The rat adenosine ELISA kit operates based on a double-antibody one-step sandwich ELISA method. The microplates are pre-coated with adenosine-specific capture antibodies. After adding the sample, standard, and HRP-labeled detection antibody sequentially, the plate is incubated and then thoroughly washed. A TMB substrate is added, which turns blue under peroxidase activity and then changes to yellow when an acid is introduced. The intensity of the color is directly proportional to the amount of adenosine in the sample. The absorbance is measured at 450 nm using a microplate reader, allowing for the calculation of the sample concentration.
Sample collection and preparation:
1. **Serum**: Collect blood in pyrogen-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Use anticoagulants like EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.
3. **Cell culture supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris.
4. **Tissue homogenate**: Homogenize tissue in physiological saline, then centrifuge at 3000 rpm for 10 minutes to obtain the supernatant.
5. **Storage**: Store samples at -20°C if not tested immediately. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use.
Required equipment:
- Microplate reader (450 nm)
- Precision pipettes: 0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL
- Incubator set at 37°C
Precautions:
- Store the kit at 2–8°C. Let it equilibrate at room temperature for 20 minutes before use. If the wash buffer crystallizes after removal from the fridge, warm it gently in a water bath before use.
- Unused plates should be returned to the ziplock bag and stored at low temperature and dry conditions.
- No dilution is needed for pre-treated samples; add 10 µL directly.
- Follow the incubation time, volume, and sequence strictly as described.
- Shake all reagents well before use.
Kit components:
- Microwells: 96-well or 48-well format
- Standards (300 ng/mL): 0.5 mL
- Standard dilutions: 6 mL (for 96-well), 3 mL (for 48-well)
- Sample diluent: 6 mL (for 96-well), 3 mL (for 48-well)
- Detection antibody-HRP: 6 mL (for 96-well), 3 mL (for 48-well)
- 20× Wash buffer: 20 mL
- Substrate A and B: 6 mL each
- Stop solution: 6 mL
- Seal film: 2 sheets
- Zip lock bags: 1 piece
Reagent preparation:
- Dilute 20× wash buffer with distilled water in a 1:20 ratio.
Washing procedure:
- Manual: Add washing solution, let sit for 1 minute, drain, and repeat 5 times.
- Automatic: Inject 350 µL of wash solution per well, soak for 1 minute, and repeat 5 times.
Procedure:
1. Remove the required number of wells from the foil pouch after 20-minute equilibration.
2. Set up standard, sample, and blank wells.
3. Add 50 µL of standard solutions to the standard wells.
4. Add 10 µL of sample and 40 µL of diluent to the sample wells.
5. Add 50 µL of HRP-labeled detection antibody to each well, seal, and incubate at 37°C for 60 minutes.
6. Wash 5 times with wash buffer.
7. Add 50 µL of TMB A and B, incubate in the dark for 15 minutes.
8. Add 50 µL stop solution and measure OD at 450 nm within 15 minutes.
Result analysis:
Plot the standard curve in Excel, with standard concentrations on the x-axis and OD values on the y-axis. Use the regression equation to calculate sample concentrations.
Kit performance:
- Accuracy: R ≥ 0.9900
- Sensitivity: <1.0 ng/mL
- Specificity: No cross-reactivity with other analogs
- Reproducibility: CV <15% between plates
- Storage: 2–8°C, away from light and moisture
- Shelf life: 6 months
- Detection range: 9.3–300 ng/mL
Disclaimer:
- This kit is for research purposes only. Not for clinical or animal testing. The user assumes all risks. The company is not liable for misuse. Always follow instructions carefully. Do not mix different batch numbers. Any deviation from the protocol will result in user responsibility.
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