8-OHDG ELISA Kit

Produktnummer ABIN2964843

Produktdetails 8-OHDG ELISA Kit

Target: 8-OHDG (8-Hydroxyguanosine (8-OHDG))

Reaktivität: Diverse Spezies

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Detektionsbereich: 0.94 ng/mL - 60 ng/mL

Untere Nachweisgrenze: 0.94 ng/mL

Applikation: ELISA

Verwendungszweck: Colorimetric detection of 8-hydroxy-2-deoxy Guanosine

Proben: Urine, Cell Lysate, Plasma, Sample Matrices

Analytische Methode: Quantitative

Spezifität: 8-hydroxy-2-deoxy Guanosine 100%
8-hydroxy Guanosine 23%
8-hydroxy Guanine 23%
Guanosine < 0.01%

Sensitivität: 0.59 ng/mL

Produktmerkmale: ELISA Kit for 8-OHdG detection in samples.
8-OHdG ELISA 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.

Bestandteile:

• 8-hydroxy-2-deoxy Guanosine : BSA Coated Plate
• 8-hydroxy-2-deoxy Guanosine Standard
• 8-hydroxy-2-deoxy Guanosine HRP Conjugated Monoclonal Antibody
• Sample and Standard Diluent
• 8-hydroxy-2-deoxy Guanosine Antibody Diluent
• Wash Buffer Concentrate
• TMB Substrate
• Stop Solution
• Plate Cover

Benötigtes Material: - A plate reader capable of measuring absorbance at 450 nm
- Adjustable pipettes and a repeat pipettor
- Deionized or distilled water
- Materials used for Sample Preparation

Anwendungsinformationen

Testdauer: 1 h

Plattentyp: Pre-coated

Protokoll:

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.

Aufbereitung der Reagenzien:

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

1X Wash Buffer Preparation

1. Prepare 1X Wash buffer by diluting 10X Wash Buffer in distilled or deionized water. For example, if preparing 500 mL of 1X Wash Buffer, dilute 50 mL of 10X Wash Buffer into 450 mL of distilled water. Mix well.
2. Store reconstituted 1X Wash Buffer at 2-8ºC for up to one (1) month. Do not use 1X Wash Buffer if it becomes visibly contaminated during storage.

8-hydroxy-2-deoxy Guanosine: HRP Conjugate Monoclonal Antibody Preparation

1. Determine the amount of Antibody Preparation required. For every strip-well used (8-wells), prepare 0.5 mL of Antibody Preparation.
2. Prepare Antibody Preparation by diluting the 8-hydroxy-2-deoxy Guanosine: HRP Conjugate Antibody Concentrate 1:100 with 8-hydroxy-2-deoxy Guanosine Antibody Diluent. For example, if 6 mL of Antibody Preparation is required (one whole plate), dilute 60 µL of Antibody in 6 mL of 8-hydroxy2-deoxy Guanosine Antibody Diluent. Mix well prior to use.

Probennahme: Urine
Interference in urine is infrequent; dilutions appropriate for this assay show a direct linear correlation between 8-OHdG immunoreactivity and 8-OHdG concentration (see figure 3). Urinary concentrations of 8-OHdG can vary considerably and can be standardized against creatinine levels if required.
Storage: Fresh urine samples should be centrifuged at 2,000 x g for 10 minutes or filtered with a 0.2 µm filter before this assay, and stored at -20°C immediately after collection.
Dilution: Dilute urine samples 1:20 (v:v) in Sample and Standard Diluent as the starting dilution prior to testing. For example: 9 µL of sample into 171 µL of Sample and Standard Diluent.

Plasma/Serum
The concentration of free 8-OHdG in plasma is very low relative to the level of DNA-incorporated 8-OHdG. Glomerular filtration results in excretion of 8-OHdG into the urine, while the DNA-incorporated 8-OHdG remains in the blood. The differing fates of free versus DNA-incorporated 8-OHdG should be considered in experimental design. If you choose to measure DNA-incorporated 8-OHdG in plasma, it is possible to purify DNA using a commercially available kit and treat the DNA with a combination of nuclease and alkaline phosphatase to liberate the individual bases. Due to the complexities of measuring 8-OHdG in plasma, urine is often a more appropriate matrix.
Storage: Collect plasma using established methods and store at -80°C.
Dilution: Serum samples may be diluted 1:20 (v:v) in Sample and Standard Diluent as the starting dilution prior to testing

Culture Media Samples
Storage: Collect culture media samples and store at -80°C.
Dilution: Fetal bovine serum contains 8-OHdG, therefore assays should either be performed in serum-free medium or PBS; these samples may be assayed directly. If the 8-OHdG concentration is high enough to dilute the sample 10-fold with Sample and Standard Diluent, the assay can be performed without any modifications. When assaying less concentrated samples (where samples cannot be diluted 1:10 with Sample and Standard Diluent), dilute the standards in the same culture medium as that used for the experiment. This will ensure that the matrix for the standards is comparable to the samples. We recommend that a standard curve be run first to ensure that the assay will perform in a particular culture medium.

Cell Lysates
Storage: Collect lysates using established methods and store at -80°C until use.
Usage: Purify DNA using a commercially available extraction kit. Digest DNA using nuclease P1 (Sigma N8630 or equivalent) following the manufacturer’s instructions. Adjust pH to 7.5-8.5 using 1M Tris. Add 1 unit of alkaline phosphatase per 100 µg of DNA and incubate at 37 °C for 30 minutes. Boil for 10 minutes and place on ice until use.

Tissue Samples
Storage: Snap-freeze tissue samples in liquid nitrogen immediately after collection. Store at -80 °C until use.
Usage: When ready to use the samples, thaw and add 5 ml of homogenization buffer (0.1 M phosphate buffer, pH 7.4, containing 1 mM EDTA) per gram of tissue. Homogenize the sample using either a Polytron-type homogenizer or a sonicator. Centrifuge at 1,000 x g for 10 minutes and purify the supernatant using a commercially available DNA extraction kit. Digest DNA using nuclease P1 (Sigma N8630 or equivalent) following the manufacturer’s instructions. Adjust the pH to 7.5-8.5 using 1 M Tris. Add 1 unit of alkaline phosphatase per 100 μg of DNA and incubate at 37°C for 30 minutes. Boil for 10 minutes and place on ice until use.

Saliva
Storage: Saliva samples should be stored at -80 °C immediately after collection. Samples may be assayed directly after appropriate dilution.
Dilution: Saliva samples can be prepared 1:8 (v:v) in Sample and Standard Diluent as a suggested starting dilution.

Aufbereitung der Proben:

Proper sample storage and preparation are essential for consistent and accurate results. Caution should be taken during sample work up, to avoid inadvertent oxidation of undamaged DNA. Please read this section thoroughly before beginning the assay.
NOTE: Prepare at least 180 µL of your diluted sample to permit assay in triplicate(approximately 50 µL/ well).

Testdurchführung:

Plate Set Up
The 96-well plate(s) included with this kit is supplied ready to use. It is not necessary to rinse the plate(s) prior to adding the reagents. NOTE: If you do not need to use all the strips at once, place the unused strips back in the plate packet and store at 2-4°C. Be sure the packet is sealed with the desiccant inside.
For statistical purposes, we recommend assaying samples in triplicate.

Addition of the Reagents

1. Add 50 µl (in triplicate) of each of the following to appropriate wells:
   • Prepared 8-hydroxy-2-deoxy Guanosine Standard (Tube #1 through Tube #7) into wells labelled S1-S7
   • Zero Standard (Tube #8- Sample and Standard Diluent, which represents 0 ng/mL)into wells labelled S8
   • Samples (previously prepared- See Sample Preparation, pages 9-11) into wells labelled 1-23
   
2. Add 50 µl of the previously diluted 8-hydroxy-2-deoxy Guanosine Antibody Preparation to each well, except the blank.
3. Add 50 µl of Standard and Sample Diluent and 50 µl of Antibody Diluent into wells labelled as the blank.

Incubate the Plate

1. Cover each plate with the plate cover and incubate 1 hour at room temperature (20-25˚C).

Plate Washing

1. Carefully remove adhesive plate cover. Gently squeeze the long sides of the plate frame before washing to ensure all strips remain securely in the frame.
2. Empty plate contents. Use a multi-channel pipette to fill each well completely (300 µl) with 1X Wash buffer, then empty plate contents. Repeat procedure three additional times, for a total of FOUR washes. Blot plate onto paper towels or other absorbent material.
   NOTE: Follow the same procedure when using an automated plate washer as well. Take care to avoid microbial contamination of equipment. Automated plated washers can easily become contaminated thereby causing assay variability.
   TMB Substrate Incubation and Reaction Stop
   
   • Only remove the required amount of TMB Substrate and Stop Solution for the number of strips being used.
   • Do NOT use a glass pipette to measure the TMB Substrate solution. Do NOT return leftover TMB Substrate to bottle. Do NOT contaminate the unused TMB Substrate. If the solution is blue before use, DO NOT USE IT.
   1. Add 100 µL of TMB Substrate into each well.
   2. Cover carefully with the second provided plate cover.
   3. Allow the enzymatic color reaction to develop at room temperature (20-25ºC) in the dark for 30 minutes. The substrate reaction yields a blue solution.
   4. After 30 minutes, carefully remove the plate cover, and stop the reaction by adding 100 µL of Stop Solution to each well. Tap plate gently to mix. The solution in the wells should change from blue to yellow.
   
   
   Absorbance Measurement
   Note: Evaluate the plate within 30 minutes of stopping the reaction.
   
   1. Wipe underside of wells with a lint-free tissue.
   2. Measure the absorbance on an ELISA plate reader set at 450 nm.

Ergebnisberechnung:

The following procedure is recommended for preparation of the data prior to graphical analysis.

1. Calculate the average Net Optical Density (OD) bound for each standard and sample by subtracting the average Blank OD from the average OD bound.

2. Plot Net OD versus Concentration of 8-OHdG for the standards. Sample concentrations may be calculated off of Net OD values using the desired curve fitting.

3. Samples that read at concentrations outside of the standard curve range will need to be re-analyzed using a different dilution. Make sure to multiply sample concentrations calculated off the curve by the dilution factor used during sample preparation to get starting sample concentration.

Testpräzision: 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 <5%. Inter-Assay Precision: Three samples of known concentration were assayed thirty times in three individual assays, the inter-assay coefficient of variation of the DNA Damage ELISA has been determined to be <5%.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C,4 °C

Informationen zur Lagerung: This kit will perform as specified if the components are stored as directed and used before the expiration date indicated on the outside of the box. All reagents are stable as supplied at 4°C, except the standard, which should be stored at -20°C. For optimum storage, the 8-OHdG Standard should be aliquoted into smaller portions and then stored appropriately. Avoid repeated freeze/thaw cycles (10 µL of Standard can prepare a triplicate standard curve.

Referenzen für 8-OHDG ELISA Kit (ABIN2964843)

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Details zu 8-OHDG

Target: 8-OHDG (8-Hydroxyguanosine (8-OHDG))

Andere Bezeichnung: DNA Damage (8-OHdG) (8-OHDG Produkte)

Substanzklasse: Chemical

Hintergrund: 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.