Topographical characterization of the DNA polymerase from Thermus aquaticus. Defining groups of inhibitor mAbs by epitope mapping and functional analysis using surface plasmon resonance.
Among 24 unique monoclonal antibodies (mAb) generated against Taq polymerase (TaqPol) 13 are potent inhibitors of polymerase activity. These antibodies have been sorted into groups defined by their topographical or functional properties using surface plasmon resonance-based methods to examine three different types of interactions. An epitope map of all the pairwise interactions among all 24 antiTaqPol antibodies revealed the surface of TaqPol as a complex space populated by isolated antigenic domains with no evident relationship to each other. 11 discrete epitopes or epitope clusters were defined and potent inhibitors bound to sites within seven of them. The second method examined the ability of antiTaqPol mAbs to bind to recombinant forms of the constituent functional domains of TaqPol, the N-terminal 5'-nuclease domain and the C-terminal polymerase domain. Most of the antibodies demonstrated a clear specificity for one domain or the other. The third method measured the ability of each mAb to block the interaction of TaqPol with a preformed, immobilized primer:template complex (PTC). Some antibodies had no effect on this interaction while others effectively blocked it. Together these latter two methods resolved many of the antibodies into five distinct groups. In addition, antibodies that bound to overlapping epitopes in the pairwise interaction analysis were members of the same group by their interaction with the polymerase fragment and PTC. Three groups of polymerase inhibitors were clearly resolved by these analyses: (1) those that recognize an epitope on the 5'-nuclease domain and have no effect on the interaction of TaqPol with PTC; (2) those that recognize an epitope on the polymerase domain and block the interaction of TaqPol and PTC; and (3) those that recognize an epitope on the polymerase domain, but have no effect on the interaction of TaqPol with PTC. The surface of TaqPol bears at least three antigenic regions that are topographically and functionally distinct and may correspond to sites for inhibition of different steps in the enzymatic activity of TaqPol.
Daiss
J L
JL
Clinical Diagnostics Research Laboratories, Johnson & Johnson Clinical Diagnostics, Rochester, NY 14650-2117, USA.
Scalice
E R
ER
Sharkey
D J
DJ
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D015374:Biosensing Techniques; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D019384:Nucleic Acid Synthesis Inhibitors; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods*; D013499:Surface Properties; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D019384:Nucleic Acid Synthesis Inhibitors; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase