Monoclonal Antibodies to Thymidine Glycol Generated by Different Immunization Techniques

Abstract

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.