Immunological quantification by high-affinity antibodies of O6-ethyldeoxyguanosine in DNA exposed to N-ethyl-N-nitrosourea.

Three immunological methods [radioimmunoassay (RIA), enzyme-linked immunosorbent assay, and radioimmunosorbent technique] were established for quantification of the potentially mutagenic O6-ethyldeoxyguanosine (O6-EtdGuo) in DNA treated with the carcinogen ethylnitrosourea in vivo or in vitro. To obtain high-affinity antibodies for specific detection of low levels of O6-EtdGuo in small amounts of DNA (cells), different schemes were applied for immunization of rabbits with the hapten O6-ethylguanosine coupled to various carrier proteins (rat serum albumin, bovine serum albumin, keyhold limpet hemocyanin). Low-dose immunization with the hapten-keyhold limpet hemocyanin conjugate resulted in antibodies with an affinity constant of 1 to 2 X 10(10) liters/mol and very low levels of cross-reactivity with normal as well as other alkylated DNA components. The RIA (the most sensitive of the three assays) detects 0.05 pmol of O6-EtdGuo at 50% inhibition of tracer (O6-ethyl[8,5'-3H]-3'-deoxyguanosine)-antibody binding. This permits quantification by RIA of O6-EtdGuo at an O6-EtdGuo:2'-deoxyguanosine molar ratio of approximately 3 X 10(-7) in a hydrolysate of 100 micrograms of ethylated DNA. By chromatographic separation of O6-EtdGuo prior to the RIA, this value can be lowered to less than 5 X 10(-8).