Development of a Monoclonal Antibody-based Immunoassay for Cyclic DNA Adducts Resulting from Exposure to Crotonaldehyde

In order to develop an immunoassay for DNA modifications resulting from exposure to crotonaldehyde, monoclonal antibodies specific for the 8R, 6R-and 85, 65-stereoisomers of 3-(2-deoxy-β-D-erythro-pentofuranosyl)-5, 6, 7, 8-tetrahydro-8-hydroxy-6-methylpyrimido[1, 2-a]purine-10(3H)one were produced. These cyclic 1, N²-propanodeoxyguanosines are formed in DNA exposed to crotonaldehyde in vitro. Three of the four antibodies were most specific for one stereoisomer while the fourth was most specific for the other stereoisomer. Fifty % inhibition of binding in an enzyme-linked immunoabsorbent assay could be achieved with 0.2 picomol of either stereoisomer. A high-pressure liquid chromatography-enzyme-linked immtmoabsorbent assay using two of these antibodies and capable of detecting 0.5 μmol of 1, N 5-propanodeoxyguanosine per mol of deoxyguanosine was developed. The method was validated by comparison to results obtained with fluorescence assay.