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High sensitivity ELISA kit for measuring UV-induced DNA damage

High Sensitivity 6-4PP/ (6-4)Photoproducts ELISA kit


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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.

 

See also  High Sensitivity CPD/ Cyclobutane Pyrimidine Dimer ELISA kit 

Background

Prolonged exposure to solar UV radiation may result in harmful acute and chronic effects to the skin (including skin cancers), eye, and immune system. These harmful effects appear to be closely related to UV-induced DNA damage. The major types of DNA damage induced by solar UV radiation are cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (6-4PPs), and Dewar photoproducts (DewarPPs), which are formed between adjacent pyrimidine nucleotides on the same DNA strand. These helix-distorting DNA lesions are repaired exclusively by a nucleotide excision repair system in humans. To better study molecular events surrounding UV-induced DNA damage and repair, Mori et al. previously developed and characterized monoclonal antibody (mAb) specific for CPDs and mAb specific for 6-4PPs (1) while Matsunaga et al. developed and characterized mAb specific for DewarPPs (2). Three of these antibodies (CPDs: clone TDM-2; 6-4PPs: clone 64M-2; DewarPPs: clone DEM-1) continue to be cited frequently in the literature, often for use in ELISA by a recommended procedure.

This High Sensitivity (6-4)photoproducts (6-4PPs) ELISA Kit is the only commercially available ELISA utilizing anti-6-4PPs clone 64M-2 and has been optimized for high sensitivity detection of 6-4PPs in DNA purified from cultured cells or from skin epidermis. This ELISA detects 6-4PPs from dipyrimidines in all DNA sequence contexts (i.e., TT, TC, CT and CC). Thus, the availability and convenience of this ELISA Kit will contribute to further understanding molecular mechanisms involved in cellular responses to UV radiation and DNA damage with applications across many research fields including cancer research, photobiology, dermatology, ophthalmology, immunology, and cosmetics science

High Sensitivity CPD ELISA kit@flyer
High sensitivity CPD ELISA Kit Flyer download [PDF]

Assay Principle

The format of this assay is ELISA with colorimetric detection. In brief, genomic DNA purified from UV-damaged cells is heat denatured and applied to microtiter wells pre-coated with protamine sulfate. 6-4PP specific monoclonal antibody clone 64M-2 (Cosmo Bio Cat. No. CAC-NM-DND-002) is then added to each well for thirty minutes and unbound antibody is removed by rinsing. The amount of 64M-2 antibody remaining in each well is then measured by sequential treatment of wells with biotinylated 2nd antibody, streptavidin-peroxidase, and o-phenylenediamine (OPD). The reaction between peroxidase, H2O2 and OPD produces a yellow orange color, the strength of which is generally proportional to the amount of 64M-2 antibody remaining bound to the plate. The color development reaction is stopped and the absorbance of each well at 492 nm is measured with a spectrophotometer.

[REACTIVITY]
• Anti-6-4PPs monoclonal antibody clone 64M-2 recognizes 6-4PPs on single-stranded DNA.
• 64M-2 binds to 6-4PPs formed each dipyrimidine sequence context (TT, TC, CT and CC).
• 64M-2 stably binds to 6-4PPs in DNA longer than eight bases.
• 64M-2 binds to 6-4PPs in UV-irradiated DNA purified from a wide range of sources the prokaryote and eukaryote irradiated with UV.

Kit Components

Item Amount
ELISA plate precoated with protamine sulfate
(12 x 8 well strips)
1 plate
Positive standard
Calf thymus DNA, UVC irradiated (10 J/m2 )
1 vial (20 μg/mL, 500 µL)
Negative standard
Calf thymus DNA, not irradiated
1 vial (20 μg/mL, 500 µL)
Assay Diluent Concentrate (10X) 1 vial (10 ml)  
Wash Buffer Concentrate (20X) 2 x 15 mL vials
Blocking Reagent Concentrate (50X) 2 vials, lyophilized.
Reconstitute with 400 µl purified water before use.
Anti-6-4PPs Monoclonal Antibody (100X)
(clone 64M-2)
1 vial, lyophilized.
Reconstitute with 150 μL purified water for a 100X working solution.
Biotinylated 2nd antibody  (100X) 1 vial (150 µL)
Streptavidin-peroxidase (100X) 1 vial (150 µL)
OPD Tablet (5mg) 3 tablets
OPD Diluent Concentrate (10X) 1 vial (1.7 mL)
Stop Solution 1 vial (12 mL)
Plate Cover Film 3 covers
Instruction Manual 1 manual

Expiration date:@less than 6 month

Experimental Examples


UV-induced 6-4PPs in DNA measured by ELISA


Formation and repair of UV-induced 6-4PPs in human cells measured by ELISA
UVC radiation induces 6-4PPs in DNA of HeLa cells in dose-dependent manner. The initial level of 6-4PPs induced by 10 J/m2 of UVC decreases over time as 6-4PPs are repaired, indicating the capacity of nucleotide excision repair in HeLa cells.

< SELECTED REFERENCES >

  • 1) Mori, T., et al., Photochem. Photobiol. 54, 225-232 (1991). PMID: 1780359
  • 2) Matsunaga, T., et al., Photochem. Photobiol. 54, 403-410 (1991). PMID: 1784641
  • 3) Potten, C.S., et al., Int. J. Radiat. Biol. 63, 313-324 (1993) PMID: 8095281
  • 4) Kobayashi, N., et al., J. Invest. Dermatol. 101, 685-689 (1993) PMID: 8228329
  • 5) Todo, T., et al., Nature 361, 371-374 (1993) PMID: 8426655
  • 6) Nakane, H., et al., Nature 377, 165-168 (1995) PMID: 7675085
  • 7) Komatsu, Y., et al., Nucleic Acids Res. 25, 3889-3894 (1997) PMID: 9380513
  • 8) Kobayashi, N., et al., J. Invest. Dermatol. 110, 806-810 (1998) PMID: 9579550
  • 9) Nakagawa, A., et al., J. Invest. Dermatol. 110, 143-148 (1998) PMID: 9457909
  • 10) Otoshi, E., et al., Cancer Res. 60, 1729-1735 (2000) PMID:10749146
  • 11) Katsumi, S., et al., J. Invest. Dermatol. 117, 1156-1161 (2001) PMID: 11710927
  • 12) Kobayashi, N., et al., Pigment Cell Res. 14, 94-102 (2001) PMID: 11310797
  • 13) Wakasugi, M., et al., J. Biol. Chem., 277, 1637-1640 (2002) PMID: 11705987
  • 14) Imoto, K., et al., J. Invest. Dermatol. 119, 1177-7782 (2002) PMID: 12445209
  • 15) Nishiwaki, Y., et al., J. Invest. Dermatol. 122, 526-532 (2004) PMID: 15009740
  • 16) Sugasawa, K., et al., Cell 121, 387-400 (2005) PMID: 15882621
  • 17) Yasuda, G., et al., Mol. Cell. Biol., 27, 6606-6614 (2007) PMID: 17682058
  • 18) Matsumoto, M., et al., J. Cell Sci., 120, 1104-1112 (2007) PMID: 17327276
  • 19) Yamamoto, A., et al., DNA Repair, 6, 649-657 (2007) PMID: 17236820

More than 200 papers using TDM-2 antibodies have been published so far.

Product List

Product Name Cat# Quantity Price

High Sensitivity 6-4PP/ (6-4) photoproduct ELISA kit DataSheet ,

CSR-NM-MA-K002

1 KIT ¥100,000
$1334

Product Name Cat# Quantity Price

High Sensitivity CPD ELISA kit DataSheet ,

CSR-NM-MA-K001

1 KIT N/A

Anti CPDs (Mouse)  DataSheet

CAC-NM-DND-001

1 VIAL ¥44,000
$587

UVC-irradiated DNA samples DataSheet ,

CSR-NM-MA-R010

5*10 UG ¥30,000
$400

PROTAMINE SULFATE COATED ELISA PLATE 96 DataSheet ,

CSR-NM-MA-P001

1 PLATE ¥2,000
$27

PROTAMINE SULFATE COATED ELISA PLATE 96 x 5 DataSheet ,

CSR-NM-MA-P002

1*5 PLATE ¥9,500
$127

PROTAMINE SULFATE COATED ELISA PLATE 96 x 10 DataSheet ,

CSR-NM-MA-P003

1*10 PLATE ¥18,000
$240

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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