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Immunoaffinity Purification of Human O⁶-Alkylguanine-DNA Alkyltransferase Using Newly Developed Monoclonal Antibodies¹

Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

As well as repairing mutagenic lesions induced by simple methylating agents, O⁶-alkylguanine-DNA alkyltransferase repairs precursors of cytotoxic interstrand cross-links induced by chloroethylating anticancer drugs. Moreover, levels of the transferase correlate with cellular resistance to these agents. Thus far, the human transferase has not been highly purified. In our quest to obtain the homogeneous protein we have produced four stable cloned hybridomas that secrete monoclonal antibodies against the alkyltransferase from human lymphoblasts. The specificity of these monoclonals was established by immunoblot analysis and immunoprecipitation. All these antibodies recognized the alkyltransferase only after its denaturation by sodium dodecyl sulfate. One of them, designated 19.2, was used in immunoaffinity chromatography to obtain the pure protein.

¹ This work was supported by Grants CA 14799, CA 36888, CA 21765, CA 11898, and NS 20023 from the NIH and by the American Lebanese Syrian Associated Charities.

Received 6/ 5/89. Revised 9/ 5/89. Accepted 10/ 3/89.

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