Description

The 8-Hydroxy-2′-Deoxyguanosine (8-OHdG) ELISA Kit is an ELISA kit for the quantitative in vitro measurement of 8-Hydroxy-2′-Deoxyguanosine (8-OHdG) concentrations in serum, plasma, and other biological fluids. This assay has high sensitivity and excellent specificity for the detection of 8-hydroxydeoxyguanosine (8-OHdG). No significant cross-reactivity or interference was observed between 8-hydroxydeoxyguanosine (8-OHdG) and its analogs.

Target: 8-hydroxy-2′-deoxyguanosine (8-OHdG)

Reactivity: General (all species)

Proven applications: ELISA

Recommended dilutions

The end-user must determine the optimal dilutions/concentrations.

Storage

Shipped at 4 ° C. Upon receipt, store the kit according to the storage instructions in the kit manual.

Validity

The validity of this kit is 6 months.

Stability

The stability of the kit is determined by the rate of loss of activity. The loss rate is less than 5% within the expiration date under proper storage conditions. To minimize fluctuations in performance, operating procedures and laboratory conditions must be strictly controlled. It is also strongly suggested that all testing be performed by the same user at all times.

• Test Range:  74.1 pg / ml – 6000 pg / ml
• Sensitivity: <26.9 pg / ml
• Standard form: Lyophilized
• Detection method: Colorimetric
• Test type: Competitive
• Test data: Quantitative
• Sample type: Serum, plasma, and other biological fluids.
• Target-type: antigen

Assay Principle

This kit is based on competitive enzyme-linked immunosorbent assay technology. An antibody is precoated in a 96-well plate. Standards, test samples, and biotin-conjugated reagent are added to the wells and incubated. A competitive inhibition reaction occurs between biotin-labeled 8-OHdG and unlabeled 8-OHdG in the pre-coated antibody. The HRP-conjugated reagent is then added and the entire plate is incubated. Unbound conjugates are removed using a wash buffer at each step.

The TMB substrate is used to quantify the HRP enzymatic reaction. After adding TMB Substrate, only wells containing enough 8-OHdG will produce a blue product, which then turns yellow after adding Acid Stop Solution. The intensity of the yellow color is inversely proportional to the amount of 8-OHdG bound to the plate. OD is measured spectrophotometrically at 450 nm in a microplate reader, from which the concentration of 8-OHdG can be calculated.

Kit components

• 96-well precoated microplate
• Standard
• Standard diluent buffer
• Wash buffer
• Detection reagent A
• Detection reagent B
• Diluent A
• Diluent B
• TMB substrate
• Stop solution
• Plate sealer

Material required but not provided

• Incubator at 37 ° C
• Sterile multi-channel and single-channel pipettes and pipette tips
• Jet bottle or automatic microplate washer
• 1.5 ml tubes
• Distilled water
• Absorbent filter papers
• Graduated cylinders of 100 ml and 1 liter
• Microplate reader (wavelength: 450 nm)
• ELISA shaker

Reagent Preparation

1. Standard: Prepare the standard with the recommended volume of Standard Diluent Buffer, to make the standard solution. Then use the standard diluent buffer to make serial dilutions of the standard solution, as indicated in the Protocol.
2. Wash buffer: dilute concentrated wash buffer with distilled water, as indicated in the Protocol.
3. Detection Reagent Preparation: Calculate the total volume of working solution required. Dilute Detection Reagent A and Detection Reagent B with Diluent A and Diluent B, respectively, 1: 100.

Test procedure

1) Establish the standard, test samples, and control wells.
2) Place 50 µl aliquots of the diluted standard into the standard wells.
3) Place 50 µl aliquots of Standard Diluent Buffer into the control (zero) well.
4) Place 50 µl aliquots of diluted samples into the sample wells.
5) Immediately place 50 µl aliquots of Detection Reagent A into each well. Incubate for 1 hour at 37 ° C.
6) Wash 3 times.
7) Aliquot 100 µl Detection Reagent B into each well. Incubate for 30 minutes at 37 ° C.
8) Wash 5 times.
9) Aliquot 90 µl TMB Substrate into each well. Incubate for 10-20 minutes at 37 ° C.
10) Aliquot 50 µl of Stop Solution.
11) Measure OD at 450 nm.

Protocol

• Equilibrate kit components and samples at room temperature (18-25 ° C) before use. It is recommended to draw a standard curve for each test.
• Place the standard, test sample, and control (zero) wells on the pre-coated plate, respectively, and then record their positions. It is recommended to measure each standard and sample at least in duplicate.
• Add 50 µL of each standard, control, and sample to the corresponding wells.
• Remove the cap and discard the liquid.
• Immediately withdraw 50 µl aliquots of Detection Reagent A working solution. Seal the plate with a lid and incubate for 1 hr at 37 ° C.
• Remove the cap and discard the solution. Wash the plate 3 times with 1X Wash Buffer.
• Add 100 µL of Detection Reagent B working solution to each well, seal, and incubate at 37 ° C for 30 min
• Discard the solution and wash the plate 5 times with wash buffer as explained in the previous step.
• Place 90 µl aliquots of TMB Substrate into each well. Seal the plate with a lid and incubate at 37 ° C for 10-20 min. Avoid exposure to light. The incubation time is for reference use only, the end-user must determine the optimal time. Do not exceed 30 min.
• Add 50 µL of Stop Solution to each well. Read at 450 nm immediately.

Calculation of results

This assay is competitive, therefore there is an inverse correlation between the concentration of 8-OHdG in the sample and the measured absorbance. Create a graph with the logarithm of the standard concentration (y-axis) and the measured mean absorbance (x-axis). Apply a trend line of best fit through the standard points. The 8-OHdG concentration of the samples can be interpolated from the standard curve.

Availability

Shipped within 5-7 business days.

Note

• This product is for research only.
• Range and sensitivity are subject to change. Contact us for the latest product information. For accurate results, sample concentrations must be diluted to half the kit range. If you need a specific range, please contact us in advance or write your request in the comments of your order.
• Please note that our ELISA and CLIA kits are optimized for the detection of native samples, rather than recombinant proteins. We cannot guarantee the detection of recombinant proteins, as they may have different sequences or tertiary structures than the native protein.