Simultaneous Establishment of Monoclonal Antibodies Specific for Either Cyclobutane Pyrimidine Dimer or (6‐4)photoproduct From the Same Mouse Immunized With Ultraviolet‐irradiated Dna

Abstract

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.

Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field

“…Fin clips were then collected at ZT23, placed into culture and exposed to 1 mJ cm À 2 of UV light for 2 s. Samples were then incubated in the dark for 24 h, and genomic DNA was extracted from UV-treated fins and untreated controls. To assess DNA damage, we performed an enzyme-linked immunosorbent assay (ELISA) using an antibody to CPD photoproducts 41 . The results show significantly lower DNA damage, and therefore higher DNA repair activity, in Pachón and Chica cavefish compared with surface fish even in the dark (Fig.…”

“…Cyclobutane pyrimidine dimer photoproducts (CPDpp) were detected by ELISA using TDM-2 monoclonal antibodies (Cosmo Bio) following a modified protocol 41 . Denatured genomic DNA (diluted to 0.2 mg ml À 1 in PBS, 10 ng per well) was bound in quadruplicate to 96-well polyvinylchloride flat-bottom plates, pre-coated with 0.003% protamine sulphate, overnight at 37°C.…”

Temperature-dependent formation and photorepair of DNA damage induced by UV-B radiation in suspension-cultured tobacco cells

“…The CPDs and 6-4PPs were quantified by enzyme-linked immunosorbent assay (ELISA) according to Mori et al [7] with monoclonal antibodies KTM53 and KTM50, respectively. Both antibodies were supplied commercially by Kamiya Biomedical Company, USA.…”

BRCA1 Transcriptionally Regulates Damaged DNA Binding Protein (DDB2) In the DNA Repair Response Following UV-Irradiation

“…The monoclonal antibodies, 64 M-2 (1/200) and TDM-2 (1/1500) were used for detection of 6-4PP and CPD, respectively. 49 Cells were treated with primary antibodies for 30 min at 37°C. Cells were incubated with goat anti-mouse immunoglobulin G (H + L) conjugated with biotin (PIERCE) at 20 µg/ml final concentration for 30 min.…”

“…49 DDB2 has been shown to play a key role in removing CPDs during GGR in vivo and it was proposed that the GGR of CPDs is initiated by the binding of DDB, leading to NER. We investigated the effect of antisense abrogation of BRCA1 on DNA repair of UV-induced photoproducts.…”