High avidity monoclonal antibody to imidazole ring-opened 7-methylguanine

A hybridoma (K1A8) secreting a high affinity antibody to imidazole ring-opened 7-methylguanine (N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine) was obtained from spleen cells of a mouse immunized with a conjugate of keyhole-limpet hemocyanin and imidazole ring-opened 7-methylguanylic acid (iro-7mGMP). The antibody recognizes the iro-7-methylguanine (iro-7mG) determinant in the BSA-iro-7mGMP conjugate, in chemically methylated, denatured DNA, and in the ring-opened 7-methylguanosine, 7-methyldeoxyguanosine and 7mGMP haptens. In the competitive ELISA of DNA-iro-7mG, 50% inhibition (I50) was observed at 4 fmol determinant per well (8 x 10(-11) M) using BSA-iro-7mGMP as the immobilized antigen. The lower limit of 7-methylguanine (7mG) detection in DNA is determined by the binding of unmodified DNA per se to the antibody. The intrinsic reaction of DNA with antibody is low; in the competitive ELISA I50 was obtained with 330 micrograms calf thymus DNA per 50 microliters well, equivalent to 4 nmol iro-7mG per mol nucleotide. The 7mG content of calf thymus DNA is 7 nmol per mol nucleotide (approximately 20 amol per micrograms DNA). The limit of detection of 7mG by competitive ELISA is quoted provisionally as 7 nmol iro-7mG per mol nucleotide, where 30% inhibition of antibody binding is obtained in the presence of 105 micrograms DNA per 50 microliters well. Nuclear DNAs of tissue culture cells treated with 0, 0.01 and 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine contained 0.18, 31 and 320 mumol, respectively, of 7-methylguanine adducts per mol of nucleotides. This report indicates that the K1A8 antibody will serve to quantify DNA alkylation in human populations exposed to low levels of methylating carcinogens.