8-OHDG ELISA 试剂盒

• 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

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.

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

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.

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.

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).

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).

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.

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.