Immunological studies of thymine dimer quantitation.
Kriste, Angela Gayle.
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Ultraviolet irradiation of DNA induces the formation of a number of mutagenic lesions. The most prolific of these is the cis-syn thymine dimer (formed maximally at 260 nm) and this has been implicated in the reaction pathways that lead to ultraviolet-induced carcinogenesis. In order that the molecular events underlying these neoplastic events be understood, it is imperative that the thymine dimers formed in ultraviolet-irradiated thymine containing systems be quantitated. In this laboratory, dimer quantitation is performed using reverse phase high performance liquid chromatography (HPLC) with ultraviolet (DV) detection and the data obtained has allowed a kinetic mechanism for lesion formation to be proposed. Such studies have used in vitro thymine containing substrates (aqueous thymine, thymidine, thymidylyl-3',5'thymidine, calf thymus DNA and pUC19 plasmid DNA) to generate the thymine dimer using DV irradiation. with the planned extension of this research to in vivo cellular systems (where DNA and hence thymine concentrations are intrinsically less than those of in vitro systems), a more sensitive technique for thymine dimer quantitation is required. An immunological approach to providing this technique was chosen. Here, DV-irradiated DNA was injected into rabbits whose immune system mounted a ' response (i.e. antibody production) to the DV-DNA antigen. Blood was drawn from the rabbits at regular intervals to obtain the antibodies. The technique of immunoblotting was chosen and developed to allow detection of the thymine dimer antigen. This involved the reaction between the UV-DNA antigen, the primary antibody (generated by the rabbit) and a secondary antibody conjugated to an enzyme, all of which were immobilized on a commercially available membrane system. Detection and quantitation of the immune complex immobilized on the membrane was performed using the technique of enhanced chemiluminescence. Upon addition of a chemiluminescent substrate (luminol) to the immune complex, the horseradish peroxidase enzyme catalysed the reaction of luminol, with one of the products being light of 425 nm to 430 nm. This light impinged on a luminescence film which was developed and printed using standard photographic techniques. The use of dilutions of the primary antibody in the immunoblotting protocol with enhanced chemiluminescent detection, allowed correlations of antibody dilutions with UV-DNA antigen to be made. This immunoblotting technique with enhanced chemiluminescent detection has been used successfully in detecting thymine dimer lesion formation at levels currently above the detection limit of the HPLC. It has also been used successfully in detecting and quantitating thymine dimers at levels undetectable by the HPLC. To this end it has proved to be 4000 to 8000 times more sensitive than the chromatographic technique. Any immunological technique requires that the antibody of interest be purified and characterized. Here, purification of the crude serum was performed using the classical technique of ammonium sulphate precipitation of proteins. As an alternative technique, affinity chromatography was performed on the crude serum using a Memsep 1000 affinity chromatography cartridge attached to a preparative HPLC system. Chromatographic data illustrating this purification are given. Characterization of the DV-DNA antigen was performed by considering the specificity of the antibody response in the laboratory animal. Support for the kinetic mechanisms previously proposed for pyrimidine dimer formation in DNA is also given in this work. Calf thymus DNA was irradiated and dimer yields obtained by immunoblotting. These were used in the computer programme CAKE together with the previously determined rate constants to determine simulated dimer yields. A good agreement between experimental and simulated data indicated the validity of the mechanism at a DNA concentration of 0.025 mg/ml.