Monoclonal antibodies to thymidine glycol generated by different immunization techniques.
Monoclonal antibodies specific for thymidine glycol in oxidized DNA were generated by immunization with thymidine glycol monophosphate (TMP-OH) or thymidine glycol (T-OH), respectively, conjugated to keyhole limpet hemocyanin (KLH) or thyreoglobulin (TG). Forty-five clones (TMP-OH) and 70 clones (T-OH) were examined upon antibody production in ELISA. Four clones secreting IgG1, kappa, were characterized further. In several studies the antibodies derived from the immunization with TMP-OH were inhibited by various inhibitors. In descending order of effectiveness, they were thymidine glycol monophosphate (TMP-OH), thymidine glycol (T-OH), thymidine monophosphate (TMP), and thymidine (Thn). After immunization with T-OH, antibodies were inhibited in following order: T-OH > TMP-OH > TMP > Thn. Inhibition by the bases thymine, adenine, cytosine, and guanine were negligible. In ISB (Immuno Slot Blot) performed with OsO4-treated DNA (Poly-[dA-dT]) and amount of 70 fmol thymidine glycol was detectable. DNA had to be irradiated at a level of at least 20 Gy to detect any damage in ELISA but at a lower level of irradiation (10 Gy) in ISB by one of these antibodies, TPS-1. The antibodies obtained after immunization with hapten-protein are therefore capable of the detection of low frequency lesions in DNA generated by free radicals after radiation or oxidative stress.
Greferath
R
R
Institut für Pathologie und Rechtsmedizin, Universität Ulm Albert Einstein.
Nehls
P
P
D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D003429:Cross Reactions; D004249:DNA Damage / Q000276:immunology*; D006241:Haptens / Q000276:immunology; D006433:Hemocyanins / Q000276:immunology; D006825:Hybridomas; D007114:Immunization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D013936:Thymidine / Q000031:analogs & derivatives* / Q000276:immunology; D013954:Thyroglobulin / Q000276:immunology
D006241:Haptens; C029390:thymidine glycol; D013954:Thyroglobulin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D013936:Thymidine