©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Library of Monoclonal Antibodies to Escherichia coli K-12 Pyruvate Dehydrogenase Complex
COMPETITIVE EPITOPE MAPPING STUDIES (*)

(Received for publication, August 19, 1994; and in revised form, June 19, 1995)

Alan J. McNally (1) (2) Lars Mattsson (3) Frank Jordan (2)(§)

From the  (1)Roche Diagnostics Systems, Inc., Somerville, New Jersey 08876-1760, (2)Rutgers University, Departments of Chemistry and Biology, Newark, New Jersey 07102, and (3)Pharmacia Biosensor, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Presented here are competitive epitope mapping studies on a monoclonal antibody library to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC1.2.4.1) subunit (E1). Several of the monoclonal antibodies had been found to inhibit PDHc from 0 to 98%. Of the 10 monoclonal antibodies that showed the greatest inhibition of PDHc, 4 were elicited by PDHc and 6 by E1. Surface plasmon resonance was used for competitive epitope mapping and revealed that these 10 monoclonal antibodies had at least 6 separate binding regions on the PDHc. The three monoclonal antibodies that demonstrated the strongest inhibition appeared to bind the same region on the PDHc. Mapping studies with the E1 antigen using an additional five monoclonal antibodies demonstrated that the two strongest inhibitory monoclonal antibodies (18A9 and 21C3) shared the same binding region on E1, whereas the third strongest inhibitor (15A9) displayed an epitope region that overlapped the previous two on the E1 subunit. Antibody 15A9 had been shown to counteract GTP regulation of PDHc. Simultaneous multiple site binding experiments confirmed that the defined epitope regions were indeed independent. Limited competitive epitope binding experiments using radiolabeled E1 confirmed the surface plasmon resonance results.


INTRODUCTION

Pyruvate dehydrogenase (PDHc) (^1)is a multienzyme complex that is found in both prokaryotic and eukaryotic cells. The enzyme complex catalyzes the oxidative decarboxylation of pyruvate in the following overall reaction (1)

REACTION 1

In the preceding manuscript, we described a library of monoclonal antibodies (MAb) generated to the Escherichia coli PDHc antigen. In a MAb library, the relationship of the individual MAbs to each other is of significance. As a prelude to direct epitope mapping, we initiated competitive epitope mapping studies. Direct studies generally use specific peptide fragments, and immunoreactivity may be detected by one of several methods such as radioimmunoassay, Western blot, and ELISA. Competitive methods, on the other hand, determine region binding areas recognized by two MAbs relative to each other. In this method, binding matrix tables can be set up, exploring binding regions relative to the number of MAbs used within the matrix. As one increases the number of MAbs in the binding matrix, better resolution of specific binding regions or epitopes becomes apparent. The competitive method, given a large binding matrix pattern, can offer a more global look at many MAbs binding to the same antigen. For this reason, competitive mapping experiments were conducted on a total of 15 of the MAbs previously described (10 for PDHc and with 5 additional antibodies from the E1 group).

A novel biosensor technique, surface plasmon resonance (SPR), was used to study these MAbs for epitope similarity. Surface plasmon resonance was first put to practical use in 1968 (2, 3) and was later described in detail in 1977 and 1991(4, 5) . The SPR technique creates an optical phenomenon that takes place at the interface of two transparent media of different refractive index. As polarized, monochromatic light is directed at the two media, whose interface has been coated with a defined thin layer of metal, at a critical angle defined by the media; the light is totally reflected back into the transparent media, which possesses the higher refractive index(6) . At angle , the incident or resonance light is totally absorbed inside the metal of high dielectric constant (Fig. SI). At angle , an electromagnetic field is created and oscillates through the metal conducting media, creating surface plasmons(7) . For resonance to occur, the vector component of the incident light momentum in the plane of the surface must match the momentum of the surface plasmons(8) . The detection unit then views that finite angle as an intensity dip in the reflected light (Fig. SII).


Figure SI: Scheme ISurface plasmon resonance phenomenon. This scheme is a visual presentation of SPR. SPR occurs under the conditions of total internal reflection depicted in the upperhalf of the diagram at a critical angle . This phenomenon uses monochromatic light directed through a prism toward another media with a lower refractive index (n(2)). In the lowerhalf of the scheme, plasmon resonance is depicted by the loss of light at a critical angle as viewed by the detector. At this critical angle, the energy is transmitted through the metal film, and an evanescent wave is formed. For further details of this phenomenon, see text. This scheme is reproduced with permission of Pharmacia and is part of the Pharmacia BIAcore Biosensor AB operator's system manual. n(1), refractive index 1; n(2), refractive index 2; d, thickness of metal film; I, monochromatic light at 760 nm.




Figure SII: Scheme IISurface plasmon resonance vector components. Surface plasmon resonance arises when the vector component of the incident light parallel to the plane of the surface matches the momentum of the surface plasmons. The magnitude of this vector component varies with incident angle.



Pharmacia has developed an instrument BIAcore Pharmacia Biosensor AB (9) and a sensor chip, CM5, that is used to perform SPR. A light-emitting diode (wavelength 760 nm), which produces a range of incident angles (66-69°), and a photodetector aligned with 16 pixel rows over the 4° degrees enable changes in the reflection angle to be observed. Given the media chosen for the CM5 chip and the wavelength of light selected, the SPR phenomenon occurs within this 4° angle change, and that angle at which SPR occurs is seen by the detector as a dip in reflected light intensity. SPR is dependent on several factors including the properties of the metal film, the wavelength of the incident light, and the refractive index of the media on both sides of the metal film. The Pharmacia instrument maintains all of these factors constant. The only change occurring during the observation is in the refractive index of the media on the flow cell side of the sensor chip. This is caused by protein binding to the surface of the chip, causing a density change that translates into a change in refractive index at that surface. This will cause the conditions for the resonance to change, resulting in a change in the resonance angle. Stenberg et al.(10) has shown a linear correlation between resonance angle shift and protein surface concentration. For proteins low in carbohydrate, the refractive index increment was determined to be constant and independent of molecular size and amino acid composition (11) . Measurements are taken over time as the ligands are reacted with the surface, and the instrument's software transforms the changing resonance angle into resonance units (RU), defined such that 0.1° angle change corresponds to 1 ng/mm^2 protein uptake on the sensor chip(10) .

The technology has recently been shown to be useful in studying antigen-antibody interactions(12, 13, 14) . This technique has advantages over other mapping techniques because it requires no purification of the MAb ascites and no labeling of the antigen. The interactions of the MAbs with their antigens are truly within their native state(15) . Fagerstam et al.(14) has used the technology for epitope mapping of nonrepetitive epitopes on the HIV core protein, p24.

Here, we show the applicability of the SPR technology on the Pharmacia BIAcore for mapping large multienzyme complexes. Our results reveal that at least 6 different epitopes are recognized by the 10 MAbs prepared to PDHc. Of these, three antibodies that gave rise to maximum inhibition were shown not only to bind the E1 subunit but also to bind in the same region on the E1 molecule with overlapping epitopes.


MATERIALS AND METHODS

ELISA plates were purchased from Costar. Removable microtiter plate wells were purchased from Dynatech Laboratories Inc. The sensor chip CM5, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, ethanolamine, affinity-purified rabbit antimouse IgG1 (RAMG1), surfactant P20, and block mouse IgG1 (Sigma) were all generously donated by Pharmacia Biotech Inc. Instrument time on the BIAcore instrument was also donated by Pharmacia.

Radiolabeling of E1

Radiolabeling of the E1 antigen was performed using the Bio-Rad enzymobead radioiodination reagent according to Morrison and Bayse(16) , and the material was placed on a 1 30-cm Sepharose G-100 column, 100 mM Tris-HCl, pH 7.5, with 0.02% NaN(3). The first radioactive peak was pooled, and 1% bovine serum albumin was added. Specific radioactivities were determined to range from 2.0 to 3.0 mCi/µg.

Surface Plasmon Resonance Epitope Mapping Studies

A CM5 sensor chip was inserted into the BIAcore instrument. The sensor chip consisted of three main layers: a flat glass slide, a thin layer of gold (50 nm), and a non-cross-linked carboxymethylated dextran, which was covalently attached to the gold via a linker layer(17) . After calibration of the detector according to the system manual, the system was equilibrated with 20 mM potassium P(i), 150 mM KCl, 0.05% P20, pH 7.0 (running buffer), for 8 minutes. To immobilize the RAMG1 on the CM5 chip, the instrument was first programmed to mix 0.4 MN-ethyl-N`-(3-dimethylaminopropyl)carbodiimide and 100 mMN-hydroxysuccinimide at a ratio of 1:1 in water and then to immediately inject 35 µl into the instrument at a flow rate of 5 µl/min for 7 min. Next, 35 µl of RAMG1 at 30 mg/ml in 10 mM sodium acetate buffer, pH 5.0, was injected at a flow rate of 5 µl/min for 7 min. This was followed by 30 µl of 1 M ethanolamine-HCl, pH 8.5, to inactivate any remaining activated carboxyl groups. To the final surface, 5 µl of 1 M formic acid was injected to prepare the surface for use, as recommended by the manufacturer(9) . The formic acid treatment eliminates all secondary MAb that is formic acid sensitive.

To generate the PDHc sensorgrams necessary for the epitope map, each ascites fluid was diluted 1:100 in the running buffer. The block IgG1 MAb was diluted to 50 µg/ml in 10 mM Hepes buffer, pH 7.0, and the PDHc antigen was diluted to 500 µg/ml in 20 mM potassium P(i) buffer with 0.1% bovine serum albumin. The instrument was programmed to run at 5 µl/min, and then 20 µl of the first MAb was injected. To block the remaining sites on RAMG1, 15 µl of the mouse IgG1 block MAb was injected. The instrument was then programmed to mix PDHc 1:10 with 10 mM MES buffer, pH 5.5, and to also inject 30 µl of the mixed antigen. Since PDHc has multiple, repeating subunits, a 30-µl reinjection of the first MAb was performed. Finally, 30 µl of the second MAb was injected, and the sensorgram was completed. The response of the secondary MAb was measured as the difference between the base line before and after the injection of the secondary MAb. To regenerate the surface for the next run, two 5-µl injections of 100 mM HCl each were followed by 10 µl of 1 M formic acid.

The generation of the E1 sensorgrams used ascites fluid at 1:50 dilution, and the mouse monoclonal IgG1 block was the same as described above. The E1 antigen was diluted in running buffer to 60 µg/ml. 10 µl of the first MAb was injected for 2 min, and this was followed by the block antibody as described above. The instrument would then inject 15 µl of E1 antigen for 3 min, followed by a 15-µl injection of the second MAb over 3 min. Once the net SPR signal from the second MAb was measured, the chip surface was regenerated with HCl and formic acid (Fig. SIII).

Competitive Epitope Mapping Binding Assay Using Radiolabeled E1

HPLC-purified MAbs were coated onto microtiter plates at 10 µg/ml, pH 7.5, for 2 h at 25 °C. The plates were washed and blocked. To each separately coated antibody plate well, 50 µl of radiolabeled E1 (approximately 10,000 cpm) and 50 µl of one purified MAb at various stated concentrations was added. Competition between the bound and the soluble MAb for the labeled E1 was allowed to occur overnight at 4 °C. The plates were then washed, each well was removed, and the contents were counted in a counter. B/B(o) plots were then generated.


RESULTS

Since all two-site binding assay cycles used common capture in the SPR technology, the first step in generating a matrix was to covalently link RAMG1 to the CM5 chip surface. Fig. 1illustrates a typical immobilization sensorgram of RAMG1. The amount of RAMG1 immobilized was readily monitored from chip to chip by observing the total RU uptake after coupling each chip. To ensure good capturing activity of the RAMG1, a single channel on any chip was not used for more than 50 cycles. The RAMG1 uptake at each covalent linkage had an average of 10,200 RU with a coefficient of variation of less than 10%.


Figure 1: Sensorgram of the immobilization of RAMG1 to the CM5 sensor chip surface. 1, finished activation of the chip with N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide; 2, finished uptake of RAMG1 to surface; 3, deactivation of the surface with ethanolamine; 4, RAMG1 surface conditioned with HCl. The arrows indicate the completion of the designated step.



Fig. 2and Fig. 3illustrate two typical, two-site binding sensorgram cycles. Each sensorgram comprised a potential sandwich assay. Each sensorgram assay used one MAb as a primary antibody, captured by RAMG1, and the other MAb as a secondary antibody, binding the PDHc antigen. Fig. 2shows binding interference; MAb 2 was not able to bind PDHc antigen because MAb 1 has its epitope in the same binding region. Fig. 3demonstrates a positive no interference binding pattern, with MAb 2 being able to bind PDHc antigen, thus having a different epitope binding region than MAb 1. The lower resonance signal observed during the IgG1 block injection was due to the lower ionic strength of blocking buffer. Sensorgrams for the E1 map were similar, and only a typical positive, no interference sensorgram is illustrated in Fig. 4. To establish a statistically valid RU cutoff value for positive second MAb binding, each primary MAb was also run as a secondary MAb against itself. The sequence of events then followed was: primary MAb, block MAb, PDHc or E1 antigen, primary MAb, and the same primary MAb previously used as a secondary MAb for the PDHc map. For the E1 map, a second injection of primary MAb was not utilized because only one antigen was present in purified form. The MAb that resulted in the highest secondary mean antibody response was utilized. A cutoff was established by taking four standard deviations above this mean. This number resulted in 290 RU for PDHc and 150 RU for E1 epitope mapping.


Figure 2: Typical negative interference PDHc sensorgram. 1, uptake of first (primary MAb 18A9); 2, uptake of block IgG1 antibody; 3, uptake of PDHc antigen by first MAb 18A9; 4, reinjection of primary MAb 18A9; 5, uptake of secondary MAb 21C3; 6 and 7, surface reactivation with HCl. The arrowsindicate the completion of the designated step.




Figure 3: Typical positive no interference PDHc sensorgram. 1, uptake of first (primary MAb 18A9); 2, uptake of block IgG1 antibody; 3, uptake of PDHc antigen by first MAb 18A9; 4, reinjection of primary MAb 18A9; 5, uptake of secondary MAb 1F2; 6 and 7, surface reactivation with HCl; 8, surface reactivation with formic acid. The arrowsindicate the completion of the designated step.




Figure 4: Typical positive no interference E1 sensorgram. 1, uptake of first primary monoclonal antibody (1G5); 2, uptake of block IgG1 antibody; 3, uptake of E1 antigen by the first MAb; 4, uptake of secondary MAb (15A9); 5, surface reactivation. The arrowsindicate the completion of the designated step.



Statistical criteria for specific binding of the primary antibody to PDHc and E1 antigen were also established. Nonspecific binding of PDHc and E1 to the matrix was established by using the blocking MAb as a primary antibody and then establishing the mean nonspecific binding of the PDHc and E1 to the matrix over several runs and several chips. To this established mean, four standard deviations were added, and this then resulted in an estimate of 1250 RU for PDHc and 45 RU for E1. All primary MAbs would then require an antigen binding greater than this predetermined number to ensure a specific binding signal. The PDHc uptake by the primary antibodies always had greater than a 3000 RU signal with a cv of less than 11 percent. In addition, the E1 uptake by the primary antibodies was always greater than a 450 RU signal with a cv of less than 4%.

The 10 10 PDHc binding matrix presented in Fig. 5was generated as described above. To ensure reproducibility, all experiments were also run in reverse order for each sandwich system. A reverse order was defined as follows: the MAb that was originally used as the capture antibody (MAb 1) became the tag, and MAb 2, previously the tag antibody, became the capture antibody. Both results for each MAb had to read above each specified cutoff to be considered separate binding regions. This principle was applied to the E1 epitope map as well. The strength of the resonance signal obtained for MAb 2, the tag, depended on 1) the antigen binding capacity of the first MAb, 2) the degree of saturation of the first MAb with antigen, 3) the concentration of the second MAb, and 4) the affinity and association rate of the second MAb when reacting with the captured antigen(14) . To evaluate whether the affinity and association rates of secondary antibody (tag) was limiting (whenever a reverse sensorgram was negative), the antibody was rerun at five times the concentration of the original antibody concentration, and the sensorgram always proved to be positive. This method of ``reverse order analysis'' ensured that the binding matrix generated was reproducible. Additionally, to confirm the binding matrix generated in Fig. 5was reproducible, multisite binding studies were also conducted. If the MAbs were indeed to different epitope regions, then injection of several different MAbs in sequence should reveal the independent binding of these MAbs. Fig. 6depicts a multisite binding sensorgram demonstrating that among those five MAbs selected, all had independent binding sites on the PDHc antigen.


Figure 5: PDHc relative epitope matrix. Ic, binding region 1 of PDHc including MAbs 18A9, 21C3, and 15A9; IIc, binding region 2 including MAbs 8C9, 7C9, and 13A8; IIIc, binding region 3 containing MAb 1F2; IVc, binding region 4 containing MAb 3G1; Vc, region five showing MAb 8G10; VIc, region six showing MAb 23H4. 1° indicates those MAbs used as a primary MAb. 2° indicates those MAbs that were used as a secondary MAb. Shadedarea means no interference, and nonshadedarea means interference.




Figure 6: Multisite binding of various monoclonal antibodies to PDHc. 1, uptake of MAb 18A9; 2, uptake of block IgG1 antibody; 3, uptake of PDHc antigen; 4, reinjection of MAb 18A9; 5, uptake of MAb 7C9; 6, uptake of MAb 23H4; 7, uptake of MAb 1F2; 8, uptake of MAb 3G1; 9-11, surface reactivation with HCl and formic acid. The arrowsindicate the completion of the designated step.



All 10 MAbs that were evaluated in the PDHc binding matrix were mapped against the E1 antigen. 5 of these 10 MAbs bound the E1 antigen (18A9, 21C3, 15A9, 1F2, and 3G1), while 5 did not (8C9, 7C9, 13A8, 23H4, and 8G10). In addition, five new MAbs (15E2, 11D9, 1G5, 12A12, and 3F11) were run to increase the resolution of the epitope region shared by the strongest inhibitors (18A9, 21C3, and 15A9). The binding matrix pattern for binding the E1 subunit is illustrated in Fig. 7. For further clarification of the enhanced resolution obtained by using additional MAbs in the E1 map depicting the overlapping regions on the E1 subunit epitope map, an interference map is presented in Fig. 8, A and B. This figure separates the two antigen maps (PDHc and E1) into separate regions defined only by those antibodies that bound their respective antigens. Again, to confirm that the proposed epitope regions on the E1 subunit were independent regions (Fig. 7), a multisite binding sensorgram was generated and is illustrated in Fig. 9. These data confirm the existence of the independent epitopes.


Figure 7: E1 relative epitope matrix. Ie, region one showing MAbs 18A9, 21C3, and 15A9; IIe, region two showing overlap of MAbs 18A9 and 21C3 with 15A9; IIIe, region three showing overlap of MAbs 15A9 with 15E2 and 11D9; IVe, region four showing MAbs 1G5 and 3G1; Ve, region five showing MAbs 12A12 and 3F11; V1e, region six showing MAb 1F2. 1° indicates those MAbs used as a primary MAb. 2° indicates those MAbs that were used as a secondary MAb. Shadedarea means no interference, and nonshadedarea means interference.




Figure 8: A, PDHc subunit relative interference map. A diagramatic representation of the PDHc epitope matrix using only those monoclonal antibodies that bound the PDHc antigen. Those MAbs that did not bind E1 are denoted with an inscribed octagon; all five except 23H4 were elicited to E1. B, E1 subunit relative interference map. A diagramatic representation of the E1 epitope matrix using five monoclonal antibodies from the PDHc map and five new additional antibodies that were known to bind E1. This shows the increased resolution obtained for the three MAbs (18A9, 21C3, and 15A9) when using the E1 antigen and five additional MAbs.




Figure 9: Multisite binding of various monoclonal antibodies to E1. 1, uptake of MAb 18A9; 2, uptake of blocked IgG1 antibody; 3, uptake of E1 antigen; 4, uptake of 1F2; 5, uptake of 3G1; 6, uptake of 12A12; 7, uptake of 15E2; 8, surface reactivation. The arrowsindicate the completion of the designated step.



Several, more classic MAb epitope mapping studies have been described in the literature. Examples include competitive mapping studies using peptide fragments(18, 19) , pairwise competitive binding assays(20) , competitive ELISA assays(21) , and ELISA double antibody binding assays (22) . To verify the first PDHc SPR binding matrix (Fig. 5), a limited competitive epitope mapping technique (23) was used. Limited competitive epitope mapping experiments were also conducted with radiolabeled E1, as described under ``Materials and Methods,'' to confirm the results obtained from the SPR. Four of the six MAbs that had been previously characterized were used (18A9, 21C3, 15A9, and 1F2). The results (Fig. 10) suggest that MAb 1F2 has a different epitope binding region from MAbs 18A9, 21C3, and 15A9, since MAb 1F2 has a B/B(o) value near unity, indicating no competition for the same epitope. The remaining three MAbs, 18A9, 21C3, and 15A9, have lower B/B(o) values, suggesting competition for the same binding site or overlapping epitope regions.


Figure 10: Competitive epitope mapping using radiolabeled E1 with monoclonal antibody 18A9 bound to the microtiter plate. B/B(o), binding of the E1 radiolabel in the presence of the respective competing MAb/binding of the radiolabel in the absence of the competing MAb. &cjs0800;, MAb 15A9; , MAb 21C3; bullet, MAb 18A9; , MAb 1F2. For experimental details, see ``Materials and Methods.''




DISCUSSION

Epitope mapping methods can be classified into two categories, direct and competitive(24) . In this paper, the competitive binding method, through the use of SPR, was first used to map our 10 MAbs exhibiting the highest enzyme inhibition relative to the entire PDHc complex. Within this map (Fig. 5, 8A) one can identify six distinct epitope binding regions on the PDHc. The term epitope region is used because competitive epitope mapping on intact proteins cannot define a specific epitope. Region Ic (c for PDHc) contained those three MAbs (18A9, 21C3 and 15A9) that gave rise to the highest inhibition. All three were elicited to the PDHc antigen, and as previously discussed (see preceding paper), these three MAbs must be bound at a vitally important region within the enzyme because they mapped to the same region, and they also extensively inhibited the enzyme. Region IIc contained three MAbs: 8C9, 7C9, and 13A8. These three MAbs were elicited to the purified E1 antigen and all inhibited the enzyme to a similar extent (between 40 and 50%). It is interesting to note that the E1 ELISA, Western blot analysis, and E1 mapping data suggest little or no binding of these three MAbs to the purified E1 antigen (data previously described in preceding paper). Regions IIIc-VIc all contain one MAb each, with descending degrees of inhibition. This SPR study also indicated that the degrees of inhibition can be correlated to different binding regions. Those MAbs with the highest degree of inhibition bound in the most critical epitope region of the enzyme vital for activity (Table 1).



Next, the 10 MAbs used in the PDHc map, plus the 5 additional MAbs known to bind E1 (15E2, 11D9, 1G5, 12A12, and 3F11), were run in an E1 binding matrix so that we could compare the E1 matrix to the PDHc matrix. The five additional MAbs were run to increase the resolution of the matrix and to resolve, even further, the epitope regions for the three strongest inhibitors (18A9, 21C3, and 15A9). 5 of the 10 MAbs that bound PDHc (18A9, 21C3, 15A9, 1F2, and 3G1) bound the E1 antigen, and 5 did not (8C9, 7C9, 13A8, 23H4, and 8G10). The five MAbs that did bind E1, plus the five additional E1 binding MAbs, offered additional epitope regional information. SPR first revealed that the three MAbs that had the highest inhibition bound the E1 subunit as previously shown by ELISA and Western blot analysis. This additional E1 mapping further resolved the epitope region into two overlapping regions: Ie (e for E1) shared by MAbs 18A9 and 21C3 and the IIe overlapping region, which includes MAb 15A9 ( Fig. 7and Fig. 8B). As in the PDHc map, MAb 1F2 was again revealed to possess its own independent binding region. The larger binding matrix along with the use of the E1 antigen helped clarify the less definitive results generated in the limited competitive epitope mapping experiments with radiolabeled E1 (Fig. 10). In addition, the E1 binding matrix-associated MAb pairs 1G5/3G1 and 12A12/3F11 were shown to have their own common epitope regions. Finally, MAbs 15E2 and 11D9 bound to the same epitope region, which overlapped the binding region of MAb 15A9.

The five MAbs that did not bind E1 in the BIAcore (8C9, 7C9, 13A8, 23H4, and 8G10, denoted by an inscribed octagon in Fig. 8A) also did not bind any of the subunits of PDHc or the purified E1 antigen in Western blot analysis (described in the preceding paper). In addition, three of the five MAbs (8C9, 7C9, and 13A8) did not bind in the E1 ELISA, whereas MAbs 23H4 and 8G10 bound in the E1 ELISA. It can be postulated that for MAbs 8C9, 7C9, and 13A8 the epitopes specific to these respective MAbs were conformational or discontinuous in nature (25) and were buried in the purified soluble antigen and plate assay, thus not allowing binding. One should recall that resolved E1 exists as a dimeric protein according to size exclusion high pressure liquid chromatography, and its conformation could be quite different from that found in holo-PDHc. These epitopes were also totally destroyed by the denaturing conditions of Western blots, which generally allow only segmental or continuous epitopes to be recognized. In contrast, MAbs 23H4 and 8G10 had epitopes that were segmental or continuous and were buried on the soluble E1 antigen but became exposed when the antigen was bound to the plate, thus allowing binding to the plate assay but not the soluble BIAcore assay. Just as for the three MAbs (8C9, 7C9, and 13A8), Western blot conditions destroyed the epitopes for 8G10 and 23H4. The possibility of contamination of the purified E1 with the highly antigenic E2 or E2-E3 subcomplex cannot easily be ruled out.

Overall, the epitope mapping data generated with the SPR methodology correlated very well with the data from the inhibition studies and is illustrated in Table 1and Table 2. Region IVe in the E1 map is the only exception. Even though the two MAbs (1G5 and 3G1) bound to the same region, it was apparent that the specific epitope occupied by MAb 3G1 played a much more important role in inhibition than did MAb 1G5.



The need for the native enzyme as an antigen to elicit MAbs that inhibit the PDHc most effectively becomes apparent when reviewing the data. All three MAbs (18A9, 21C3, and 15A9) that produce greater than 60% inhibition were generated from the complex. ELISA plate data to the E1 antigen, Western blot analysis, and now epitope mapping to the PDHc and E1 subunit demonstrated that these three MAbs bound the E1 subunit and had overlapping epitope regions. The PDHc and E1 binding matrix clearly demonstrated that the three MAbs with the highest inhibition had the same or overlapping binding regions, whereas those MAbs derived from the E1 antigen had different binding regions, strongly supporting epitope dissimilarity.

The value of a large epitope matrix becomes apparent when reviewing the radiolabeling epitope mapping data. Four MAbs were competed against each other (18A9, 21C3, 15A9, and 1F2) in a plate binding assay. Although the radiolabeling method is very tedious, the results obtained confirmed that 1F2 has a different epitope than the other three MAbs. With this method, it was difficult to confirm whether 18A9, 21C3, and 15A9 have the same or overlapping epitope regions. The SPR technique can generate larger epitope matrix binding patterns in shorter, less tedious time.

In summary, presented here is the first reported epitope mapping studies for a library of MAbs to PDHc in E. coli. These studies have indicated that the 10 MAbs that exhibit the highest inhibition bound to six separate binding regions within the PDHc, and those three MAbs that display the highest degree of inhibition also bound to two overlapping regions on the E1 subunit. MAbs with such high degree of specificity, particularly 18A9, which provided inhibition of greater than 98%, would be predicted to be directed at a vitally important area within the E1 subunit, very likely at the active center of the enzyme. Several additional epitope regions were defined by these MAbs, and a good correlation was found between the extent of inhibitory efficacy and epitope similarity. Since these MAbs are now shown to have separate, distinct epitope regions that can be related to function, this MAb library offers the ability to probe the PDHc enzyme complex further to reveal more detailed information about its structure and function, specifically about the E1 active center, and the GTP binding site.

The successful correlation of the SPR epitope map with the inhibition data demonstrates the utility of SPR in epitope mapping of large multicopy, multisubunit complexes and offers distinct advantages over more conventional techniques such as radioimmunoassay or ELISA. There is no need for labeling the antigen or antibody, no purification of ascites fluid is required, and a universal capturing system is used. These advantages allow the antigens and antibodies to more readily mimic their native structure, whereas bound or labeled antigens or antibodies may have epitope regions destroyed or distorted.


FOOTNOTES

*
This work was supported by National Science Foundation Grant DMB9112795, National Institutes of Health Grant GM-50380, the Rutgers University Busch Grant, and Roche Diagnostics Systems, Inc., Branchburg, NJ. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Rutgers University, Dept. of Chemistry, 73 Warren St., Newark, NJ 07102.

(^1)
The abbreviations used are: PDHc, pyruvate dehydrogenase multienzyme complex; MAb, monoclonal antibody; SPR, surface plasmon resonance; RAMG1, affinity-purified rabbit anti-mouse IgG1; ELISA, enzyme-linked immunosorbent assay; RU, resonance units.


ACKNOWLEDGEMENTS

We thank Ellyn Fischberg for technical advice and usage of the monoclonal facility, Dr. Katherine Korkidis at Pharmacia for initially introducing A. McNally to the technical aspects of the BIAcore, to Jim Richey for allowing use of the instruments, and lastly Russ Granzow for the technical support related to the BIAcore data interpretation.


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