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 UVB (280-315 nm, component of sunlight) and by UVC (200-280nm) are cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), which are formed between adjacent pyrimidine nucleotides on the same strand of DNA.
Approximately 70-80% of UV-induced DNA damage is CPDs and the remaining is 6-4PPs and Dewar isomer of 6-4PPs. These types of DNA lesions are repaired by nucleotide excision repair (NER) system in normal human cells. Mori et al (19) have established monoclonal antibodies specific for CPDs or 6-4PPs.
These 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 or the skin using indirect immunofluorescence (IIF). This technology would contribute to understanding of molecular mechanisms of cellular responses to UV and DNA damage 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. This hybridoma (clone 64M-2) 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 6-4PPs in single-stranded DNA.
2) The antibodies bind to 6-4PPs formed in every dipyrimidine sequence (TT, TC, CT and CC).
3) The antibodies stably bind to 6-4PPs formed in oligonucleotides consisting of more than eight bases.
The antibodies can bind to 6-4PPs in denatured DNA from all organisms from bacteria to human.
React specifically with damaged DNA
Research applications include ELISA and IC
Useful for research into DNA damage repair mechanisms
Visualizes the DNA damage repair process
Applicable to a broad range of research interests:photobiology, optical medicine, cancer, etc.
Western blotting; Not tested
Immunoprecipitation; Not tested
Immunohistochemistry; Not tested
Flow cytometry; Not tested
Induction and repair of UV-induced DNA damage in normal human cells measured by ELISA
Visualization of UV-induced DNA damage by using immunofluorescence
Visualization of UV-induced 6-4PP and its repair
Cosmo Bio's new 6-4PP/ (6-4)Photoproducts ELISA Kit uses the highly sensitive and specific monoclonal anti-6-4PPs antibody clone 64M-2, established by Mori et al. 64M-2 is widely cited in the literature and is considered to be the gold standard antibody for 6-4PPs detection and quantification.Cosmo Bio's 6-4PPs ELISA Kit is the first and only commercially available ELISA kit using 64M-2. All kits components are optimized for high sensitivity 6-4PPs detection from cultured cells or skin epidermis.
High Sensitivity 6-4PP/ (6-4) photoproduct ELISA kit
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.