**Rat D-Dimer (D2D) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual**
This reagent is intended for research purposes only. The kit is designed to quantify the levels of D-dimer (D2D) in rat serum, plasma, and other biological fluids.
**Principle of the Assay**
The Rat D-Dimer ELISA Kit utilizes a double-antibody sandwich immunoassay. A microplate is pre-coated with purified anti-D2D antibodies. After incubation with the sample, D-dimer binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then recognizes the captured antigen. Following washing steps, TMB substrate is added, and the enzyme reaction produces a color change. The intensity of the color, measured at 450 nm, is directly proportional to the D-dimer concentration in the sample. A standard curve is generated to calculate the unknown concentrations.
**Kit Components**
- 48-well or 96-well configuration
- Sealing film: 2 pieces (48-well), 2 pieces (96-well)
- Coated plate: 1 × 48, 1 × 96
- Standard: 0.5 ml × 1 bottle (5400 pg/ml)
- Standard diluent: 1.5 ml × 1 bottle
- Enzyme reagent: 3 ml × 1 bottle
- Sample diluent: 3 ml × 1 bottle
- TMB A and B solutions: 3 ml × 1 bottle each
- Wash buffer: 20 ml × 20 times (48-well), 20 ml × 30 times (96-well)
- Storage: 2–8°C
**Sample Preparation and Handling**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge similarly.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell culture supernatant**: Centrifuge to remove debris. For intracellular components, lyse cells by freezing and thawing before centrifugation.
- **Tissue**: Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
- **Storage**: Samples should be tested immediately after collection. If not used right away, store at -20°C. Avoid repeated freeze-thaw cycles.
**Important Notes**
- Do not use samples containing NaN3, as it inhibits HRP activity.
- Always prepare a standard curve with duplicate wells.
- Dilute high-concentration samples if OD values exceed the standard range.
- Use separate pipettes for each step to prevent cross-contamination.
- Keep all reagents away from light.
**Procedure Summary**
1. Prepare standards and samples.
2. Add samples and standards to the plate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add HRP-labeled antibody and incubate again.
6. Add TMB substrate and develop color.
7. Stop the reaction with stop solution.
8. Measure absorbance at 450 nm.
9. Calculate concentrations using the standard curve.
**Calculation**
Plot the standard curve using concentration vs. OD values. Determine the sample concentration from the curve and multiply by the dilution factor.
**Performance**
- Linear range: 1000–4500 pg/ml
- Intra-batch and inter-batch CV <9% and <11%, respectively
- Correlation coefficient (R²) ≥ 0.990
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
**Safety and Disposal**
All samples, reagents, and waste should be handled as biohazardous materials. Follow local regulations for disposal.
This manual provides detailed instructions for accurate and reliable detection of D-dimer in rat samples. Ensure all steps are followed precisely for optimal results.
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