Monoclonal Antibodies against DNA Damage [PDF]
DNA damage in cells exposed to ultraviolet (UV) radiation plays significant roles in cell-cycle arrest, activation of DNA repair, cell killing, mutation, and neoplastic transformation. The major types of DNA damage induced by solar UV radiation are cyclobutane pyrimidine dimers (CPDs), (64) photoproducts (6-4PPs), and Dewar valence isomers of 6-4PPs (Dewar photoproducts; DewarPPs), which are formed between adjacent pyrimidine nucleotides on the same strand of DNA. DewarPPs are produced by the photoisomerization of 6-4PPs by solar UV radiation with the highest efficiency around 325 nm. These helix-distorting DNA lesions are repaired by nucleotide excision repair (NER) system in normal human cells. Matsunaga et al (9) have established monoclonal antibodies specific for DewarPPs. The antibodies enable one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay (ELISA) and to visualize and measure photoproducts in DNA in cultured cells using indirect immunofluorescence (IIF). This technology would contribute to understanding of molecular mechanisms of cellular responses to DewarPPs in many research fields including cancer research, photobiology, dermatology, ophthalmology, immunology, and cosmetology
This hybridoma was established by fusion of mouse myeloma cells with Balb/c mouse splenocytes immunized with methylated BSA conjugated with calf thymus DNA which was irradiated with UVC and then with 313 nm UV. This hybridoma (clone DEM-1) culture supernatant was collected and precipitated with ice-cold ammonium sulfate. After centrifugation, the pellet dissolved in small volume of double-distilled water was dialysed against PBS. The dialysate was then lyophilized.
1) The antibodies bind to DewarPPs in single-stranded DNA.
2) The antibodies bind to DewarPPs formed in TC, TT and CC dipyrimidine sequences.
3) The antibodies stably bind to DewarPPs formed in oligonucleotides consisting of more than eight bases
The antibodies can bind to DewarPPs in denatured DNA from all organisms from bacteria to human.
Western blotting; Not tested
Immunoprecipitation; Not tested
Immunohistochemistry; Not tested
Flow cytometry; Not tested
Solar UV-induced DewarPPs are detected by ELISA >
The exposure- dependent induction of Dewar photoproducts (DewarPPs) in solar UV-irradiated calf thymus DNA was measured by ELISA with NMDND003. The typical ELISA result was presented.
Fluorescent image of DewarPPs in normal human fibroblasts
Cells were cultured in a 35-mm glass-bottom dish for 24 hours. Immediately after solar UV irradiation for 3 hours or mock irradiation, cells were fixed and permeabilized. After denaturation of DNA, DewarPPs (yellow) were visualized using immunofluorescence with NMDND003. Nuclear DNA (blue) was counterstained with DAPI.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.