Immunochemical detection of UV-induced DNA damage and repair.
The application of an antiserum to ultraviolet radiation (UVR)-damaged DNA is presented. A novel experimental system was employed to ascertain the limits of detection for this antiserum. Using a DNA standard containing a known amount of dimer, the limits of detection were found to be 0.9 fmol of dimer. This was compared to a limit of 20-50 fmol dimer using gas chromatography-mass spectrometry (GC-MS). Induction of thymine dimers in DNA following UVR exposure, as assessed using this antiserum in an enzyme-linked immunosorbent assay (ELISA), was compared with GC-MS measurements. The ELISA method successfully demonstrated the induction of lesions in DNA irradiated either with UVC or UVB, although despite high sensitivity, no discernible binding was seen to UVA-irradiated DNA. The antiserum was also shown to be applicable to immunocytochemistry, localising damage in the nuclei of UVR exposed keratinocytes in culture. The ability of the antiserum to detect DNA damage in skin biopsies of individuals exposed to sub-erythemal doses of UVR was also demonstrated. Moreover, the subsequent removal of this damage, as evidenced by a reduction in antiserum staining, was noted in sections of biopsies taken in the hours following irradiation.
Cooke
Marcus S
MS
Oxidative Stress Group, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, PO Box 65, Leicester LE2 7LX, UK. msc5@le.ac.uk
Podmore
Ian D
ID
Mistry
Nalini
N
Evans
Mark D
MD
Herbert
Karl E
KE
Griffiths
Helen R
HR
Lunec
Joseph
J
D000818:Animals; D000941:Antigens / Q000032:analysis; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000276:immunology / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007120:Immunochemistry / Q000379:methods*; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000378:metabolism / Q000528:radiation effects; D018613:Microscopy, Confocal; D011740:Pyrimidine Dimers / Q000032:analysis / Q000276:immunology / Q000528:radiation effects; D012867:Skin / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects*
D000941:Antigens; D011740:Pyrimidine Dimers; D004247:DNA