ELISA/assay

DNA Damage (8-OHdG) ELISA Kit

EK7114

96 wells/kit, with removable strips.

Enabled

DNA Damage (8-OHdG) ELISA Kit

Picokine ELISA Kits, Products

0.94 - 60 ng/mL

1. A plate reader capable of measuring absorbance at 450 nm. . 2. Adjustable pipettes and a repeat pipettor. . 3. Deionized or distilled water. . 4. Materials used for Sample Preparation.

No

Colorimetric detection of DNA Damage (8-OHdG). 96wells/kit, with removable strips.

Colorimetric detection of DNA Damage (8-OHdG). 96wells/kit, with removable strips.

Introduction . Boster's ELISA Kit is a competitive assay that can be used for the quantification of 8-OHdG in urine, cell culture, plasma, and other sample matrices. The ELISA utilizes an 8-hydroxy-2-deoxy Guanosine-coated plate and an HRP-conjugated antibody for detection which allows for an assay range of 0.94 - 60 ng/mL, with a sensitivity of 0.59 ng/mL. The other highlights of this kit are a quick incubation time of 60 minutes, stable reagents, and an easy to use protocol. It is important to note that the 8-OHdG antibody used in this assay recognizes both free 8-OHdG and DNA-incorporated 8-OHdG. Since complex samples such as plasma, cell lysates, and tissues are comprised of mixtures of DNA fragments and free 8-OHdG, concentrations of 8-OHdG reported by ELISA methodology will not coincide with those reported by LC-MS where the single nucleoside is typically measured. This should be kept in mind when analyzing and interpreting experimental results.

Standard Preparation (S1-S8) . NOTE: The Standard should be aliquotted into smaller portions before use to ensure product integrity. Avoid freeze/thaw cycles. (10 µL of Standard can prepare a triplicate standard curve). . 1. Centrifuge the 8-hydroxy-2-deoxy Guanosine Standard vial before removing the cap. This process with assure that all of the standard is collected and available for use. . 2. Label seven (7) polypropylene tubes, each with one of the following standard values: 60 ng/mL, 30 ng/mL, 15 ng/mL, 7.5 ng/mL, 3.75 ng/mL, 1.875 ng/mL and 0.94 ng/mL. . 3. Add 500 µL of Sample and Standard Diluent to Tube #1. . 4. Add 250 µL of Sample and Standard Diluent to Tube #2, 3, 4, 5, 6 and 7. . 5. Add 10 µL of the 3.06 µg/mL 8-hydroxy-2-deoxy Guanosine Standard to Tube#1 for a concentration of 60 ng/mL. Mix well. . 6. Transfer 250 µL from Tube #1 to Tube #2. Mix well. . 7. Similarly, complete the dilution series to generate the remaining standards (250 µL from Tube #2 to Tube #3, mix well, etc.) up to and including Tube #7. . 8. Finally, add 250 µL Sample and Standard Diluent to another 1.5mL polypropylene tube (Tube #8), which is the zero standard (0 ng/mL). 1X Wash Buffer Preparation . Prepare 1X wash buffer by adding 50 ml of Wash Buffer Concentrate to 450 ml deionized or distilled water to prepare 500 mL of Wash Buffer. 8-hydroxy-2-deoxy Guanosine: HRP Conjugate Monoclonal Antibody Preparation . It is recommended to prepare this reagent immediately prior to use by diluting 8-hydroxy-2-deoxy Guanosine: HRP Conjugate Monoclonal Antibody 1:100 with 8-hydroxy-2-deoxy Guanosine Antibody Diluent. Prepare 100 µl by adding 1 µl of 8-hydroxy-2-deoxy Guanosine: HRP Conjugate Monoclonal Antibody to 99 µl of 8-hydroxy-2-deoxy Guanosine Antibody Diluent for each well. Mix gently and thoroughly and use within 2 hours of generation. Assay Overview . 1. Prepare standard and samples in the Sample and Standard Diluent. . 2. Add 50 µL of prepared standards and samples in triplicate to appropriate wells. . 3. Add 50 µL of the diluted antibody preparation to the appropriate wells. . 4. Cover plate with Plate Cover and incubate at room temperature (20-25°C) for 1 hour. . 5. Wash plate 4 times with 1X Wash Buffer. . 6. Add 100 µL of TMB Substrate to each well. . 7. Cover plate and develop the plate in the dark at room temperature for 30 minutes. . 8. Add 100 µL of Stop Solution to each well. . 9. Measure absorbance on a plate reader at 450 nm. . 10. Plot the standard curve and calculate sample concentrations.

96 wells/kit, with removable strips.

No

Store at 4 C and -20 C. (All reagents are stable as supplied at 4 C, except the standard , which should be stored at -20 C. )

8-Hydroxy-2-deoxy Guanosine (8-OHdG): 100%. 8-Hydroxy Guanosine (8-OHG): 23%. 8-Hydroxy Guanine (8-oxoG): 23%. Guanosine: <0.01%.

0.59 ng/mL

Description Quantity 8-hydroxy-2-deoxy Guanosine : BSA Coated Plate 1 Plate 8-hydroxy-2-deoxy Guanosine Standard 1 vial/ 100uL 8-hydroxy-2-deoxy Guanosine HRP Conjugated Monoclonal Antibody 1 vial/75uL Sample and Standard Diluent 1 vial/50mL 8-hydroxy-2-deoxy Guanosine Antibody Diluent 1 vial/13mL Wash Buffer Concentrate 1 vial/50mL TMB Substrate 1 vial/ 13mL Stop Solution 1 vial/ 13mL Plate Cover 2 covers

Cell lysates, Plasma, Sample matrices, Urine

ELISA

All species

Typical Standard Curve for the DNA Damage (8-OHdG) ELISA Kit (Enzyme-Linked Immunosorbent Assay)–EK7114. Assay Type: Competitive ELISA. Detection Method: Colorimetric Assay. Assay Range: 0.94 – 60 ng/ml. Chemical Equation of the Oxidation of Guanosine Diagram of the 8-OHdG Competitive ELISA Urine Spike Assay Diagram of the Preparation of the 8-OHdG Standards Diagram of the Triplicate Sample Plate Format Preview of the Calculations Worksheet

8-hydroxy-2-deoxy Guanosine (8-OH-dG) is produced by the oxidative damage of DNA by reactive oxygen and nitrogen species and serves as an established marker of oxidative stress. Hydroxylation of guanosine occurs in response to both normal metabolic processes and a variety of environmental factors (i.e., anything that increases reactive oxygen and nitrogen species). Increased levels of 8-OH-dG are associated with the aging process as well as with a number of pathological conditions including cancer, diabetes, and hypertension. In complex samples such as plasma, cell lysates, and tissues, 8-OH-dG can exist as either the free nucleoside or incorporated in DNA. Once the blood enters the kidney, free 8-OH-dG is readily filtered into the urine, while larger DNA fragments remain in the bloodstream. Because of the complexity of plasma samples, urine is a more suitable matrix for the measurement of free 8-OH-dG than plasma. Urinary levels of 8-OH-dG range between 2.7-13 ng/mg creatine, while plasma levels of free 8-OH-dG have been reported to be between 4-21 pg/ml as determined by LC-MS.

8-OH-dG; 8OHG; 80G; 8 hydroxyguanine; 8-OHdG; DNA Damage

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Precision . Intra-Assay Precision: Three samples of known concentration were assayed thirty times on one plate; the intra-assay coefficient of variation of the DNA Damage ELISA has been determined to be

1 hour

4/12/19 8:20