with bovine anti-RSV antiserum followed by anti-bovine peroxidase conjugate and substrate. The results are shown in Table II. With the exception of clone 13-1, a total protein concentration of 2 /~g/ml capture antibody resulted in maximum binding of RSV antigen. Inputs of 20 ~tg/ml capture antibody did not result in increased antigen binding, even though the antigen concentrations used were in excess, as determined by ELISA with a polyclonal capture antibody. The ability of clones 6-3, 16-2, and 26-2 to bind purified RSV nucleocapsid is consistent with previously published data concerning their specificity for the nucleoprotein antigen (Cote et al., 1981). Clones 13-1, 18-3, and 10-F show no specific binding of nucleoprotein when compared with either control heterologous mouse monoclones (2Hx-2 and MOPC 21) or no capture antibody. Titration of viral antigens by monoclonal capture ELISA A titration of unpurified RSV antigen was performed with each of the monoclonal capture antibodies to determine the sensitivity of the assay (Table III). The low absorbances seen with mock-infected HEp-2 antigens illustrate the specificity of the assay for viral antigens. The degree of non-specific binding of RSV antigens to the solid phase is shown in wells in which either 2Hx-2 or no capture antibody is used. Although a low level of non-specific binding can be seen at the highest antigen

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Fig. 1. Measurement of mouse monoclonalantibody binding to polyvinylchloride by ELISA.

20.0 2.0 0.2

20.0 2.0 0.2

HEp-2

0.027 0.026 0.038

0.604 0.670 0.119 0.021 0.013 0.005

0.032 0.024 0.017

0.147 0.138 0.071

0.027 0.030 0.051

0.628 0.676 0.168

0.715 0.816 0.240

0.010 0.009 0.005

0.560 0.460 0.037

0.429 0.490 0.075

0.018 0.023 0.030

0.040 0.033 0.040

0.133 0.144 0.071

10-F

0.054 0.038 0.021

0.577 0.506 t).067

1.014 0.970 0.184

HaRS

0.031 t).029 0.031

0.039 0.042 0.051

0.085 0.074 0.086

2ttx-2

0.035 0.029 0.027

0.048 1/.037 0.t)45

0.081 0.089 0.091

M O P C 21

0.017

/).043

0.074

None

1.665 0.551 0.079 0.015 0.003 0.017

0.541 0.133 0.033 0.013 0.000 0.008

1.679 0.553 0.090 0.018 0.002 0.012

1.103 0.324 0.081 0.013 0.006 0.011

6-3

0.42 ! 0.098 0.016 0.006 0.006 0.016

10-F

1.436 0.841 0.219 0.061 0.028 0.1)32

HaRS

Boldface type indicates positive test. ~' 1:50 dilution of indicated antigens. RSV: unpurified infected cell cultures: HEp-2: unpurified uninfected cell cultures.

1.379 0.344 0.072 0.029 0.015 0.013

26-2

5 50 1 : 500 1 : 5 000 1:5

1 :

l :

18-3

RSV RSV RSV RSV None HEp-2

16-2

A b s o r b a n c e at 490 nm with indicated capture a n t i b o d i e s

Dilution

Antigen b

13-1

RSV A N T I G E N S IN M O N O C I . O N A L C A P T U R E E L I S A "

TITRATION OF UNPURIFIED

T A B L E Ill

0.151 0.038 0.013 0.013 0.019 0.021

2Hx-2

0.173 0.037 0.012 0.008 0.005 0.019

None

~' Boldface type indicates positive test. b 1:50 dilution of indicated antigens. RSV: unpurified infected cell cultures: NC: purified RSV nucleocapsids: l t E p - 2 : unpurified uninfected cell cultures. Total protein concentration of i m m u n o g l o b u l i n fractions a d d e d per well.

0.025 0.015 0.007

0.078 0.079 0.050

0.681 0.770 0.153

6-3

NC

0.323 0.435 0.609

26-2

20.0 2.0 0.2

18-3

RSV

16-2

A b s o r b a n c e at 490 n m with indicated capture a n t i b o d i e s

Capture concentration c ( I1g / m l )

Antigen b

13-1

C A P T U R E A N T I B O D I E S BY E L I S A ~

TITRATION OF MONOCLONAL

T A B L E II

253

concentration tested, specific binding by the anti-RSV monoclones can be clearly discerned in all cases. When compared with the heterologous monoclone (2Hx-2) or controls with no capture antibody, the polyclonal capture antibody (HaRS) detects a 1 : 500 dilution of unpurified RSV antigen. Clones 6-3, 13-1, 16-2, and 26-2 all detect RSV antigen diluted as much as 1 : 100. Clones 18-3 and 10-F are capable of detecting approximately a 1 : 10 dilution of unpurified RSV antigen. Similar results were obtained with a panel of 8 different anti-G and 7 different anti-F monoclonal capture antibodies in ELISA (data not shown). From the data in Table III, it is possible to determine for each monoclone the concentration of antigen at which capture antibody binding is saturated. This determination was necessary for the competitive binding assay. Competitive binding E L I S A in a rnonoclonal capture antibody system An ELISA was constructed to examine the ability of antibody to interfere with the binding of RSV antigen to capture antibody. Pre-incubation of limiting amounts of antigen with antibody results in antigen-antibody complexes that are then undetectable in the monoclonal capture ELISA system. This is presumably because the complexes are unable to bind to capture antibody that recognizes the same antigenic site. Antigen binding in this system is then measured by sequential incubations with a polyclonal bovine anti-RSV serum followed by a goat anti-bovine peroxidase conjugate and enzyme substrate. Fig. 2 shows the results of experiments in which limiting amounts of unpurified RSV antigen (1 : 50 final dilution) were pre-incubated with varying concentrations of competing antibodies. The antigen-antibody mixture was added to microtiter plates containing 2 ~ g / m l of capture antibody, and the amount of bound RSV antigen was subsequently measured as outlined above. In Fig. 2A, the mean absorbance of duplicate wells is plotted against the competing antibody concentration with 13-1 used as the capture antibody. Only the homologous competing antibody 13-1 and the polyclonal serum show inhibition of RSV binding when 13-1 is used as capture antibody. Monoclonal competing antibodies against either RSV nucleoprotein (6-3, 16-2, 25-2, 26-2), G protein (18-3, 10-F) , or adenovirus hexon (2Hx-2) show no effect on antigen binding by 13-1 capture antibody at the concentrations tested. Furthermore, the absorbances obtained with the heterologous competing monoclones are not significantly different from those seen in the absence of any competing antibody, indicating that non-specific binding of heterologous RSV antigens by 13-1 capture antibody is not detectable. A similar experiment was performed with an antinucleoprotein monoclonal capture antibody, 26-2 (Fig. 2B). In this case, both the homologous (26-2) and heterologous anti-nucleoprotein (6-3, 16-2, 25-2) competing monoclones show similar competitive inhibition of antigen binding by clone 26-2 capture antibody, confirming the antigen specificity of these monoclones. The polyclonal competing antibody also inhibits binding of antigen by 26-2 capture antibody, although to a lesser extent, as in Fig. 2A. Competing monoclonal antibodies against heterologous RSV antigens as well as adenovirus hexon showed no effect on antigen binding.

254

Reciprocal experiments with each of the antinucleoprotein antibodies as capture antibody and the same panel of competing antibodies all gave similar patterns (data not shown). In a third experiment, horse anti-RSV hyperimmune immunoglobulin was used as capture antibody (Fig. 2C). Only the homologous polyclonal competing antibody

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A, COMPETING ANTIBODY

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.550 250 150 0

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255

,35T ¸

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Fig. 3. Results of competitive binding ELISA with monoclone 1446 competing antibody.

was able to inhibit antigen binding completely, although monoclone 13-1 inhibited binding by approximately 40%. All other competing antibodies showed little or no effect on antigen binding by HaRS capture antibody.

Identification of monoclonal antibody specificity by capture competition ELISA The use of competitive binding ELISA to examine the antigenic specificity of a monoclone relative to antibodies of known specificity is demonstrated by the similar competitive binding curves obtained with all 4 anti-NP monoclones (Fig. 2B). The assay was also applied to characterize a monoclonal antibody that recognizes an antigenic site distinct from those seen by NP, G, or F monoclones (Fig. 3). In this assay various capture antibodies were adsorbed to microtiter plates, and a limiting dilution of unpurified RSV antigen was preincubated with dilutions of a single competing monoclonal antibody of unknown specificity. To eliminate differences in the binding capacity of the different monoclones (as seen in Table III), the mean absorbance in the absence of competing antibody for each capture was assumed to be 100%. The binding as a percent of control (no competing Ab) was computed as ( A , / A o ) × 100, where A x equals the A490 at each dilution of competing antibody and A o equals the A490 in the absence of competing antibody. A monoclonal antibody designated monoclone 1446, thought to recognize the 33 K MW putative phosphoprotein of RSV, was used as the competing antibody (Fig. 3). Significant inhibition of binding was seen only with the homologous combination, indicating that monoclone 1446 recognized an antigenic determinant distinct from those recognized by the monoclones against NP, F, or G proteins. Subsequent immunoprecipitation of [3H]leucine-labeled RSV infected cell extracts with clone 1446 showed a 33 K MW band by SDS-PAGE (data not shown). Discussion

We have shown that monoclonal antibodies can be used as solid-phase immunosorbents to measure viral antigens in unpurified preparations. By addition of a

256

competitive binding step, the antigen specificity of unlabeled monoclonal antibodie~s can be determined. The test is simple and does not require labeled material or harsh treatment of the antigens involved. In general, the specificity of solid-phase sandwith immunoassays has not depended solely on the specificity of the capture antibody. An exception is the IgM capture assay, by which serum IgM antibody to virus or other microorganisms is measured (Isaac and Payne, 1982). We have demonstrated that specificity at this level is possible with the RSV system and is, moreover, useful for a virus in which the individual proteins cannot be readily purified. Monoclonal capture competition ELISA was used to examine the antigen specificity of monoclonal antibodies (Figs. 2 and 3). Cote and Gerin (1983) and Cote et al. (1984) have described a similar method that uses an 1251-labeled monoclonal antibody as detector to map antigenic sites on woodchuck hepatitis virus surface antigen and found good agreement with conventional competitive binding radioimmunoassays. The assay described here differs in that an unlabeled polyclonal antiserum followed by a peroxidase-labeled anti-species conjugate is used to measure antigen binding. Antibody specificity can be determined in the positive case by the observation of competition with an antibody of known specificity, as in the case of the anti-nucleoprotein monoclones (Fig. 2B). Because most proteins contain multiple epitopes, some of which may be non-overlapping, antibody specificity in negative cases is more difficult to determine. The lack of competition with monoclones of known specificity (Fig. 3) cannot be used to determine absolutely the protein specificity of a given monoclone. Through such an analysis it is possible only to infer that the competing monoclone binds an epitope distinct from those bound by the panel of capture antibodies. The assay is specific for both unpurified viral antigens and purified nucleocapsids (Table It). Low signals are observed when heterologous capture antibodies or mock-infected antigen preparations are used. When unpurified viral antigens are titrated by monoclonal capture EL1SA (Table III), similar evidence for specific antigen binding is apparent, although differences in the relative amount of antigen measured can be seen among the different capture antibodies used. Several factors may account for these differences. Although the avidity of solid-phase antibody and the variable kinetics of solid vs. fluid-phase interactions may play a role (Rubin et al., 1980; Kennel, 1982), a panel of 8 anti-G monoclones all exhibited a similar 10-fold decrease in the amount of specific antigen detected, compared with a panel of 7 anti-F monoclones (data not shown). Therefore, the lower specific G signal in unpurified preparations is probably due to the lower relative concentration. A final consideration is that the detector antiserum must be of high avidity, specific for a broad range of antigenic determinants, and free from non-specific reactions, particularly when crude antigen preparations are used. Further evidence of the antigen binding specificity of monoclonal capture antibody ELISA is demonstrated by competitive binding assays (Fig. 2). In Fig. 2A, 13-1 competing antibody strongly inhibits antigen binding to 13-1; HaRS does so to a lesser extent. The apparent 10-fold difference between 13-1 and H a R S inhibition of binding is likely a result of the lower proportion of antigen-specific antibody in the

257

hyperimmune serum. Similar differences are apparent between the homologous competing anti-nucleoprotein monoclone, 26-2, and HaRS (Fig. 2B). The similar competition patterns of the other 3 nucleoprotein monoclones (6-3, 16-2, 25-2) (Fig. 2B) and in reciprocal competition experiments (not shown) indicate that these monoclones recognize the same antigenic site on the nucleoprotein. When polyclonal capture antibody (HaRS) is used (Fig. 2C), monoclonal competing antibodies appear to have little or no effect on antigen binding, with the exception of clone 13-1. The decreased antigen binding by HaRS capture in the presence of 13-1 competing antibody may be due to interference by 13-1 with detector or capture antibody binding or both. In either case, this would suggest that antibodies of 13-I-like specificity constitute a significant proportion of the total in our polyclonal sera. A possible limitation of the monoclonal capture ELISA is that only polyvalent antigens can be detected, since bound antigen is measured by a polyclonal detector antiserum. For efficient binding of capture monoclones to the solid phase, immunoglobulin preparations should be used. We have found that a single ammonium sulfate precipitation gives satisfactory results with little or no loss of antigen binding capacity. Allosteric enhancement and inhibition due to antibody binding have been described with monoclonal antibodies (Lubeck and Gerhard, 1982; Heinz et al., 1984) and may affect the results obtained in this assay. Furthermore, some monoclonal antibodies preferentially recognize antigen in either the solid or liquid phase (Lehtonen, 1981; Kennel, 1982; Cote et al., 1984; Micrendorf and Dimond, 1984) and therefore may be unable to either capture or compete in this assay. Although the specificity of the capture antibody-antigen binding seems well demonstrated, contamination of specifically captured antigens with adherent proteins that can be recognized by detector antibody cannot be ruled out by our data. In systems designed to examine the purity of captured antigens, as described for murine leukemia virus proteins (Tamura et al., 1984), such contamination may affect assay specificity. In the operational context of the assays for viral antigens or antibodies described here, however, the purity of the captured antigens does not seem to affect the results obtained. If heterogeneous complexes are indeed captured by the monoclonal antibodies, then competing antibodies might be expected to block determinants recognized by the detector antibody and thereby decrease the specific signal. This clearly did not occur in the experiments described here. In no instance did heterologous competing antibodies decrease absorbance when compared with wells with no competing antibody (Figs. 2 and 3). Thus, while the possible impurity of captured antigens represents a theoretical argument against the specificity of competitive inhibition at the solid-phase monoclonal antibody level, this did not in fact prove to be true in our experience. The advantages of this assay include the ability to specifically bind and detect specific antigens in crude preparations with good sensitivity. When applied to the study of viral antigens, it can allow relative quantitation of specific viral antigens from cell culture or clinical specimens. Furthermore, the assay can be used to examine strain-specific epitopes of viral proteins that would be undetectable with conventional polyclonal antisera. In competitive binding assays the main advantages of the assay are that the

258 c o m p e t i n g a n t i b o d i e s n e e d n o t b e l a b e l e d a n d u n p u r i f i e d a n t i g e n c a n b e used. A single unlabeled polyclonal serum combined with an enzyme-labeled antispecies c o n j u g a t e c a n b e u s e d to p e r f o r m t h e a s s a y in a m i c r o t i t e r f o r m a t . T h e a s s a y is r a p i d a n d e a s y to p e r f o r m , a n d l a r g e n u m b e r s of s a m p l e s c a n b e a n a l y z e d w i t h o u t t h e n e e d for r a d i o i s o t o p e s o r e l e c t r o p h o r e t i c s e p a r a t i o n s . We are currently using monoclonal capture ELISA and the accompanying c o m p e t i t i v e b i n d i n g a s s a y to e x a m i n e t h e q u a n t i t a t i v e a n d q u a l i t a t i v e v i r a l a n t i g e n c o n t e n t o f c l i n i c a l s p e c i m e n s f r o m p a t i e n t s d u r i n g i n f e c t i o n w i t h R S V , to e x a m i n e t h e n a t u r e o f t h e s e c r e t o r y a n t i b o d y r e s p o n s e to s p e c i f i c v i r a l a n t i g e n s a n d to e x a m i n e s t r a i n v a r i a t i o n s a m o n g v i r a l isolates. M o n o c l o n a l c a p t u r e E L I S A s h o u l d significantly increase the resolution of antigenic analysis of RSV and other microo r g a n i s m s w h e r e s u i t a b l e m o n o c l o n a l a n t i b o d i e s are a v a i l a b l e .

Acknowledgements This work was supported by Public Health Service Contracts NOI-AI-92617 NOI-AI-22665 from the National Institutes of Health. W e t h a n k W a l t e r S i e d l e c k i f o r a s s i s t a n c e in p r e p a r a t i o n o f t h e m a n u s c r i p t .

and

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