Journal of Immunological Methods, 109 (1988) 75-84 Elsevier
75
JIM04719
A rapid and simple ELISA for the determination of duplicate monoclonal antibodies during epitopeanalysis of antigens and its application to the study of CI-INH Jochem Alsenz and Michael Loos Institute of Medical Microbiology, Johannes Gutenberg University of Mainz, A ugustusplatz/ Hochhaus, 6500 Mainz, F.R.G. (Received 26 October 1987, accepted 27 November 1987)
A rapid and simple ELISA has been developed for identifying the specificities of two monoclonal antibodies recognizing either similar or distinct epitope(s) of an antigen. The method utilizes microtiter plates coated with one of the monoclonal antibodies either by direct adsorption of the purified antibody to the plastic or by immobilization of the antibody from ascites or hybridoma supernatants via immobilized polyclonal anti-mouse immunoglobulin antibodies. After preincubation of the antigen with the second monoclonal antibody, the mixture is added to the surface-immobilized first antibody. The amount of antigen bound to the first antibody is subsequently measured by rabbit polyclonal antibodies to the antigen and peroxidase-conjugated anti-rabbit immunoglobulin antibodies. Binding of antigen to the first antibody is only observed when the second monoclonal antibody binds to a distinct epitope. The major advantages of this procedure are its simplicity, rapidity and independence of radioisotopes. Using this method a library of monoclonal antibodies against human CI-INH has been tested and several duplicate monoclonal antibodies have been identified. Furthermore, the above analytical procedure was capable of detecting conformational changes of the CI-INH molecule induced either by binding of a monoclonal antibody to CI-INH or by enzymatic cleavage of CI-INH. Key words: Epitope analysis; Monoclonal antibody; Duplicate elimination; ELISA; CI-INH
Introduction
Monoclonal antibodies (MAb) provide powerful tools for the structural and functional analysis of proteins. The initial identification of hybridomas secreting the desired antibodies is usu-
Correspondence to: J. Alsenz, Institute of Medical Microbiology, Johannes Gutenberg University of Mainz, Augustusplatz/Hochhaus, 6500 Mainz, F.R.G. Abbreviations: CI-INH, human complement C1 esterase inhibitor: Clg, human complement C1 esterase; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; MAb, monoclonal antibody; PBS, phosphate-buffered saline.
ally very simple, although some of the antigen-presenting screening methods have certain drawbacks (Friguet et al., 1984; Brennand et al., 1986; Smith et al., 1986). A more critical step during the production of monoclonal antibodies is distinguishing between different MAbs. Extensive labor and redundant culturing can be avoided when hybrids secreting antibodies directed to the same epitope can be eliminated early in the MAb development process. To date, several methods have been developed to identify MAbs recognizing the same epitope of an antigen but each method has its limitations. Isolation of various regions of an antigen after en-
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
76 zymatic or chemical cleavage and testing of antibody binding is the oldest and probably most reliable method, but also the most difficult and laborious (Kaminsky, 1965). Nevertheless this approach has been considerably improved recently (Dowse et al., 1987). An alternative method, the ELISA additivity test, takes advantage of the fact that the amount of monoclonal antibody bound to an antigen is higher when a pair of MAbs bind to distinct epitopes, compared to the uptake when two MAbs recognize the same epitope (Friguet et al., 1983). More recently a new method for epitope analysis was described using FPLC Superose 12 gel filtration (Karand et al., 1987). Here radiolabeled antigen was mixed with the MAbs and the size of the resultant immune complex, determined by gel filtration, indicated whether the two MAbs recognized the same or different epitope(s) of the antigen. The most commonly employed method, however, is a competition assay in which the binding of a labeled antibody to an antigen is blocked by a second labeled antibody. This method requires the purification and labeling of at least one of the two antibodies (Tsu and Herzenberg, 1980; Stahli et al., 1983). To circumvent some of the difficulties and drawbacks of the various assays we have developed a novel method for the identification of duplicate MAbs. The new screening ELISA is simple and rapid, requires no labeled antigen or antibody, works with purified MAbs as well as with hybridoma supernatants or with ascites and uses soluble antigen to minimize the risk of antigen denaturation. In this paper we illustrate the efficacy and practicability of the new ELISA with MAbs to human Cl-esterase inhibitor (CI-INH) and present an application of the method for the detection of conformational changes of the C1INH molecule.
Materials and methods
Reagents All chemicals were analytical grade. 96-well microtiter plates were obtained from NUNC-Immuno (cat. no. 439454, Wiesbaden, F.R.G.). Rabbit anti-Cl-INH antibodies were purchased from Dako (Copenhagen, Denmark). Purified mouse-
IgG (cat. no. 5381) and diisopropyl fluorophosphate (DFP) were from Sigma Chemical Co. (Munich, F.R.G.). Affinity-purified goat antimouse-IgG Fc was from Jackson Immuno Research Laboratories (Dianova, Hamburg, F.R.G.). Affinity-purified goat anti-rabbit gamma globulins conjugated with peroxidase were purchased from Bio-Rad Laboratories (Munich, F.R.G.). Elastase from procine pancreas was obtained from Serva (116 U / m g ) (Heidelberg, F.R.G.).
Purification of human CI-INH Human C I - I N H was purified and quantitated as reported by Alsenz and Loos (1987). In addition the protein concentration was determined by the method of Lowry et al. (1951) and by the absorbancy of the preparations at 280 nm using specific extinction coefficients (Elcm) 1~ (Cooper, 1985). Elastase-cleaved C I - I N H was obtained by incubating 1 ml of C I - I N H (400/xg/ml PBS) with 1 ml of elastase (40 /~g/ml PBS) for 10 min at 37 ° C. The reaction was stopped by the addition of 1 mM DFP. Monoclonal antibodies Cell lines producing MAbs to human C I - I N H were generated and identified as previously described (Alsenz and Loos, 1987). MAbs were purified from ascites by 45% ammonium sulfate precipitation. Precipitates were resuspended and dialyzed against 10 mM ammonium hydrogen carbonate buffer p H 8.0 (1 mS) for 18 h at 4°C. The sample was applied to fast protein liquid chromatography (FPLC) Mono Q H R 5 / 5 column, equilibrated in the above buffer and adsorbed MAbs were eluted with a linear gradient of starting buffer and 600 mM ammonium hydrogen carbonate buffer pH 8.0 (40 mS). The protein concentration of the purified MAbs were determined by the method of Lowry et al. (1951) and titers estimated as follows. Microtiter plates were coated with 20/~1 of C I - I N H (5/~g/ml PBS) for 2 h at 4 ° C . 20 /~1 of serially diluted purified MAb, culture supernatant or ascites were added for 30 min and bound MAbs were determined with 20 /~1 of a 1 in 1000 dilution of peroxidase labeled goat anti-mouse IgG. For comparative studies the antibody titer was defined as the dilution of MAb at which 50% of maximal
77 binding occurred. The subclass of the MAbs were identified as described elsewhere (Alsenz and Loos, 1987).
ELISA The binding of MAbs to CI-INH-CI~ complexes and the influence of anti-Cl-INH MAbs on CT-INH-CI~ interactions was tested using the methods previously described for human anti-C1INH antibodies (Alsenz et al., 1987). Human serum and rabbit anti-human IgG conjugated to peroxidase used in these studies were replaced by MAbs to CI-INH and by peroxidase-labeled goat anti-mouse IgG (1 in 1000 dilution), respectively. For the detection of duplicate monoclonal antibodies in culture supernatants microtiter plates were coated with 20 #1 of goat anti-mouse IgG (1 in 300) for 2 h or overnight at 4 ° C. Plates were saturated, washed and 20 #1 of hybridoma supernatant added. After 30 min, the supernatants were removed and 20 /~1 of purified mouse IgG (20 /zg/ml) were added to each well for 30 min. When purified MAb to CI-INH was used as the first MAb, microtiter plates were directly coated with 20 /~1 of MAb (5 #g/ml) for 2 h or overnight at 4 ° C. 20/~1 of a constant amount of CI-INH were then preincubated with 20 #1 of serially diluted second MAb (supematant, ascites or purified antibody) in a V-shape microtiter plate for 30 rain and 20/~1 of the mixture were subsequently transferred to the microtiter plates coated with the first MAb. After incubation for 30 min, the plates were washed, and CI-INH bound to the first MAb was detected by 20 ~tl of rabbit anti-Cl-INH antibody (1 in 200 dilution) followed by 20 #1 of goat anti-rabbit IgG conjugated to peroxidase (1 in 1000 dilution). All other experimental conditions used in this study (e.g., buffers, incubation times and temperature) have been previously described (Alsenz and Loos, 1987).
Results
Initial characterization of monoclonal antibodies 13 out of 60 positive anti-Cl-INH clones were further analyzed in this study. The MAbs designated as 84E5, 86H7, 88G2, 90G1, 91D7, 91F8,
140, 128, 155, 13E1, 94C7, 95G7 and 95Fll were all found to be IgG1 antibodies. Measuring the influence of these MAbs on CI-INH functional activities and the binding of the MAbs to CI-INH under various conditions, three different groups of MAbs were identified. The first group of MAbs (MAb 140, 88G2, 95G7 and 95Fll) was found to strongly inhibit CI-INH binding to its target protease C1L In contrast, the second group of MAbs (MAbs 84E5, 86H7, 91F8, 155 and 13El) had no influence on CI-INH functional activity at all. However, both groups of MAbs recognized epitopes accessible in preformed CT-INH-CI~ complexes. The last category of MAbs (MAb 90G1, 91D7, 128 and 94C7) weakly inhibited CI-INH-CI~ interactions and poorly recognized CI-INH bound to CI~.
ELISA for the elimination of duplicate MAbs The ELISA was based on the theoretical consideration that an antigen preincubated with a MAb (second MAb) will not be recognized by a surface-attached first MAb, when the first MAb has the same epitope specificity as the second. A schema of the ELISA system is shown in Fig. 1A. In order to set up and optimize the ELISA system, the amount of first MAb required for precoating the microtiter plates was determined. After coating the ELISA plates with serial dilutions of purified MAb, CI-INH was added. Bound CI-INH was detected by polyclonal anti-Cl-INH antibody and peroxidase-conjugated anti-rabbit IgG antibody. From the resulting dilution curve, the optimal concentration of MAb (20 /~l/well) attached to the plate was found to be 3-5/~g/ml (results not shown). Next, the concentration of CI-INH antigen required was estimated. Microtiter plates were coated with 20/~1 of MAb 140 (5/~g/ml) for 2 h at 4°C. 20 #1 of various concentrations of CI-INH were preincubated with 20 /~1 of serially diluted MAb 140 (50 /xg/ml) for 30 min at 22°C and subsequently added to the microtiter plate for 30 min at 22 ° C. CI-INH captured by the immobilized MAb was finally detected by the addition of 20/zl of a 1 in 200 dilution of polyclonal rabbit anti-Cl-INH antibody followed by incubation with 20/~1 of a 1 in 1000 dilution of peroxidase-conjugated goat anti-rabbit IgG (Fig. 1B). To ensure that the
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Fig. 1. A: Diagrammatic representation of the ELISA for the detection of duplicate monoclonal antibodies. Microtiter plates were coated with the first MAb to CI-INH preincubated with the second MAb to CI-INH was then added, d - I N H bound to first MAb was detected by polyclonal rabbit anti-Cl-INH-antibody and peroxidase conjugated anti-rabbit-IgG antibody. B: Estimation of the optimal amount of Ci-INH antigen. Serial dilutions of MAb 140 (50/~g/ml) were preincubated with 4/~g (x), 2 / t g (o) or 1/Lg (zx) of CI-INH or with buffer CA). The mixture was added to microtiter plates coated with MAb 140 (5/~g/ml) and adsorbed CI-INH was determined as described above CA).
MAbs were competing for the same antigenic site of the C I - I N H molecule, the same MAb was used in this experiment for coating the plates and for preincubation of Ci-INH. The results shown in Fig. 1B demonstrate that the sensitivity of the ELISA was dependent on the amount of second MAb, as well as on the concentration of CI-INH. To obtain clear inhibition when using identical MAbs, even at low concentrations of the second MAb, the amount of C I - I N H was kept as low as possible, although high enough to guarantee detection by polyclonal anti-Cl-INH antibody. Therefore, a concentration of C I - I N H of 2 /~g/ml was used in all subsequent experiments.
An example of the application of the ELISA is given in Fig. 2A. Microtiter plates were coated with MAB 140 (5 /~g/ml) and MAbs 140, 88G2, 84E5 and 9 5 F l l were tested for binding to different or identical determinants on C ] - I N H as described above. Except for the hybridoma supernatant 9 5 F l l , all MAbs were purified and adjusted t o 100/xg I g G / m l . The antibody titer of the supernatant 9 5 F l l was about 10 times lower than that of the purified antibodies when tested with Cl-INH-coated microtiter plates. Compared to MAb 140 that served as a positive control only MAB 9 5 F l l inhibited C I - I N H binding to immobilized MAb 140, the antibody titers of the two
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Fig. 2. A: Ci-INH was preincubated with serial dilutions of purified MAb 140 (x), 88G2 (o) or 84E5 (zx)or with supernatant of hybridoma clone 95Fll (O). The mixture was added to microtiter plates coated with purified MAb 140 (5 /zg/ml) and CI-INH binding was monitored as described in Fig. 1A. B: Determination of the epitope specificities of monoclonal antibodies in hybridoma supernatants. Microtiter plates were coated with goat polyclonalanti-mouse IgG and incubated with the supernatant of hybridoma clone 95G7. Purified mouse-IgGwas added followedby Ci-INH preincubated with hybridoma supernatants 95G7 (x), 95Fll (zx)or 94C7 (o). CI-INH bound to microtiter plates was detected as described in Fig. 1A.
MAbs corresponding to the extent of their inhibitory capacity. Therefore, antibodies 140 and 9 5 F l l presumably recognized the same epitope. Indeed, this was not unlikely since both MAbs originated from a common positive well after the fusion. The other two MAbs, 88G2 and 84E5 did not inhibit C I - I N H binding and therefore were clearly distinct from MAb 140. For MAb 84E5 this result was not suprising since functional studies had already revealed differences. In contrast, MAb 88G2 had always reacted in a similar fashion to MAb 140 in functional assays and was, apparently, indistinguishable from it. In the ELISA described above, microtiter plates were always coated with purified MAb. In the early phase of monoclonal antibody production, however, only culture supernatants are available. Therefore, for direct screening of duplicate MAbs in two hy-
bridoma supernatants, the ELISA was slightly modified. In order to immobilize the first MAb from the culture supernatants, microtiter plates were precoated with 20/~1 of a 1 in 300 dilution of goat anti-mouse IgG antibody for 2 h at 4 ° C. 20 /~1 of hybridoma supernatant were added and incubated for 30 min at 22 ° C. Remaining binding sites of the anti-mouse I g G were then blocked by the addition of 20 #1 of purified mouse IgG (20 /~g/ml) for 30 min at 22 ° C. Afterwards the ELISA was processed as before. Some of the results obtained with culture supernatants are shown in Fig. 2B. Binding of C1I N H to MAb 95G7 was inhibited when C I - I N H was preincubated with the hybridoma supernatants 9 5 F l l and 95G7. The more efficient inhibition by MAb 9 5 F l l compared to MAb 95G7 can be explained by the three times higher anti-
80 TABLE I SCREENING FOR DUPLICATE MONOCLONAL ANTIBODIES BY ELISA Purified MAbs were analyzed for binding to the same or different antigenic site(s) as presented in Fig. 1A. For each pair of antibodies the reciprocal of the dilution of the second MAb (100/~g/ml) corresponding to a 50% inhibition of Ci-INH binding to the first MAb is shown. ELISA plate coated
CI-INH preincubated with MAb
with first MAb (20/~g/ml)
84E5
86H7
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.
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. . .
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b o d y titer of that s u p e r n a t a n t . I n c o n t r a s t n o i n h i b i t i o n was f o u n d with M A b 94C7, i n d i c a t i n g that this a n t i b o d y was n o t identical to M A b s 9 5 F l l or M A b 95G7. U s i n g the E L I S A described above, ten of the M A b s to C I - I N H were analyzed for b i n d i n g to different or identical epitopes. T h e results are s u m m a r i z e d in T a b l e I. All M A b s were purified a n d adjusted to 1 0 0 / t g ( I g G ) / m l . Each M A b was used for coating the plates (5 /~g M A b / m l ) , as well as for p r e i n c u b a t i o n with C I - I N H (2 /~g C I - I N H / m l ) . Therefore, T a b l e I includes two estimates for each pair of M A b s . T h e results in T a b l e I show that M A b s 84E5, 155 a n d 1 3 E l strongly i n h i b i t e d the b i n d i n g of each other, i n d i c a t i n g that these a n t i b o d i e s recognize the same epitope. Similar results were obtained with M A b s 90G1, 91D7 a n d 128. A strong i n h i b i t i o n of C I - I N H b i n d i n g was also f o u n d with M A b s 86H7 a n d 91F8. These two M A b s , however, p r e s u m a b l y did n o t have the same epitope specificity, since M A b 91F8 showed a n a d d i t i o n a l i n h i b i t i o n of d - I N H b i n d i n g to M A b s 90G1, 91D7 a n d 128 that was n o t f o u n d with M A b 86H7. T h e reason for the observed i n h i b i t i o n m a y have been steric h i n d r a n c e b e t w e e n the two antibodies caused b y the proximity of their respective antigenic d e t e r m i n a n t s .
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Detection of conformational changes in C-[-INH Some pairs of M A b s shown in T a b l e I revealed a n u n e x p e c t e d i n h i b i t i o n pattern. F o r example, p r e i n c u b a t i o n of C I - I N H with M A b 88G2, strongly i n h i b i t e d C I - I N H b i n d i n g to surface attached M A b s 90G1, 91D7 a n d 128. However, in the reverse system u s i n g i m m o b i l i z e d M A b 88G2, n o n e of these M A b s h a d a n y i n h i b i t o r y effect o n C I - I N H b i n d i n g . A n o t h e r interesting finding, n o t s h o w n i n T a b l e I, was a n increase in C I - I N H b i n d i n g to M A b s 90G1, 91D7 a n d 128 after preinc u b a t i o n of C I - I N H with M A b s 84E5, 86H7, 91F8, 140, 155 a n d 1 3 E l . These u n u s u a l results were o b t a i n e d o n l y w h e n the M A b s 90G1, 91D7 or 128 were attached to the surface. T o o b t a i n m o r e d a t a b e a r i n g o n the m e c h a n i s m of these p h e n o m e n a we first c o m p a r e d the b i n d i n g of M A b s 140 a n d 128 to i m m o b i l i z e d a n d to soluble C I - I N H . As shown i n Figs. 3A a n d 3B, M A b s 140 a n d 128 (10 t t g / m l ) b o u n d equally well to surface attached C I - I N H (2 /~g/ml). However, soluble C I - I N H (2 / x g / m l ) was recognized b y i m m o b i lized M A b 140 m o r e t h a n 100 times better t h a n b y M A b 128 ( 5 / t g / m l ) . T h e most likely e x p l a n a t i o n for these findings appears to be c o n f o r m a t i o n a l changes of the C I - I N H molecule. M A b 128 appeared to recognize a n epitope of C I - I N H partially h i d d e n w h e n the p r o t e i n was in solution b u t
81
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Fig. 3. Application of the ELISA to the analysis of CI-INH conformational changes induced by binding of monoclonal antibodies to CI-INH or enzymatic cleavage of Ci-INH. A: Binding of MAb 140 (zx, A) and 128 (o, O) to native d - I N H (zx, o) and elastase-cleaved CI-INH (A, o) immobilized on microtiter plates. B: Binding of native CI-INH (A, ©) and elastase-cleaved CI-INH (A, o) to microtiter plates coated with MAb 140 (zx, A) or MAb 128 (o, O). C: Influence of preincubation of CI-INH with serial dilutions of MAbs 86H7 (12), 140 (zx), 128 (o) or 88G2 (v) on the binding of native CI-INH to MAb 128 adsorbed to microtiter plates. D: Binding of elastase-cleaved CI-INH to MAb 128 coated microtiter plates after pretreatment of CI-INH with serially diluted MAb 86H7 (IN), 140 (A), 128 (O) or 88G2 (v).
b e t t e r e x p o s e d w h e n t h e C I - I N H w a s a d s o r b e d to t h e plastic. T h e i n c r e a s e d a v a i l a b i l i t y o f t h e b i n d i n g site o f M A b 128 in a d s o r b e d C I - I N H p r e -
sumably reflected a partial denaturation of the molecule. S e v e r a l o t h e r m e t h o d s a r e k n o w n to d e n a t u r e
82 antigens, including cleavage with enzymes. We therefore tested the binding of MAbs 140 and 128 to C I - I N H pretreated with elastase (enzyme: substrate = 1:10, 10 min, 37 ° C). A comparison between immobilized elastase-cleaved and native CiI N H showed no differences in antibody binding (Fig. 3A). In contrast, the binding of elastasecleaved C I - I N H to surface-bound MAb 128 was increased about 20-fold compared to native C]I N H (Fig. 3B). An increase in C I - I N H binding to immobilized MAb 128 was also found when native C I - I N H (2 /~g/ml) was preincubated with other MAbs, e.g., MAbs 84E5 and 140 (100/~g/ml) (Fig. 3C). This effect was markedly dependent on the concentration of the second MAb and peaked at ratios of antigen to antibody between 1 : 1 and 1 : 5. Higher or lower amounts of antibody were inefficient. When elastase-cleaved C I - I N H was used in the same experiment, the second MAb induced no enhanced binding of C I - I N H to MAb 128, presumably because the epitope recognized by MAb 128 was already exposed by the enzymatic cleavage of C I - I N H (Fig. 3D). Fig. 3C and Fig. 3D also show that binding of MAb 88G2 (100/~g/ml) to C I - I N H or elastase cleaved C I - I N H inhibited its binding to microtiter plates coated with MAb 128 (5 /zg/ml). The observed inhibition did not result from identical antibodies, since MAb 88G2 recognized soluble C I - I N H about 100 times better than MAb 128 (not shown) and since MAb 128 did not inhibit C I - I N H binding to MAb 88G2 (Tab. I). Therefore, the epitope recognized by MAb 128 seems to be poorly accessible in the native molecule. Immobilization of CI-INH, enzymatic cleavage of C I - I N H or binding of MAbs 140 or 84E5 to C I - I N H apparently induced a better exposure of the binding site for MAb 128. In contrast binding of MAb 88G2 to C i - I N H seemed to cause a significant decrease in the availability of the binding site for MAb 128.
Discussion
The ELISA screening test described in this report was shown to be a convenient and reliable method for the detection of MAbs recognizing the same or different epitope(s) on antigens. Using
this simple and rapid assay, 13 out of 60 MAbs to human C i - I N H were compared and MAb 84E5, MAb 128 and MAb 140 were found to have similar epitope specificities as MAbs 13E1/155, MAbs 9 0 G 1 / 9 1 D 7 / 9 4 C 7 and MAbs 9 5 F l l / 95G7, respectively. Compared to other methods reported, the ELISA system described has several distinct advantages. The assay neither requires labeled antigen nor labeled antibody, which is a prerequisite for the gel filtration method reported by Karande et al. (1987) and for the blocking solid-phase RIA described by Stahli et al. (1983). Moreover, purification of the MAbs is not necessary and ascites or culture supernatants can be used equally well in this assay. Thus, screening for duplicate MAbs can be performed very early after the fusion. In addition, because of the specificity of the monoclonal and polyclonal antibodies, crude antigen preparations can be utilized instead of highly purified antigen (not shown). This might be important in cases where the antigen is rare or difficult to purify. Other major advantages of our method result from the fact that the antigen is used in its soluble form. Results obtained with solid-phase systems such as dot immunoblot systems (Freeman et al., 1985), solid-phase RIA (Stahli et al., 1983) or the ELISA additivity test (Friguet et al., 1983) are not always reliable, since antigens immobilized by solid-phase techniques have been shown by several authors to undergo some denaturation (Friguet et al., 1984; Brennand et al., 1986). Antigenic sites available in the native antigen can be hidden after direct immobilization and conversely hidden determinants can be exposed as shown, for example, in this study for the binding site of MAb 128 in C I - I N H (Figs. 3A and 3B). In contrast, conformational alterations of the antigen are negligible in our assay, since both the first and second MAb are incubated with the antigen in solution. Furthermore, the ELISA is not limited by one MAb-binding site per antigen but also functions with oligomeric antigen, since preincubation of the antigen with the second MAb in the fluid phase results in complete saturation of all identical antigenic determinants. Therefore, the method circumvents the disadvantage of the classic double antibody system (Tsu and Herzenberg, 1980) where
83
sites of the same specificity may remain available to the second MAb after the antigen is bound to the insolubilized first MAb. In addition, it can be assumed that steric hindrance between two MAbs recognizing very close antigenic sites in an antigen is less in our system than in others. Compared to assays where the second MAb has to bind to an already immobilized antigen, the antigen complexed with one of the MAbs is still mobile when being incubated with the second, fixed MAb and therefore is more flexible in its binding reactions. An additional advantage of the new method is that there is almost no limitation in the molecular weight of the antigen to which the MAbs are raised. Experiments using MAbs developed against formaldehyde-inactivated leptospira have shown that the assay is not restricted to soluble antigens, but also functions with whole bacteria (not shown). Although the new ELISA has several obvious advantages, it shares with all other previously described methods the ambiguity that an inhibition of antigen binding to the first MAb by the second MAb can still be interpreted in three ways: that the MAbs recognize identical antigenic structures, that they bind to overlapping structures or alternatively that the MAbs are totally different and the inhibition results from a steric hindrance of antibody binding. Bearing this in mind, the data resulting from the new assay system have to be analysed very carefully and critically. Identity of two MAbs can only be assumed when both MAbs inhibit the antigen binding to each other and when the two MAbs reveal the same inhibition pattern when being tested with other MAbs (e.g., MAbs 155 and 13El). In the case where two MAbs block the binding of each other but show different inhibition pattern with other MAbs, it is very likely that the MAbs are different and that the inhibition between the two MAbs results from a steric hindrance of antibody binding (e.g., MAbs 86H7 and 91F8). When only one of the two MAbs alters the binding of the antigen to the other surface attached MAb, it is very likely that this MAb induces conformational changes of the antigen resulting in a less accessible or, alternatively, more exposed binding site for the second MAb. Indeed, some of the MAbs analyzed in this study showed such effects, as, e.g., MAb 88G2 that inhibited and MAb 140 that increased binding of
d - I N H to MAb 128. The assay system can, therefore, also be used to detect and analyze conformational changes of antigens induced, e.g., by antibody binding or by enzymatic cleavage (Fig. 3). In summary, a new method for the rapid and easy detection of duphcate monoclonal antibodies has been developed. The ELISA does not require labeled antigen or antibody and uses soluble antigen to minimize antigen denaturation or steric hindrance. In addition, the size of the antigen is not limiting and soluble monomeric or oligomeric antigens or bacteria can be tested equally well. The ELISA works with purified MAbs, ascites and culture supernatants and, therefore, can be used early in the development of MAbs.
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