**Elastase ELISA Kit Instructions for Use**
This Elastase ELISA Kit is intended for research use only and is designed to quantify the level of bovine elastase in bovine serum, plasma, and other related liquid samples. The method employed is a double-antibody sandwich ELISA, ensuring high specificity and accuracy.
The procedure involves coating microtiter plate wells with purified bovine elastase-specific antibodies. After incubation with the sample, the target elastase binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured elastase, forming an antibody-antigen-enzyme complex. Following washing steps, TMB substrate is added, which turns blue under HRP catalysis and then changes to yellow upon acid termination. The intensity of the color is directly proportional to the elastase concentration in the sample.
The optical density (OD) at 450 nm is measured using a microplate reader, and the results are compared against a standard curve to calculate the actual elastase concentration in the sample.
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**Kit Components (48-well or 96-well configurations):**
- Microtiter Plate (1 × 48 or 1 × 96)
- Standard (54 U/L) – 0.5 mL × 1 bottle
- Standard Diluent – 1.5 mL × 1 bottle
- Enzyme Label Reagent – 3 mL × 1 bottle (or 6 mL for 96-well)
- Sample Diluent – 3 mL × 1 bottle (or 6 mL for 96-well)
- TMB Substrate A & B – 3 mL × 1 bottle each
- Wash Buffer (20×) – 20 mL × 20 times (or 20 mL × 30 times)
- Sealing Film – 2 pieces (for 48-well), 2 pieces (for 96-well)
- Sealed Bag – 1
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of manufacture.
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**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant; if precipitate forms, re-centrifuge.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge similarly. Collect supernatant.
3. **Urine:** Collect in sterile tubes, centrifuge, and collect supernatant.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
5. **Tissue Samples:** Weigh the tissue, homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C after thawing.
6. **General Notes:** Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freezing and thawing. Do not use samples containing NaN3 due to enzyme inhibition.
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**Procedure Summary:**
1. **Standard Dilution:** Prepare a 5-fold serial dilution of the standard (36, 24, 12, 6, 3 U/L).
2. **Sample Addition:** Add 40 μL of sample diluent and 10 μL of sample to each well (final dilution 5×).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Rinse 5 times with diluted wash buffer.
5. **Enzyme Addition:** Add 50 μL of HRP-labeled antibody to each well (except blank).
6. **Second Incubation:** Repeat incubation at 37°C for 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 OD at 450 nm within 15 minutes.
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**Important Notes:**
- Equilibrate the kit to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Ensure accurate pipetting and avoid light exposure during TMB development.
- Always prepare a standard curve and run duplicates for better precision.
- Follow all safety protocols when handling biological samples and waste.
**Calculation Method:**
Plot the standard curve using OD values vs. concentrations. Calculate the sample concentration using linear regression and multiply by the dilution factor (e.g., ×5 for 5× dilution).
**Performance Characteristics:**
- Correlation coefficient (R²) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
- Detection range: 1–45 U/L
This kit is a reliable tool for researchers studying elastase levels in various biological matrices. Always refer to the latest version of the manual for any updates or changes.
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