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High sensitivity ELISA kit for measuring UV-induced DNA damage

High Sensitivity CPD/ Cyclobutane Pyrimidine Dimer ELISA kit

Cosmo Bio's new Cyclobutane Pyrimidine Dimer (CPD) ELISA Kit uses the highly sensitive and specific monoclonal anti-CPD antibody clone TDM-2, established by Mori et al. TDM-2 is widely cited in the literature and is considered to be the gold standard antibody for CPD detection and quantification.Cosmo Bio's CPD ELISA Kit is the first and only commercially available ELISA kit using TDM-2. All kits components are optimized for high sensitivity CPD detection from cultured cells or skin epidermis.

 

See also  Renewed High Sensitivity CPD

See also  High Sensitivity  6-4PP/ (6-4)Photoproducts 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 (CPD: clone TDM-2; 6-4PP: clone 64M-2; DewarPP clone DEM-1) continue to be cited frequently in the literature, often for use in ELISA by a recommended procedure.

This High Sensitivity Cyclobutane Pyrimidine Dimers (CPDs) ELISA Kit is the only commercially available ELISA utilizing anti-CPD clone TDM-2 and has been optimized for high sensitivity detection of CPDs in DNA purified from cultured cells or from skin epidermis. This ELISA detects CPDs 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.

Cyclobutane pyrimidine dimer (CPD)

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

ASSAY PRINCIPLE

The format of this assay is direct 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. CPD specific monoclonal antibody clone TDM-2 (Cosmo Bio Cat. No. CAC-NM-DND-001) is then added to each well for thirty minutes and unbound antibody is removed by rinsing. The amount of TDM-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 TDM-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-CPD monoclonal antibody TDM-2 recognizes CPDs on single-stranded DNA.
 ・ TDM-2 binds to CPDs formed each dipyrimidine sequence context (TT, TC, CT and CC).
 ・ TDM-2 stably binds to CPDs in DNA longer than eight bases.
 ・ TDM-2 binds to CPDs 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),lyophilized.
Reconstitute with 100 μL purified water before use.
Negative standard
Calf thymus DNA, not irradiated
1 vial (20 μg), lyophilized.
Reconstitute with 100 μL purified water before use.
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 each vial with 200 μL purified water before use.
Anti-CPDs Monoclonal Antibody
(clone TDM-2)
1 vial, lyophilized.
Reconstitute with 150 μL purified water for a 100x working solution.
Biotinylated 2nd antibody  1 vial, lyophilized.
Reconstitute with 150 μL purified water for a 100x working solution. 
Streptavidin-peroxidase (100X) 1 vial, lyophilized.
Reconstitute with 150 μL purified water for a 100x working solution.
OPD Tablet (2mg) 2 tablets
OPD Diluent Concentrate (10X) 2 x 600 μL vials
Stop Solution 1 x 12 mL vial
Plate Cover Film 3 covers
Instruction Manual 1 manual

EXAMPLE OF RESULTS


UV-induced CPDs in DNA measured by ELISA


Formation and repair of UV-induced CPDs in human cells measured by ELISA
UVC radiation induces CPDs in DNA of HeLa cells in dose-dependent manner. The initial level of CPDs induced by 10 J/m2 of UVC gradually decreases over time as CPDs 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 CPD ELISA kit Ver.2

CSR-NM-MA-K003 1KIT

¥ 80,000
$ 1334
€ 1000

Anti CPDs

CAC-NM-DND-001 1VIAL

¥ 44,000
$ 587
€ 440

UVC-irradiated DNA samples

CSR-NM-MA-R010 5*10UG

¥ 24,000
$ 400
€ 300

PROTAMINE SULFATE COATED ELISA PLATE 96

CSR-NM-MA-P001 1PLATE

¥ 2,000
$ 27
€ 20

PROTAMINE SULFATE COATED ELISA PLATE 96 x 5

CSR-NM-MA-P002 1*5PLATE

¥ 7,600
$ 127
€ 95

PROTAMINE SULFATE COATED ELISA PLATE 96 x 10

CSR-NM-MA-P003 1*10PLATE

¥ 14,400
$ 240
€ 180

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

CSR-NM-MA-K002 1KIT

¥ 80,000
$ 1334
€ 1000

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.

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

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