Monoclonal antibody-based immunoanalytical methods for detection of carcinogen-modified DNA components.
Hybridoma cell lines secreting monoclonal antibodies (Mab) directed against the products formed by reaction of alkylating N-nitroso carcinogens with DNA have been established by fusion of rat or mouse myeloma cells, respectively, with spleen cells of rats or mice immunized either with conjugates of various alkyl-ribonucleosides with suitable carrier proteins, or with alkylated DNA electrostatically complexed to carrier proteins. Due to their high affinity and specificity, some of these Mab detect very low amounts of the respective alkyl-deoxynucleosides (e.g., O6-methyl-2'-deoxyguanosine, O6-ethyl-2'-deoxyguanosine, O6-n-butyl-2'-deoxyguanosine, O6-isopropyl-2'-deoxyguanosine, O4-methyl-2'deoxythymidine, O4-ethyl-2'-deoxythymidine) and can be used in various types of immunoassays. With a competitive radioimmunoassay (RIA), specific DNA alkylation products can be quantitated in hydrolysates of cellular DNA, in body fluids, or in urine. The RIA is routinely applicable, reproducible, and sufficiently sensitive to permit the quantitation of femtomole amounts of modified nucleosides in small samples of DNA. When the alkyl-deoxynucleosides in question are separated from bulk DNA by high-performance liquid chromatography prior to analysis by RIA, very low levels of modification in DNA can be detected. The immuno-slot-blot (ISB), a noncompetitive solid-phase immunoassay, is more sensitive than the RIA. For analysis by ISB, alkylated DNA is heat-denatured and immobilized on nitrocellulose filters prior to exposure to the respective Mab and subsequent binding of a second (125I-labelled or biotinylated) antibody. In immunocytological analysis (ICA), the binding of Mab to alkyl-deoxynucleosides is visualized in individual cells by immunostaining of denatured nuclear DNA in situ (direct immunofluorescence; peroxidase-staining).(ABSTRACT TRUNCATED AT 250 WORDS)
D000911:Antibodies, Monoclonal; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000276:immunology; D007163:Immunosorbent Techniques; D008854:Microscopy, Electron / Q000379:methods; D011863:Radioimmunoassay; D013936:Thymidine / Q000031:analogs & derivatives* / Q000276:immunology
D000911:Antibodies, Monoclonal; D003849:Deoxyguanosine; D013936:Thymidine