Monoclonal antibody-based immunoassay for the determination of cellular enzymatic activity for repair of specific carcinogen–DNA adducts (O6-alkylguanine)

Abstract

We describe a rapid and sensitive, monoclonal antibody (mAb)-based immunoassay that permits the quantitation of cellular enzymatic activity for repair of specific carcinogen– DNA adducts. The assay was established on the basis of the observation that complexes between an anti (O⁶-ethyl–2' -deoxyguanosine) mAb (mAb ER–6) and O⁶-ethylguanine (O⁶–EtGua) residues in double-stranded DNA can be immobilized on nitrocellulose filters. Circular pSV2gpt plasmid DNA was linearized by digestion with EcoRI and reacted with N-ethyl-N-nitrosourea in vitro to a level of about one O⁶-EtGua residue per DNA molecule, labeled with ³²P, and subsequently incubated with extracts prepared from L929 mouse fibroblasts and with mAb ER-6. The amount of repaired O⁶-EtGua is proportional to the decrease in ³²P activity retained on the filters. Maximum O⁶-alkyl- guanine repair activity requires preparation of the cell extracts in the presence of high (∼0.5 M) NaCl concentrations, whereas the repair reaction per se is markedly inhibited by NaCl. Under the conditions used, the detection limit of the assay is ∼1 × 10⁻¹⁶ mol of repaired O⁶-alkylGua. The repair reaction followed second-order kinetics, indicating that O⁶-EtGua is predominantly repaired by an enzyme activity inactivated by the repair reaction (e.g. via an alkyl group transfer mechanism analogous to that exhibited by the alkyl group accepting cysteine residues of the ‘suicidal’ bacterial ogt and ada proteins). The data obtained from the assay can be used for computation of the relative enzymatic repair activity of a given cell extract and of the rate constant K (I × mol⁻¹ × s⁻¹ of the repair reaction.