**Human Matrix Lysin (ST3) ELISA Kit Instruction Manual**
**Kit Specification:**
This Human Matrix Lysin (ST3) ELISA Kit is available in 48-well or 96-well configurations. It includes the following components:
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Standard Reagent: 3 mL × 1 bottle (for 48-well) or 6 mL × 1 bottle (for 96-well)
- Sealing Film: 2 pieces (for 48-well) / 2 pieces (for 96-well)
- Standard: 0.5 mL × 1 bottle, concentration: 2700 ng/L
- Sample Diluent: 3 mL × 1 bottle (for 48-well) or 6 mL × 1 bottle (for 96-well)
- Developer A: 3 mL × 1 bottle (for 48-well) or 6 mL × 1 bottle (for 96-well)
- Chromogen B: 3 mL × 1 bottle (for 48-well) or 6 mL × 1 bottle (for 96-well)
- Stop Solution: 3 mL × 1 bottle (for 48-well) or 6 mL × 1 bottle (for 96-well)
- Concentrated Washing Solution: 20 mL × 20 times (for 48-well) or 20 mL × 30 times (for 96-well)
**Storage Conditions and Expiration:**
- Storage: 2–8°C
- Shelf Life: 6 months from the date of manufacture
**Purpose:**
This kit is designed for the quantitative determination of Human Matrix Lysin (ST3) in human serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Principle of Operation:**
The kit utilizes a sandwich ELISA method. The microtiter plate is pre-coated with a specific antibody against ST3. After incubation with the sample, the ST3 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex. TMB substrate is used for color development, which changes from blue to yellow under acidic conditions. The intensity of the color is directly proportional to the concentration of ST3 in the sample. The absorbance is measured at 450 nm, and the concentration is calculated based on a standard curve.
**Sample Preparation and Requirements:**
1. **Serum:** Collect blood at room temperature, allow it to clot for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant. If precipitates form, re-centrifuge before use.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge as above.
3. **Urine:** Collect using a sterile tube, centrifuge, and collect the 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:** Homogenize in PBS, centrifuge, and collect the supernatant. Store at 2–8°C after thawing.
6. **General Notes:** Process samples as soon as possible. If not tested immediately, store at -20°C and avoid repeated freezing/thawing. Do not use samples containing NaN3 as it may inhibit HRP activity.
**Operation Steps:**
1. **Standard Dilution and Loading:** Prepare a 5-point standard curve (1800, 1200, 600, 300, 150 ng/L).
2. **Loading:** Add 40 µL of sample diluent and 10 µL of sample to each test well (final dilution: 5×).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute washing solution and wash 5 times.
5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well (except blank wells).
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.
**Notes and Warnings:**
- Allow the kit to equilibrate at room temperature for 15–30 minutes before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Ensure accurate pipetting and correct dilution.
- Make a standard curve for each run, preferably in duplicate.
- Keep all reagents away from light.
- Follow the manufacturer's instructions strictly.
- All waste should be treated as biohazardous material.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Performance:**
- Linear regression correlation coefficient (R) ≥ 0.95
- Intra-batch variation < 9%, Inter-batch variation < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
We offer free technical support during working hours and sample testing services to ensure optimal experimental results.
**Delivery Period:** From payment to delivery.
This kit is ideal for researchers conducting studies related to matrix metalloproteinases and their role in tissue remodeling and disease progression.
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