MEASUREMENT OF BENZO(A)PYRENE-DNA ADDUCTS BY ENZYME IMMUNOASSAYS AND RADIOIMMUNOASSAY

Abstract

Ultrasensitive enzymatic radioimmunoassay (USERIA) was compared to radioimmunoassay and enzyme-linked immunosorbent assay in determining the amount of benzo(a)pyrene [B(a)P] metabolite covalently bound to quanine in [calf thymus] DNA. In USERIA, DNA with or without B(a)P metabolite modification was adsorbed on the wells of microtiter plates and rabbit antiserum to B(a)P metabolite-modified DNA [B(a)-DNA] was then added. Antibodies reacted specifically with the B(a)P-DNA attached to the plate surface. After reaction between goat anti-rabbit IgG conjugated to alkaline phosphatase and rabbit IgG bound to the solid phase, this specific antigen-antibody reaction was enzymatically amplified by alkaline phosphatase conversion of [3H]adenosine 5''-monophosphate to [3H]adenosine. Following chromatographic separation, [3H]adenosine was measured by liquid scintillation counting. The amount of [3H]-adenosine formed was linearly related to the amount of B(a)P-DNA in 10 ng DNA attached to the solid phase. As little as 3 f(femto)mol of bound B(a)P metabolite can be detected by noncompetitive USERIA, while 10 fmol of the adducts in 25 .mu.g DNA can be measured by the competitive USERIA approach. Under standard competitive procedure with 1 .mu.g DNA in the antigen-antibody reaction mixture, USERIA is .apprx. 500-fold more sensitive than radioimmunoassay and 5-fold more sensitive than enzyme-linked immunosorbent assay for the detection of B(a)P-DNA adducts. These highly sensitive assays should be extremely useful for studies in DNA damage and repair by B(a)P metabolites as well as in studies on environmental exposure to this ubiquitous carcinogen.