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Quantitative immunofluorescence assay for cyclobutyldithymidine dimers in individual mammalian cells |
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| Authors: | Lesko, S.A. Li, W. Zheng, G. Callahan, D. Kaplan, D.S. Midden, W.R. Strickland, P.T. |
| Affiliation: | 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 |
| Abstract: | An indirect immunofluorescence procedure was developed for themeasurement of cyclobutyl dithymidine dimers in DNA of individualSyrian hamster embryo cells using a specific monoclonal antibody.A fluorescein-labeled secondary antibody and a fluorochromewhich binds to DNA were used to measure the photoproduct andtotal DNA in the same nucleus. Fluorescence intensity was quantitatedwith a computer-assisted microfluorometric system which wascalibrated with a uranyl oxide impregnated glass slide. Similardose-response curves, i.e. normalized fluorescence intensityplotted as a function of dose of germicidal irradiation, wereobtained with two different cell types. Normalized fluorescenceintensity per nucleus was related to thymidine dimer contentwith a competitive enzyme-linked imnmunosorbent assay usingDNA isolated from cells given doses of germicidal irradiationidentical to those used in the immunofluorescence assay. Thymidinedimer levels produced by 10 J/m2 of germicidal irradiation ( 8x105/nucleus)and which allow for 1530% cell survival can readily bedetected. The specific monoclonal antibody was labeled withtritium and used in the immunofluorescence assay to relate thenumber of antibodies bound to the number of thynudine dimersper cell. The data revealed that 45% of the thymidine dimersin cells exposed to 100 J/m2 of germicidal irradiation and essentiallyall the T<>T in cells receiving 20 J/m2 were being detectedin the indirect immunofluorescence assay. This technique canprovide a sensitive means for measuring various types of DNAdamage in individual cells given that the appropriate probesare available. It can be especially useful for monitoring occupationallyor environmentally exposed populations where usually only smallsamples of cells or tissues are available. |
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