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Production and Characterization of Antipeptide Antibodies against Human O⁶-Methylguanine-DNA Methyltransferase¹

Department of Pathology [L. E. O., C. N. P., P. A. H., X. H., D. D. B.] and the Preuss Laboratory for Brain Tumor Research [D. D. B.], Duke University Medical Center, Durham, North Carolina 27710; Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 [M. A. v. W., T. P. B.]; and University of Tennessee Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 [S. S., S. M.]

Four synthetic peptides from the sequence of human O⁶-methylguanine-DNA methyltransferase (MGMT), three corresponding to different hydrophilic regions and one corresponding to the sequence containing the alkyl acceptor residue cysteine 145, were used to immunize rabbits. The antibody against Peptide III (residues 171–184) was highly specific, and MGMT protein could be detected on Western blots of soluble protein extracts containing as little as 1 fmol of active MGMT. Antibodies against all of the peptides were able to immunoprecipitate denatured MGMT, while only the antibody against Peptide III was able to react with active enzyme. The antibody against Peptide III did not cross-react with methyltransferase from mice. The use of synthetic peptides has led to the production of a highly sensitive, specific antibody that recognizes native and denatured human MGMT. This antibody should prove useful in studies involving the detection, purification, and characterization of this enzyme.

¹ Supported by NIH Grants CA 11898, CA 32672. NS 20023, CA 14799, CA 217651, CA 23099, CA 36888, and 31721; Bristol-Meyers Grant 100-R18; and the American Lebanese Syrian Associated Charities. Research performed at the Oak Ridge National Laboratory was supported by DOE Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.

² To whom requests for reprints should be addressed, at P. O. Box 3156, Department of Pathology, Duke University Medical Center, Durham, NC 27710.

Received 1/18/91. Accepted 4/16/91.

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