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 andgroup-specific antibodies against 7-alkylguanines (7-alkGua)are described. A compound-specific antibody against 7-methylguaninewas prepared using a hapten bound to carrier protein throughthe N² position. In a competitive enzyme-linked immunosorbantassay (ELISA) 7-methyl-guanine (7-MeGua) showed 50% inhibition(I_50% at 10 pmol/ well at room temperature, but the inhibitionwas found to be 40 times better at 4°C (I_50% at 250 fmol/well).When the antibody was bound to protein A-Sepharose CL4B 7-MeGuawas retained in immunoaffinity columns. A group-specific antibodyto 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua)bound to carrier protein via the carboxyl group. In a competitiveELISA, this antibody cross- reacted 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. Immunoaffinitycolumns prepared from this antibody retained a number of 7-alkGuaof diverse structure. 7-EtGua in calf thymus DNA treated withdiethyl sulphate and ethylnitrosourea was isolated by immunoaffinitypurification and quantified by HPLC-fluorescence. These resultsillustrate the potential of immunoaffinity purification forboth individual DNA adducts and groups of adducts.