Anal. Chem. 2002, 74, 3714-3719
Binding Stoichiometry of DNA Adducts with Antibody Studied by Capillary Electrophoresis and Laser-Induced Fluorescence Hailin Wang, James Xing, Woei Tan, Mike Lam, Trevor Carnelley, Michael Weinfeld,* and X. Chris Le*
Department of Public Health Sciences and Cross Cancer Institute, University of Alberta, Edmonton, Alberta T6G 2G3, Canada
Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE-16mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE-16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange. Immunoassays have broad application, ranging from small molecules to proteins and whole cells.1 The commonly used monoclonal antibodies (MAb) for immunoassays are immunoglobulin G (IgG) molecules that are bidentate, capable of binding to two antigen (Ag) or hapten molecules:
MAb + Ag ) MAb-Ag + MAb(Ag)2
(1)
However, in most immunoassays, whether antibody binds to one or two molecules is not described although both the MAb-Ag and MAb(Ag)2 complexes may be formed. Most heterogeneous immunoassays are performed using solid phases, such as microtiter plates, membranes, beads, and tubes, * Corresponding authors. Phone: (780) 492-6416. Fax: (780) 492-7800. E-mail:
[email protected];
[email protected]. (1) Immunoassays; Gosling, P. J., Ed; Oxford University Press: Oxford, U.K., 2000.
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typically in the form of an enzyme-linked immunosorbent assay (ELISA). Sequential washes are applied in the ELISA approach to remove the unbound antibody or antigen from the complexes that are immobilized on the solid phase. Information on binding stoichiometry is not available because the washing steps cannot efficiently distinguish complexes of different stoichiometry. More recently, immunoassays have been coupled with highly efficient separation techniques, such as high-performance liquid chromatography (HPLC)2,3 and capillary electrophoresis (CE).4-10 The CE-based immunoassays11-14 have been used for the evaluation of binding constants, binding stoichiometry, specific activity of different protein isoforms,15,16 and complex formation between viruses and monoclonal antibodies.17 With the improved separation it is possible to resolve multiple complexes arising from antibody and antigen binding. The separation of the complexes between receptors and ligands with different stoichiometry has been demonstrated.6,18 In the past few years, many competitive immunoassays using capillary electrophoresis and laser-induced fluorescence (CE/LIF) have been developed.4,7,12,13,19-21 These assays make use of highly (2) De Frutos, M.; Paliwal, S. K.; Regnier, R. E. Methods Enzymol. 1996, 270, 82-101. (3) Schmalzing, D.; Nashabeh, W.; Yao, X. W.; Mhatre, R.; Regnier, F. E.; Afeyan, N. B.; Fuchs, M. Anal. Chem. 1995, 67, 606-612. (4) Schultz, N. M.; Kennedy, R. T. Anal. Chem. 1993, 65, 3161-3165. (5) Shimura, K.; Karger, B. L. Anal. Chem. 1994, 66, 9-15. (6) Chu, Y.-H.; Avila, L. Z.; Gao, J.; Whitesides, G. M. Acc. Chem. Res. 1995, 28, 461-468 and references therein. (7) Chen, F.-T. A. J. Chromatogr., A 1994, 680, 419-424. (8) Heegaard, N. H. H.; Nilsson, S.; Guzman, N. A. J. Chromatogr., B 1998, 715, 29-54 and references therein. (9) Wan, Q. H.; Le, X. C. Anal. Chem. 1999, 71, 4183-4189. (10) Wan, Q. H.; Le, X. C. Anal. Chem. 2000, 72, 5583-5589. (11) Wan, Q. H.; Le X. C. J. Chromatogr., A 1999, 853, 555-562. (12) Chen, F.-T. A.; Sternberg, J. C. Electrophoresis 1994, 15, 13-21. (13) Lam, M. T.; Wan, Q. H.; Boulet, C. A.; Le, X. C. J. Chromatogr., A 1999, 853, 545-553. (14) Le, X. C.; Xing, J. Z.; Lee, J.; Leadon, S. A.; Weinfeld, M. Science 1998, 280, 1066-1068. (15) Chu, Y.-H.; Avila, L. Z.; Biebuyck, H. A.; Whitesides, G. M. J. Med. Chem. 1992, 35, 2915-2917. (16) Rao, J. H.; Colton, I. J.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 9336-9340. (17) Okun, V. M.; Ronacher, B.; Blaas, D.; Kenndler, E. Anal. Chem. 2000, 72, 4634-4639. (18) Chu, Y.-H.; Lees, W. J.; Stassinopoulos, A.; Walsh, C. Biochemistry 1994, 33, 10616-10621. (19) Ye, L.; Le, X. C.; Xing, J. Z.; Ma, M.; Yatscoff, R. J. Chromatogr., B 1998, 714, 59-67. (20) Schmalzing, D.; Buonocore, S.; Piggee, C. Electrophoresis 2000, 21, 39193930. 10.1021/ac0201979 CCC: $22.00
© 2002 American Chemical Society Published on Web 06/25/2002
sensitive LIF detection for the analysis of nonfluorescent compounds, such as therapeutic drugs. The analytes are usually small molecules, generally less than 1000 Da. The separation of the unbound fluorescent probe (e.g., 1000 Da) from the antibodybound probe (∼151 000 Da) is relatively easy using CE because of the large mobility differences between the free and antibodybound probes. However, the separation between the MAb-Ag (∼151 000 Da) and the MAb(Ag)2 (∼152 000 Da) complexes is more difficult. In this study, we have taken advantage of the fact that both size and charge of the molecules contribute to CE separation. If additional charges can be introduced to the complex due to binding, then the separation of the multiple complexes becomes possible. We decided to design fluorescent oligonucleotide probes that contain a single base adduct that can be recognized by an antibody.22 These probes introduce large mobility changes to the antibody when bound to the probe because of the highly negative charge of the probe. With these probes, we are able to study the binding stoichiometry between oligonucleotides and the antibody. We chose to study DNA adducts of benzo[a]pyrene diol epoxide (BPDE) because BPDE is a known chemical carcinogen, reacting primarily with the guanine groups in the DNA to form adducts.23 The parent compound, benzo[a]pyrene, is an ubiquitous environmental carcinogen, which is a product of incomplete combustion of fossil fuels and cigarette smoke among other sources. We previously reported24 a competitive immunoassay for BPDE-DNA adducts and determined the levels of BPDE-DNA adducts in A549 human lung carcinoma cells that were incubated with various concentrations of BPDE (9.4-300 µM). The assay relied on the use of a fluorescently labeled BPDE-modified oligonucleotide probe and a commercially available monoclonal IgG antibody. With the carefully designed fluorescent probe containing a single BPDE adduct and high-affinity monoclonal antibody for BPDE-DNA adducts, we now describe a detailed study on binding stoichiometry between the antibody and the DNA adducts. This paper demonstrates the formation of binary and tertiary complexes between DNA adducts and the antibody. We provide direct information on antibody binding stoichiometry and reveal ligand redistribution/exchange between the fluorescently labeled and unlabeled oligonucleotides in the antibody complexes. EXPERIMENTAL SECTION Reagents. Oligonuleotides were synthesized by the Department of Biochemistry DNA synthesis laboratory, University of Alberta, or by Integrated DNA Technologies (Coralville, IA). All oligonucleotides were purified by sequencing polyacrylamide gel electrophoresis prior to use. Purity of the modified oligonucleotides was confirmed by gel electrophoresis and 32P-postlabeling. Tetramethylrhodamine (TMR)-labeled oligonucleotide was synthesized by University Core DNA Services, (University of Calgary, AB, Canada). (()-r-7,t-8-Dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(()-anti-BPDE] was supplied by the National (21) Guijt-van Duijn, R. M.; Frank, J.; van Dedem, G. W. K.; Baltussen, E. Electrophoresis 2000, 21, 3905-3918. (22) Carnelley, T. J.; Barker, S.; Wang, H.; Tan, W. G.; Weinfeld, M.; Le, X. C. Chem. Res. Toxicol. 2001, 14, 1513-1522. (23) Szeliga, J.; Dipple, A. Chem. Res. Toxicol. 1998, 11, 1-11. (24) Tan, W. G.; Carnelley, T. J.; Murphy, P.; Wang, H.; Lee, J.; Barker, S.; Weinfeld, M.; Le, X. C. J. Chromatogr., A 2001, 924, 377-386.
Cancer Institute Chemical Carcinogen Reference Standard Repository (Midwest Research Institute, Kansas City, MO). (Caution: BPDE is carcinogenic. Appropriate care should be exercised when handling BPDE). Mouse monoclonal antibody 8E11 was purchased from BD PharMingen (San Diego, CA). Polyclonal rabbit IgG antibody was purchased from Calbiochem (La Jolla, CA). Solvents and other biochemicals were supplied by Sigma (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), or VWR Canlab (Mississauga, ON, Canada). BPDE-DNA Adducts. Two 16-mers with the sequence 5′CCCATTATGCATAACC-3′ were synthesized and reacted with BPDE to yield the BPDE-N2 deoxyguanosine (dG) adduct.25,26 One of the 16-mer oligonucleotides was labeled with TMR at the 5′ end, and the other was not labeled. The formation of BPDEoligonucleotide was based on the procedure described by Margulis et al.,25 with slight modifications. The 16-mer was diluted in 20 mM phosphate buffer (pH 11) containing 1.5% triethylamine, to a concentration of 60 µM in a volume of 400 µL. To the oligonucleotide solution was added 40 µL of 3 mM BPDE in DMSO. This corresponded to a BPDE/oligonucleotide ratio of 5:1. The reaction was carried out at room temperature for 20 h, in the dark with gentle shaking. The double-stranded TMRBPDE-90-mer and BPDE-90-mer were constructed through ligation of BPDE-16-mer with five other oligonucleotides.22,24 The concentrations of the TMR-BPDE-16-mer and TMR-BPDE90-mer were estimated using absorbance at 260 nm. The use of the TGC sequence in the 16-mer encouraged maximum yield of BPDE-dG adduct.25,26 Although the ratio of dA to dG in the 16-mer oligonucleotide was 5:1, the major adduct formed was with dG. This is in agreement with previous findings by others where the yield of dG adducts was found to be 5-7fold larger than dA adducts in a 13-mer oligonucleotide containing 4-fold more dA than dG.27 In DNA damage studies involving incubation of cells with benzo[a]pyrene, over 90-95% of the adducts formed were the BPDE-N2 deoxyguanosine (dG) adduct.23,28 Various reaction products including the four isomers, (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-BPDE-N2 deoxyguanosine adducts, have been extensively characterized previously using HPLC, circular dichroism, and nuclear magnetic resonance techniques.23,25,27-30 The trans/cis ratios were 7:1 in the case of (+)-BPDE and 2:1 in the case of (-)-BPDE.30 We separated these compounds using reversed-phase HPLC as described previously.22 We collected the fraction corresponding to the trans(+)-BPDE-dG adduct, the major product. The single isomer of BPDE-dG 16-mer was used for the subsequent studies of binding stoichiometry. Capillary Electrophoresis with Laser-Induced Fluorescence. Characterization of the DNA-BPDE adducts and their binding with the antibody was carried out using a laboratory-built capillary electrophoresis/laser-induced fluorescence (CE/LIF) (25) Margulis, L. A.; Ibanez, V.; Geacintov, N. E. Chem. Res. Toxicol. 1993, 6, 59-63. (26) Funk, M.; Ponten, I.; Seidel, A.; Jernstrom, B. Bioconjugate Chem. 1997, 8, 310-317. (27) Ponten, I.; Seidel, A.; Graslund, A.; Jernstrom, B. Chem. Res. Toxicol. 1996, 9, 188-196. (28) Meehan, T.; Straub, K.; Calvin, M. Nature 1977, 269, 725-727. (29) Sayer, J. M.; Chadha, A.; Agarwal, S. K.; Yeh, H. J. C.; Yagi, Hl; Jerina, D. M. J. Org. Chem. 1991, 56, 20-29. (30) Cosman, M.; Ibanez, V.; Geacintov, N. E.; Harvey, R. G. Carcinogenesis 1990, 11, 1667-1672.
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system as previously described.10,31 Uncoated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with 20 µm i.d., 150 µm o.d., and 30 cm in length were used for separation, and a voltage of 15 kV was applied. Samples were electrokinetically injected into the capillary at 15 kV for 5-10 s. The running buffers were either 1× Tris-glycine (25 mM Tris, 192 mM glycine, pH 8.3) or 0.5× Tris-glycine (12.5 mM Tris and 96 mM glycine, pH 8.3). The capillary was washed approximately every three injections with 0.02 M NaOH electrophoretically at 15 kV for 7 min followed by electrophoresis using water and the running buffer for 7 min each. Fluorescence was detected at 580 nm upon excitation at 543.5 nm with a green HeNe laser. Complex Formation of TMR-Labeled BPDE-DNA Adducts and Antibody. TMR-BPDE-16-mer (single stranded) and TMR-BPDE-90-mer (double stranded) samples were diluted to appropriate concentrations in running buffer (Tris-glycine, pH 8.3). The double-stranded BPDE-90-mer was denatured by heating to 95 °C for 5 min in a heating block. It was then placed on ice to prevent reannealing. Appropriate dilutions of antibody stock solutions were prepared immediately before use and kept on ice. After addition of antibody to the BPDE-oligonucleotide solutions, the samples were gently vortexed and incubated at the room temperature for 5-10 min, and then analyzed by CE/LIF. The total sample volume was typically 20 µL. Simultaneous Binding of two BPDE-DNA Adducts to the Antibody. Both TMR-labeled and unlabeled BPDE adducts were allowed to bind with the antibody. The freshly diluted BPDE16-mer (16mer) and TMR-BPDE-16-mer (16mer*) solutions were mixed together before addition of the antibody. Concentrations of the TMR-BPDE-16-mer and the antibody were kept constant at 9.6 and 33.3 nM, respectively, and the unlabeled BPDE-16-mer was varied from 2.5 nM to 5.0 µM. The sample mixtures were incubated for 5 min at room temperature and then analyzed by CE/LIF. Similarly, the antibody was added to mixtures of the TMR-BPDE-16-mer and BPDE-90-mer to study the competitive binding and ligand redistribution/exchange. RESULTS AND DISCUSSION Stoichiometry of Antibody Binding with TMR-BPDE-16mer (16mer*) Oligonucleotide. Figure 1 shows a series of electropherograms from CE/LIF analyses of mixtures containing 24 nM 16mer* and varying concentrations of mouse MAb to BPDE, from 0.5 to 16.0 µg/mL. In these mixtures, two complexes between the MAb and the TMR-labeled BPDE-16-mer (16mer*) can be expected as follows:
MAb + 16mer* ) MAb(16mer*) + MAb(16mer*)2 (2) Figure 1 shows that the free (unbound) 16mer* (peak 3) is well resolved from the two complexes. The unbound 16mer* oligonucleotide and the antibody-bound 16mer* complexes are negatively charged. Under the free zone electrophoretic conditions, the direction of their electrophoretic mobility (µep) is opposite to that of the electroosmotic flow (EOF). Among the three fluorescent species, the unbound 16mer* has the highest (31) Le, X.; Scaman, C.; Zhang, Y.; Zhang, J.; Dovichi, N. J.; Hindsgaul, O.; Palcic, M. M. J. Chromatogr., A 1995, 716, 215-220.
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Figure 1. Capillary electrophoresis analyses of mixtures containing 24 nM 16mer* and varying concentrations of mouse MAb, showing the presence of antibody complexes (peaks 1 and 2) and the unbound 16mer* (peak 3). CE separation was carried out using a fused-silica capillary (30 cm long, 20 µm i.d., and 150 µm o.d.) and a Tris-glycine buffer (pH 8.3). Laser-induced fluorescence (λex ) 543.5 nm and λem ) 580 nm) was detected from the TMR-labeled species. Peak 1 corresponds to MAb(16mer*) complex (1:1 stoichiometry), and peak 2 corresponds to MAb(16mer*)2 complex (1:2 stoichiometry).
negative effective charge. It has the highest electrophoretic mobility toward the positive (injection) end and, thus, the longest migration time (3.4 min). When the 16mer* binds to an antibody molecule, the reduction of the effective charge results in a smaller electrophoretic mobility and thus a shorter migration time (2.1 min, peak 1). When the MAb binds to two 16mer* molecules, the charge density of the complex is between those of the MAb(16mer*) complex and the unbound 16mer*. Therefore, a smaller mobility shift is expected. Indeed, we observed the MAb(16mer*)2 complex at 2.4 min (peak 2). The two complexes (peaks 1 and 2) are baseline resolved from each other. It is evident that formation of the two complexes depends on the relative concentrations of the 16mer* and the antibody (MAb) (Figure 1). The intensity of the MAb(16mer*) complex (1:1 stoichiometry, peak 1) increases with increasing concentration of the antibody, whereas the MAb(16mer*)2 complex (1:2 stoichiometry, peak 2) reaches a maximum at an antibody concentration of 1.0 µg/mL and then decreases gradually with increasing concentrations of the antibody. Relative peak area of the complexes compared to the total peak area as a function of antibody concentration is illustrated in Figure 2. The binary complex of the 1:1 stoichiometry increases with increasing concentration of the antibody. When the concentration of the antibody is 8-16 µg/mL, approximately 80-85% of the total 16mer* oligonucleotide is present as the binary complex with the antibody (1:1 stoichiometry). Formation of the tertiary complex, MAb(16mer*)2 (1:2 stoichiometry), is favored at lower concentrations of the antibody, when the 16mer* oligonucleotide is in excess. The amount of the tertiary complex reaches a maximum
Figure 2. Relative peak areas of two complexes (1:1 and 1:2 stoichiometry) as a function of antibody concentration. Peak areas of the two complexes and the unbound 16mer* were measured. Percentage of the peak area of a complex over the total peak area of the three fluorescent species was plotted as the relative peak area. The same conditions as shown in Figure 1 were used.
Figure 3. Relative fluorescence intensity of the binary and tertiary complexes between mouse MAb (2.7 nM) and the varying concentrations of 16mer*. Both the binary [MAb(16mer*)] and tertiary [MAb(16mer*)2] complexes were separated with CE and detected with LIF. The same conditions as shown in Figure 1 were used for CE/LIF analyses.
at the antibody concentration of 1 µg/mL, when 50% of the total 16mer* is present as the tertiary complex (Figure 2). Although the MAb(16mer*)2 tertiary complex is observed throughout the entire antibody concentration range studied, from 0.02 (∼0.14 nM) to 16 µg/mL (∼100 nM), it is predominant only when the antibody concentration is below 2 µg/mL (∼14 nM) and is lower than the concentration of the 16mer* (24 nM). The binary complex dominates when the concentrations of antibody (4 µg/mL or 27 nM) and the 16mer* (24 nM) are similar, suggesting that the complex with one binding site is preferred. We have determined the binding constants for the binary complex [(2.5 ( 0.2) × 108 M-1] and the tertiary complex [(0.9 ( 0.3) × 108 M-1]. The higher binding constants for the binary complex than the tertiary complex is consistent with the above observations. To further study the tertiary complex, varying concentrations of the 16mer* (2.4-19.2 nM) were mixed with a fixed concentration of the antibody (0.4 µg/mL or ∼2.7 nM), and the mixtures were analyzed using CE/LIF. Fluorescence intensity of the tertiary and binary complexes as a function of the concentration of the fluorescently labeled BPDE-16-mer (16mer*) is shown in Figure 3. At a lower concentration of the 16mer* (2.4 nM) than the antibody concentration (2.7 nM), the intensity of the binary complex is comparable to that of the tertiary complex. When the 16mer* concentration is higher (4.8-19.2 nM) than the antibody concentration (∼2.7 nM), the tertiary complex dominates. The fluorescence intensity of the tertiary complex increases with increasing amounts of the 16mer* until it reaches a plateau at ∼17 nM, when the amount of the antibody presumably becomes the limiting factor. Most previous studies on immunoassays have not been able to address the binding stoichiometry although multiple complexes might have formed. The present study clearly shows the formation
of binary and tertiary complexes. The behavior may be qualitatively explained by a two-binding-site model. The mouse MAb is an IgG, which has two specific binding sites for antigen (hapten), in this case, BPDE-16-mer oligonucleotide. When the antibody is in excess of antigen, the formation of binary complex (1:1 stoichiometry) is favored (Figure 1). Only when the binding sites are limited, as in the case of lower antibody concentration, does the tertiary complex (1:2 stoichiometry) dominate (Figure 3). These results suggest that binding to the second sites of antibody is less favored probably because of steric hindrance by the binding on the first site. Binding of the Antibody with TMR-BPDE-90-mer (90mer*). To confirm the preferential binding to the first site and the possible hindrance to the secondary binding, we further compared binding of the antibody with a larger oligonucleotide, the 90mer*. Consistent with the binding of the 16mer*, we observed the formation of both binary and tertiary complexes and demonstrated their dependence on the relative concentrations of the antibody and the 90mer* (data not shown). The ratio of the binary and tertiary complexes was also found to depend on the relative concentrations of the antibody and the 90mer*, similar to that shown in the 16mer* experiments described above. The binary complexes of the antibody with both the 90mer* and the 16mer* were found to be much more stable than the tertiary complex. The results support our suggestion that the primary binding is stronger than the secondary binding. Despite the identical sequence and structure of the two binding sites of the monoclonal IgG antibody, it is likely that the secondary binding is affected by the primary binding on the first site. This may be caused by a conformational change of the antibody structure after binding to one site. Alternatively, steric hindrance of the molecule bound to one site of the antibody may affect the Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
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binding of another molecule on the second site of the same antibody molecule. The 90mer* is ∼28 000 Da, ∼5 times larger than the 16mer* (∼5000 Da). A comparison of antibody binding with the 16mer* and 90mer* indicates that the secondary binding with the larger 90mer* is less favored. This is supported by the fact that the tertiary complex of the antibody with the 90mer* is less stable than the tertiary complex of the same antibody with the 16mer*. These results suggest that steric hindrance plays an important role in the formation of the tertiary complex between an antibody and an antigen. In the case of oligonucleotides, there is also a possibility of charge repulsion between the two oligonucleotide molecules bound on the same antibody molecule. Competitive Binding of Antibody with TMR-BPDE-16mer and Unlabeled BPDE-16-mer. The commonly used competitive immunoassays are based on competitive binding of two ligands to a limiting amount of antibody. Typically, one ligand is labeled and is used as a detection probe. The target analyte is usually the unlabeled ligand. In the traditional competitive assay format, the analyte (unlabeled ligand) can only be indirectly determined through monitoring the relative intensity of signals produced from the labeled ligand. In this study, we found that the binary and tertiary complexes of the antibody with the ligands can be separated. Thus, we decided to further study the binding of multiple ligands to the antibody, with a possibility of developing new approaches to binding assays. The present CE/LIF technique allows the separation of two antibody complexes when a labeled ligand (L*) (e.g., 16mer*) is mixed with the monoclonal antibody (MAb).
MAb + L* ) MAbL* + MAbL/2
(3)
To allow for detection of an unlabeled ligand (L), competitive immunoassay approaches rely on the competition of unlabeled ligand with the labeled ligand for the limiting amount of antibody (MAb). The following species may be formed:
MAbL* + L ) MAbL + L*
(4)
MAbL* + L ) MAbL*L
(5)
MAbL/2 + L ) MAbL*L + L*
(6)
MAbL/2 + 2L ) MAbL2 + 2L*
(7)
The species that are fluorescent and can be detected include the free ligand L*, the binary complex MAbL*, and the tertiary complexes MAbL*L, and MAbL/2. It is clear that the end products are a redistribution of L* and L in the complexes although L* and L compete for the same antibody. Figure 4 shows relative changes of the binary and tertiary complexes with varying concentrations of unlabeled 16mer when it is incubated with 9.6 nM TMR-labeled 16mer* and 33 nM (5 µg/mL) antibody. When the concentration of the unlabeled 16mer is below 10 nM, the binary complex [MAb(16mer*)] dominates and its amount remains almost constant with increasing concentration of the unlabeled 16mer up to 10 nM. The total ligand concentration (16mer* and 16mer) is lower compared with that of the antibody, and there are excess antibody binding sites 3718
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Figure 4. Concentration of the binary and tertiary complexes as a function of unlabeled BPDE-16-mer concentration. Solutions containing 9.6 nM 16mer*, 33 nM (5 µg/mL) MAb, and varying concentrations of unlabeled 16mer were analyzed. The same CD/LIF conditions as shown in Figure 1 were used.
available. Thus, no significant competition between the two ligands takes place. Increasing the competing 16mer concentration from 10 to 200 nM results in the reduction of the binary complex in a manner similar to that commonly observed for conventional competitive assays. Further increase of the unlabeled 16mer concentration to above 250 nM results in disappearance of the binary MAb(16mer*) complex, probably due to the displacement of the 16mer* by the excess 16mer:
MAb(16mer*) + 16mer ) MAb(16mer) + 16mer* (8) The tertiary complexes initially increase with increasing concentration of the unlabeled 16mer and reach a maximum when the concentration of the 16mer is 100 nM. This behavior is different from that of traditional competitive immunoassays where only the mixture of antibody complexes is commonly detected and the separation of the 1:1 and 1:2 complexes is not available for examination. Further increase of the 16mer concentration (100-1000 nM) results in a gradual decrease of the tertiary complex. Only at this high concentration range (100-1000 nM) of the competing 16mer was the competitive immunoassay behavior observed. When the tertiary complex and the unlabeled BPDE-16-mer are plotted logarithmically, two linear lines are observed (Figure 5). A linear regression coefficient (r2) of 0.970 and a positive slope are observed between the tertiary complex and the concentration of the 16mer below 100 nM. This region probably corresponds to redistribution of the ligands. Above 100 nM, a linear regression coefficient (r2) of 0.997 and a negative slope are observed. This may indicate competition with limited amount of antibody. These two linear lines clearly distinguish the competition and redistribution and can be used as quantitative curves for different concentration zones.
Figure 5. Logarithmic correlation between the tertiary complex and the concentration of unlabeled BPDE-16-mer. The same conditions as shown in Figure 4 were used.
It is expected that two types of the tertiary complexes could be formed: one antibody bound to two TMR-BPDE-16-mer [MAb(16mer*)2] and one antibody bound to one TMR-BPDE16-mer and one BPDE-16-mer [MAb(16mer*)(16mer)]. These two tertiary complexes of 16-mer cannot be separated since there is only a slight difference between the TMR-labeled and unlabeled 16-mer oligonucleotides. However, the two tertiary complexes can be observed using a different competing oligonucleotide as described below. Competitive Binding between TMR-BPDE-16-mer and Unlabeled BPDE-90-mer. To observe two types of tertiary complexes, we further devised a binding system using unlabeled BPDE-90-mer (90mer) to compete with the labeled 16mer* for a limiting amount of the antibody. The resultant electropherograms are shown in Figure 6. In the absence of the 90mer (bottom trace), the antibody forms two complexes with the 16mer*: MAb(16mer*) and MAb(16mer*)2. In the presence of the 90mer (top electropherogram), the 90mer competes with the 16mer* for the antibody binding sites. Several complexes containing the 90mer may be formed, including MAb(90mer), MAb(90mer)2, and MAb(16mer*)(90mer). Among these complexes, only MAb(16mer*)(90mer) is fluorescent and can be detected (Figure 6, peak 4). The observation of MAb(16mer*)(90mer) and the accompanying decrease of the MAb(16mer*)2 (peak 2) in the presence of the 90mer clearly demonstrate the binding stoichiometry and redistribution of the two ligands. The redistribution of ligands between two binding sites of the antibody cannot be observed in traditional binding assays that are based on measurement of total bound ligands. In conclusion, we demonstrated that the DNA adduct and the antibody formed two complexes with 1:1 and 2:1 stoichiometry. Binding of the antibody with a mixture of the TMR-labeled and unlabeled BPDE-oligonucleotides showed a typical competitive binding behavior when a high concentration of the unlabeled BPDE-oligonucleotide was used. With a lower concentration of
Figure 6. Electropherograms showing the presence of two tertiary complexes. The bottom electropherogram was obtained from CE/ LIF analyses of a mixture containing 30 nM 16mer* and 6.7 nM (1 µg/mL) MAb. The top electropherogram was obtained from CE/LIF analyses of a mixture containing 30 nM 16mer*, 6.7 nM (1 µg/mL) MAb, and 40 nM unlabeled 90mer. The 90mer oligonucleotide and antibody were incubated in 2-fold diluted Tris-glycine buffer (pH 8.3), and this buffer was used for CE separation. Peak 1 corresponds to the binary complex, MAb(16mer*). Peaks 2 and 4 correspond to the tertiary complexes, MAb(16mer*)2 and MAb(16mer*)(90mer), respectively. Peak 3 corresponds to the unbound 16mer* probe.
the unlabeled BPDE-oligonucleotide, the TMR-labeled BPDEoligonucleotide was partially distributed into the tertiary complex. The binding constant for the binary complex [(2.5 ( 0.2) × 108 M-1] was higher than that for the tertiary complex [(0.9 ( 0.3) × 108 M-1]. The results suggest that the two binding sites of the mouse monoclonal antibody are dependent upon each other and that the primary binding (binary complex) has a higher apparent affinity than the secondary binding (tertiary complex). Steric hindrance and charge repulsion may also contribute to the weaker binding of the tertiary complex than the binary complex. These observations have not been made possible previously with traditional immunoassays because the binary and tertiary complexes would not be differentiated with traditional immunoassays. The present study provides insights to the binding stoichiometry that is important to a better understanding of binding assays. ACKNOWLEDGMENT This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), National Cancer Institute of Canada, the Canada Research Chairs Program, and the Canadian Water Networks NCE.
Received for review March 22, 2002. Accepted May 17, 2002. AC0201979 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
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