PREPARATION OF COMPOUND-SPECIFIC AND GROUP-SPECIFIC ANTIBODIES TO 7-METHYLGUANINE AND RELATED 7-ALKYLGUANINES AND THEIR USE IN IMMUNOAFFINITY PURIFICATION

The preparation and characteristics of compound-specific and group-specific antibodies against 7-alkylguanines (7-alkGua) are described. A compound-specific antibody against 7-methylguanine was prepared using a hapten bound to carrier protein through the N-2 position. In a competitive enzyme-linked immunosorbant assay (ELISA) 7-methylguanine (7-MeGua) showed 50% inhibition (I-50%) at 10 pmol/well at room temperature, but the inhibition was found to be 40 times better at 4 degrees C (I-50% at 250 fmol/well). When the antibody was bound to protein A-Sepharose CL4B 7-MeGua was retained in immunoaffinity columns. A group-specific antibody to 7-alkGua was prepared using 7-(2-carboxy-ethyl)guanine (7-CEGua) bound to carrier protein via the carboxyl group. In a competitive ELISA, this antibody crossreacted well with 7-CEGua, 7-ethylguanine (7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine (7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinity columns prepared from this antibody retained a number of 7-alkGua of diverse structure. 7-EtGua in calf thymus DNA treated with diethylsulphate and ethylnitrosourea was isolated by immunoaffinity purification and quantified by HPLC-fluorescence. These results illustrate the potential of immunoaffinity purification for both individual DNA adducts and groups of adducts.