**Rat D-Dimer (D2D) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual**
This reagent is intended for research purposes only. It is designed to quantitatively determine the concentration of D-dimer (D2D) in rat serum, plasma, and other biological fluids.
**Principle of the Assay**
The kit utilizes a double-antibody sandwich ELISA method to detect D-dimer levels in the sample. A microplate is pre-coated with purified anti-rat D-dimer antibodies. After adding the sample, D-dimer binds to the immobilized antibodies. Subsequently, horseradish peroxidase (HRP)-labeled goat anti-human antibody is added, forming an immune complex. After washing, TMB substrate is introduced, which changes color under the catalytic action of HRP. The reaction is stopped with an acidic solution, and the final color intensity is directly proportional to the D-dimer concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the D-dimer concentration is calculated from a standard curve.
**Kit Components**
The kit includes:
- 1 × 48 or 1 × 96 pre-coated enzyme immunoassay plates
- 2 sealing films (for 48-well format) / 2 sealing films (for 96-well format)
- 1 bottle of standard (5400 pg/ml), 0.5 ml
- 1 bottle of standard diluent, 0.5 ml
- 1 bottle of enzyme-labeled reagent, 3 ml
- 1 bottle of sample diluent, 3 ml
- 1 bottle of TMB A solution, 3 ml
- 1 bottle of TMB B solution, 3 ml
- 1 bottle of stop solution, 3 ml
- 1 bottle of concentrated wash buffer (20×), 20 ml × 20 times (48-well) or 30 times (96-well)
**Storage Instructions**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months when properly stored.
**Sample Preparation and Handling**
1. **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. If precipitate forms, re-centrifuge.
2. **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
3. **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
4. **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells by repeated freeze-thaw cycles, then centrifuge again.
5. **Tissue Samples**: Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge, and collect supernatant. Store at 2–8°C if not used immediately.
6. **General Notes**: Process samples as soon as possible after collection. Avoid repeated freezing and thawing. Do not use samples containing NaN₃, as it may inhibit HRP activity.
**Procedure**
1. **Standard Dilution**: Prepare serial dilutions of the standard on the plate.
2. **Sample Addition**: Add 40 μL of sample diluent followed by 10 μL of sample to each well.
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing**: Wash the plate 5 times with diluted wash buffer.
5. **Enzyme Addition**: Add 50 μL of enzyme-labeled reagent to each well except blank wells.
6. **Incubation**: Incubate at 37°C for another 30 minutes.
7. **Color Development**: Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction**: Add 50 μL of stop solution to each well.
9. **Measurement**: Read absorbance at 450 nm within 15 minutes.
**Notes**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Always prepare a standard curve and run duplicates for accuracy.
- Handle all reagents and waste as biohazardous materials.
- Never mix reagents from different batches.
**Calculation**
Plot the standard curve using OD values vs. concentrations. Determine the unknown sample concentration by interpolation or linear regression. Multiply the result by the dilution factor.
**Performance**
- Intra-batch and inter-batch variation <9% and <11%, respectively.
- Linear range: 1000–4500 pg/mL.
- Correlation coefficient (R²) ≥ 0.990.
**Important:** This manual is for reference only. Always follow the manufacturer's instructions for accurate results.
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