IMMUNOCHEMICAL DETECTION OF ADDUCTS OF SULFUR MUSTARD TO DNA OF CALF THYMUS AND HUMAN WHITE BLOOD-CELLS

As part of a program to develop methods for dosimetry of exposure to sulfur mustard, we developed immunochemical methods for the detection of the major adduct, N7-[2-[(hydroxyethyl)thio] ethyl] guanine (N7-HETE-Gua), formed after alkylation of DNA with sulfur mustard. After immunization of rabbits with calf thymus DNA treated with sulfur mustard, we obtained the antiserum W7/10 with a high specificity for DNA adducts of sulfur mustard. With this serum, a competitive enzyme-linked immunosorbent assay was developed in which sulfur mustard adducts to DNA could be detected with a minimum detectable amount of 1-5 fmol per well and a selectivity that allows detection of one N7-HETE-Gua among 5 X 10(6) unmodified nucleotides in single-stranded DNA. The complications that arise to isolate double-stranded DNA from biological samples and to make the DNA single-stranded without destruction of the sulfur mustard adducts result in about a 20-fold higher limit for adduct detection in DNA from human blood than in single-stranded DNA. Presently, adducts in white blood cells can be detected after exposure of human blood to sulfur mustard concentrations greater than or equal to 2 mu M. We synthesized N7-HETE-GMP for use as a hapten to generate monoclonal antibodies against this adduct. After immunization of mice with this adduct coupled to the carrier protein keyhole limpet hemocyanin we obtained several hybridomas producing monoclonal antibodies that recognize N7-HETE-Gua, containing an intact imidazolium ring. The sensitivity of the competitive ELISA with the monoclonal antibodies was comparable to that of the assays performed with the rabbit antiserum. The adduct in DNA can be detected at a 3 orders of magnitude lower level than the free GMP adduct. Furthermore, the antibodies have some cross-reactivity toward N7-methyl-Gua, free N7-HETE-Gua and O-6-HETE-Gua. No cross-reactivity was observed with GMP.