Rat 8-hydroxydeoxyguanosine (8-OHdG) elisa detection kit operation - Database & Sql Blog Articles

Rat 8-hydroxydeoxyguanosine (8-OHdG)

ELISA Test

Kit

Operating Instructions

This kit is for research use only and not intended for human or animal diagnostic purposes.

Experimental Principle

The ELISA kit uses a sandwich immunoassay method to quantify 8-hydroxydeoxyguanosine (8-OHdG) in rat samples. The microplate is pre-coated with a specific monoclonal antibody against 8-OHdG. After adding the sample, the antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured antigen, forming an immune complex. Following incubation and washing, the substrate TMB is added. Under the catalytic action of HRP, TMB turns blue and then yellow upon acid termination. The intensity of the color is directly proportional to the concentration of 8-OHdG in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the concentration is determined by comparing the OD value to a standard curve.

Kit Composition

1. 130× Washing Solution – 20ml × 1 bottle

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

4. Standard (200ng/L) – 0.5ml × 1 bottle

5. Enzyme-Labeled Coating Plate – 12 wells × 8

6. Sample Diluent – 6ml × 1 bottle

7. TMB Substrate A – 6ml × 1 bottle

8. TMB Substrate B – 6ml × 1 bottle

9. Standard Dilutions – 1.5ml × 1 bottle

10. Instruction Manual – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If testing is delayed, store at -20°C. Avoid repeated freeze-thaw cycles.

2. Sodium azide (NaN₃) must not be present in the sample, as it can inhibit HRP activity and lead to false results.

Procedure

1. Prepare standards by diluting the original stock according to the provided dilution table.

2. Load 50μL of standard and 40μL of sample diluent into each well, followed by 10μL of the sample (final 5-fold dilution).

3. Seal the plate and incubate at 37°C for 30 minutes.

4. Wash the plate 5 times using the diluted washing solution.

5. Add 50μL of enzyme-labeled reagent to each well (except blank wells).

6. Incubate again at 37°C for 30 minutes.

7. Wash the plate again 5 times.

8. Add 50μL of TMB A and 50μL of TMB B, mix gently, and incubate at 37°C for 15 minutes.

9. Add 50μL of stop solution to terminate the reaction. The color will turn from blue to yellow.

10. Measure the absorbance at 450 nm within 15 minutes of stopping the reaction.

Calculation

Plot the OD values of the standards against their concentrations to create a standard curve. Use this curve to determine the sample concentration based on its OD value. Multiply the result by the dilution factor (5x) to obtain the actual sample concentration.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened components in a sealed bag.

2. If the washing solution crystallizes, warm it gently in a water bath before use.

3. Use accurate pipettes and maintain consistent timing during sample loading.

4. Always prepare a standard curve with duplicate wells. If the sample OD exceeds that of the highest standard, dilute the sample before testing.

5. Do not reuse sealing films to prevent cross-contamination.

6. Keep the substrate away from light.

7. Follow all instructions carefully and verify results with a microplate reader.

8. Treat all waste materials as biohazardous.

9. Do not mix components from different batches.

10. In case of discrepancies, the English manual takes precedence.

Storage Conditions & Expiration

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.