High Sensitivity CPD (Cyclobutane pyrimidine dimer) ELISA kit

Transcription

1 Instruction manual High sensitivity ELISA kit for measuring UV-induced DNA damage High Sensitivity CPD (Cyclobutane pyrimidine dimer) ELISA kit Catalog Number : NM-MA-K001(96 tests) For research use only, Not for diagnostic use. - Please.. read.. all.. the.. package.. insert.. carefully.. before.. beginning the.. assay.-

2 INTRODUCTION Prolonged exposure to solar UV radiation may result in acute and chronic health effects to the skin, eye, and immune system, including skin cancers. These harmful effects are suggested to be closely related to 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 strand of DNA. These helix-distorting DNA lesions are repaired exclusively by a nucleotide excision repair system in humans. Mori et al. have developed and characterized monoclonal antibodies specific for CPDs and for 6-4PPs (1). Matsunaga et. al. have established and characterized monoclonal antibodies against DewarPPs (2). Cyclobutane pyrimidine dimers (CPDs) ELISA kit is optimized for high sensitivity detection of CPDs in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay (ELISA). This system can detect CPDs formed in every dipyrimidine sequence (TT, TC, CT and CC) in DNA. Thus, this technology will contribute to understanding the molecular mechanisms of cellular responses to UV light and DNA damage in many research fields including cancer research, photobiology, dermatology, ophthalmology, immunology, and cosmetology. Figure 1: Structures of DNA damage produced by UV light. HS CPD ELISA kit Cat#: NM-MA-K001 2/8 Version#:

3 ASSAY PRINCIPLE To measure DNA damage in DNA, we use ELISA (enzyme-linked immunosorbent assay) using Anti-CPDs (Clone: TDM-2). Genomic DNA is purified from UV-damaged cells and heat denatured DNA is coated on wells of 96-well plate. The binding of TDM-2 to DNA damage is detected by sequential treatment with biotinylated 2nd antibody and streptavidin-peroxidase. Then, the absorbance of colored products derived from OPD is measured at 492 nm. Figure 2: ELISA method REACTIVITY 1) Anti-CPDs bind to CPDs in single-stranded DNA. 2) Anti-CPDs bind to CPDs formed in every dipyrimidine sequence (TT, TC, CT and CC). 3) Anti-CPDs stably binds to CPDs formed in oligonucleotides consisting of more than eight bases. HS CPD ELISA kit Cat#: NM-MA-K001 3/8 Version#:

4 KIT COMPONENTS Reagents PROTAMINE SULFATE COATED ELISA PLATE 96 (8X12) Positive CPD Standard using Calf thymus DNA (UVC irradiation : 10 J/m 2 ) Negative CPD Standard using Calf thymus DNA (UVC irradiation : 0 J/m 2 ) Assay Diluent (10X) Wash Buffer (20X) Blocking Reagent (50X) Anti-CPDs (100X) Biotinylated 2nd antibody (100X) Streptavidin-peroxidase (100X) OPD Tablet (2mg) OPD Diluent (10X) Stop Solution Cover Film Quantity 1 plate 10 μg/ml, 100 μl/vial,1 vial (Lyophilized) 10 μg/ml, 100 μl/vial,1 vial (Lyophilized) 10 ml, 1 bottle (Liquid) 15 ml, 2 bottles (Liquid) 200 μl/vial, 2 vials (Lyophilized) 150 μl/vial, 1 vial (Lyophilized) 150 μl/vial, 1 vial (Lyophilized) 150 μl/vial, 1 vial (Lyophilized) 2 tablets 600 μl, 2vials (Liquid) 12 ml, 1 bottle (Liquid) 3 sheets Instruction manual 1 MATERIALS TO BE SUPPLIED BY THE USERS DNA samples DNA Purification Kit (Reccomended is a QIAamp Blood Kit (QIAGEN, Cat. No or 51106)) Heating Block Pure water. 10 μl to 1000 μl adjustable single channel micropipetters and disposable tips. 50 μl to 150 μl adjustable multichannel micropipetters and disposable tips. Reservoir for Wash Solution 1.5 ml tubes for diluting samples. 15 ml or 50 ml tubes for dilution Incubator (37 ) Microplate reader capable of monitoring the absorbance at 492 nm. STORAGE 1. For unopened kit: The kit should be stored at After opening: Reconstituted form should be stored at -20. Others should be stored at 4. PROTAMINE SULFATE COATED ELISA PLATE can be stored at room temperature in the dark. HS CPD ELISA kit Cat#: NM-MA-K001 4/8 Version#:

5 PREPARATION OF REAGENTS 1. PROTAMINE SULFATE COATED ELISA PLATE 96 Bring to room temperature (18-25 ) before use. Please return the unused wells to the foil pouch. 2. Positive and Negative CPD Standards Standards are lyophilized form. Reconstitute with 100 μl of pure water. The concentration of the standard solution is 10 μg/ml. 3. Assay Diluent Dilute 10 ml of Assay Diluent Buffer concentrate (10X) with 90mL of pure water to make 100 ml of Assay Diluent (1X). 4. Wash Buffer Dilute 15 ml of Wash Solution concentrate (20X) with 285 ml of pure water to make 300 ml of Wash Solution (1X). 5. Blocking Reagent This Blocking Reagent is lyophilized form. Reconstitute with 200 μl of pure water. The solution is a 50X concentrate. Dilute (1:50) it to the working concentration with Assay Diluent. 6. Anti-CPD This antibody is lyophilized form. Reconstitute with 150 μl of pure water. The Anti-CPDs solution is a 100X concentrate. Dilute (1:100) it to the working concentration with Assay Diluent. 7. Biotinylated 2nd antibody This 2nd antibody is lyophilized form. Reconstitute with 150 μl of pure water. The Biotinylated 2nd antibody solution is a 100X concentrate. Dilute (1:100) it to the working concentration with Assay Diluent. 8. Streptavidin-peroxidas This is lyophilized form. Reconstitute with 150 μl of pure water. The solution is a 100X concentrate. Dilute (1:100) it to the working concentration with Assay Diluent. 9. Substrate solution (OPD solution) Dilute 500 μl of OPD Diluent concentrate (10X) with 4.5 ml of pure water to make 5 ml of substrate buffer (1X). Resolve an OPD tablet with 5 ml of substrate buffer to make substrate solution. It should be prepared immediately before use. HS CPD ELISA kit Cat#: NM-MA-K001 5/8 Version#:

6 ASSAY PROTOCOLS A. Cell culture and UV irradiation 1. Plate cells in 10 cm dishes and culture one or two days. 2. Wash cells once by Dulbecco s PBS (DPBS) and irradiate cells with UV (for example ; 0, 2.5, 5, 7.5, 10 J/m 2 of 254 nm UV). To study DNA repair, following UV irradiation with 10 J/m 2, incubate cells for a variety of times (for example ; 1, 3, 8, 24 hours) to allow to repair. 3. Wash cells by 10 ml of DPBS and then cells were harvested by a cell scraper from the dishes and centrifuged at 10,000 x g for 15 seconds at 4 4. Cell pellets were stored at 80 until processing. B. DNA isolation 5. Genomic DNA was purified using a QIAamp Blood Kit (QIAGEN, Cat. No or 51106). DNA concentrations were calculated from the absorbance at 260 nm. C. DNA sample coating to the PROTAMINE SULFATE COATED ELISA PLATE 6. Prepare sample DNA or CPDs DNA Standard solutions in 1X Assay Diluent at the concentration of 0.2 μg / ml 7. To denature DNA, heat DNA solutions in a hot plate at 100 for 10 minutes and chill rapidly in an ice bath for 15 minutes. 8. Distribute 50 μl / well of each denatured DNA solution to PROTAMINE SULFATE COATED ELISA PLATE (use 2 wells for each sample) and dry completely overnight at 37. D. DNA damage detection 9. Wash the DNA-coated plates 5 times with 150 μl/ well of X1 Wash Buffer. 10. Distribute 150 μl/ well of X1 Blocking Reagent to each well to prevent non-specific antibody binding. 11. Incubate 30 minutes at Wash the plates 5 times with 150 μl/ well of X1 Wash Buffer. 13. Distribute 100 μl / well of 1:100 Anti-CPDs (Clone: TDM-2) diluted with X1 Assay Diluent to each well and incubate 30 minutes at Wash the plates 5 times with 150 μl/ well of X1 Wash Buffer. 15. Distribute 100 μl / well of 1:100 Biotinylated-2nd antibody diluted with X1 Assay Diluent to each well and incubate 30 minutes at Wash the plates 5 times with 150 μl/ well of X1 Wash Buffer. 17. Distribute 100 μl / well of 1:100 Peroxidase-Streptavidin diluted with X1 Assay Diluent to each well and incubate 30 minutes at Wash the plates 5 times with 150 μl/ well of X1 Wash Buffer. 19. After throwing the buffer away, distribute 100 μl / well of the Substrate solution to each well and incubate 30 minutes at Distribute 50 μl / well of Stop solution to each well and stop enzyme reaction. 21. After gentle mixing, determine the absorbance at 492 nm of each well by a spectrophotometer. HS CPD ELISA kit Cat#: NM-MA-K001 6/8 Version#:

7 EXAMPLE OF RESULTS Figure 3: UV-induced DNA damage measured by CPDs ELISA Figure 4: Induction and repair of UV-induced DNA damage in human cells measured by ELISA By measuring the absorbance of colored products at 492 nm, we can examine the induction and repair of CPDs. In this case of human cells, DNA damage increased with increasing UV doses and the initial damage number gradually decreased with increasing repair times. SELECTED REFERENCES 1) Mori, T., et al., Photochem. Photobiol. 54, (1991). 2) Matsunaga, T., et al., Photochem. Photobiol. 54, (1991). 3) Potten, C.S., et al., Int. J. Radiat. Biol. 63, (1993) 4) Kobayashi, N., et al., J. Invest. Dermatol. 101, (1993) 5) Todo, T., et al., Nature 361, (1993) 6) Nakane, H., et al., Nature 377, (1995) 7) Komatsu, Y., et al., Nucleic Acids Res. 25, (1997) 8) Kobayashi, N., et al., J. Invest. Dermatol. 110, (1998) 9) Nakagawa, A., et al., J. Invest. Dermatol. 110, (1998) 10) Otoshi, E., et al., Cancer Res. 60, (2000) 11) Katsumi, S., et al., J. Invest. Dermatol. 117, (2001) 12) Kobayashi, N., et al., Pigment Cell Res. 14, (2001) 13) Wakasugi, M., et al., J. Biol. Chem., 277, (2002). 14) Imoto, K., et al., J. Invest. Dermatol. 119, (2002) 15) Nishiwaki, Y., et al., J. Invest. Dermatol. 122, (2004) 16) Sugasawa, K., et al., Cell 121, (2005). 17) Yasuda, G., et al., Mol. Cell. Biol., 27, (2007). 18) Matsumoto, M., et al., J. Cell Sci., 120, (2007). 19) Yamamoto, A., et al., DNA Repair, 6, (2007). More than 200 papers using TDM-2 antibodies have been published so far. HS CPD ELISA kit Cat#: NM-MA-K001 7/8 Version#:

8 RELATED PRODUCT Product Name Maker Cat# Anti cyclobutane pyrimidine dimers (CPDs) Monoclonal Antibody (Clone:TDM-2) CAC NM-DND-001 Anti (6-4) photoproducts (6-4PPs) Monoclonal Antibody (Clone:64M-2) CAC NM-DND-00 Anti Dewar photoproducts (DewarPPs) Monoclonal Antibody (Clone:DEM-1) CAC NM-DND-003 Anti Acetylaminofluorene(AAF)-DNA adducts Monoclonal Antibody (Clone:AAF-1) CAC NM-MA-001 UVC irradiated DNA sample (0, 2.5, 5, 7.5, 10 J/m2 ) CSR NM-MA-R010 PROTAMINE SULFATE COATED ELISA PLATE 96 CSR NM-MA-P001 PROTAMINE SULFATE COATED ELISA PLATE 96 x 5 CSR NM-MA-P002 PROTAMINE SULFATE COATED ELISA PLATE 96 x 10 CSR NM-MA-P003 Anti XPA Monoclonal Antibody (Clone:A-2) CAC KUP-TM-M01 Anti XPA Monoclonal Antibody (Clone:5F12) BAM Anti XPF Monoclonal Antibody (Clone:A-2) CAC KUP-TM-M02 Anti XPG Monoclonal Antibody (Clone:A-2) CAC KUP-TM-M03 Anti ERCC1Monoclonal Antibody (Clone:A-2) CAC KUP-TM-M04 Anti DDB1 Monoclonal Antibody (Clon: ) CAC KUP-TM-M05 For research use only, Not for diagnostic use. TOYO 2CHOME, KOTO-KU, TOKYO, , JAPAN URL: export@cosmobio.co.jp [Outside Japan] Phone : [ 国内連絡先 ] Phone : FAX : FAX : HS CPD ELISA kit Cat#: NM-MA-K001 8/8 Version#: