© 1989 Oxford University Press
research-article |
Quantitative immunofluorescence assay for cyclobutyldithymidine dimers in individual mammalian cells
1Division of Biophysics, The Johns Hopkins University, School of Hygiene and Public Health 615 N. Wolfe Street, Baltimore, MD 21205-2179, USA
2Division of Occupational Medicine, The Johns Hopkins University, School of Hygiene and Public Health 615 N. Wolfe Street, Baltimore, MD 21205-2179, USA
3Division of Environmental Chemistry, The Johns Hopkins University, School of Hygiene and Public Health 615 N. Wolfe Street, Baltimore, MD 21205-2179, USA
4Present address: Food and Drug Administration/Center for Devices and Radiological Health/Division of Mechanics and Material Science (HFZ-150) 12200 Wilkins Avenue, Rockville, MD 20852, USA
5Present address: Centre for Photochemical Sciences, Chemistry Department, Bowling Green State University Bowling Green, OH 43403, USA
6To whom reprint requests should be sent
An indirect immunofluorescence procedure was developed for the measurement of cyclobutyl dithymidine dimers in DNA of individual Syrian hamster embryo cells using a specific monoclonal antibody. A fluorescein-labeled secondary antibody and a fluorochrome which binds to DNA were used to measure the photoproduct and total DNA in the same nucleus. Fluorescence intensity was quantitated with a computer-assisted microfluorometric system which was calibrated with a uranyl oxide impregnated glass slide. Similar dose-response curves, i.e. normalized fluorescence intensity plotted as a function of dose of germicidal irradiation, were obtained with two different cell types. Normalized fluorescence intensity per nucleus was related to thymidine dimer content with a competitive enzyme-linked imnmunosorbent assay using DNA isolated from cells given doses of germicidal irradiation identical to those used in the immunofluorescence assay. Thymidine dimer levels produced by 10 J/m2 of germicidal irradiation (
8x105/nucleus) and which allow for 15–30% cell survival can readily be detected. The specific monoclonal antibody was labeled with tritium and used in the immunofluorescence assay to relate the number of antibodies bound to the number of thynudine dimers per cell. The data revealed that 45% of the thymidine dimers in cells exposed to 100 J/m2 of germicidal irradiation and essentially all the T<>T in cells receiving 20 J/m2 were being detected in the indirect immunofluorescence assay. This technique can provide a sensitive means for measuring various types of DNA damage in individual cells given that the appropriate probes are available. It can be especially useful for monitoring occupationally or environmentally exposed populations where usually only small samples of cells or tissues are available.