Immunological Quantification by High-Affinity Antibodies of O6-Ethyldeoxyguanosine in DNA Exposed to N-Ethyl-N-nitrosourea

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