Development of an avidin-biotin amplified enzyme-linked immunoassay for detection of DNA adducts of the human bladder carcinogen 4-aminobiphenyl.

4-Aminobiphenyl (ABP) is a known human urinary bladder carcinogen which is present in tobacco smoke and may be ubiquitous in the environment. As a biological monitor of carcinogen exposure, we have developed an immunological method for measuring the predominant carcinogen-DNA adduct of ABP, N-(deoxyguanosin-8-yl)-ABP (dG-C8-ABP). Rabbits were immunized with keyhole limpet hemocyanin (KLH) conjugate prepared by a periodate oxidation and coupling of N-(guanosin-8-yl)-ABP (rG-C8-ABP) to the protein. The resulting polyclonal antisera was systematically characterized using dual inhibitor methodology augmented by specialized computer and software support; and a competitive avidin-biotin enzyme-linked immunoassay (A-B ELISA) assay employing polyclonal rabbit anti-KLH-(rG-C8-ABP) was developed. Under the assay conditions described, the detection limit for dG-C8-ABP was 18 fmol/well. The relative lack of reactivity toward ABP, N-acetyl-4-aminobiphenyl, N-(deoxyadenosin-8-yl)-ABP, N-(deoxyguanosin-8-yl)-2-aminofluorene and deoxyguanosine as inhibitors indicated that primary specificity involves epitopes found on the purine and biphenyl rings. Results emphasize the need to define polyclonal anti-adduct sera operationally in the context of the antigen/assay system used to evaluate it. Assay sensitivity was achieved by decreasing the amount of antibody and solid-phase antigen in the competitive portion of the assay and the use of avidin-biotin as well as enzymatic amplification. This methodology is a useful alternative to other ultrasensitive techniques and should be directly applicable to the detection of ABP-DNA adducts in exposed human populations.