**Human Matrix Lysin (ST3) ELISA Kit Instruction Manual**
**Kit Specification:**
This ELISA kit is available in 48-well or 96-well configurations. It includes the following components:
- **Standard Dilution:** 1.5 ml × 1 vial
- **Enzyme Standard Reagent:** 3 ml × 1 vial (for 48-well) / 6 ml × 1 vial (for 96-well)
**Calculation Method:**
To determine the concentration of ST3 in the sample, plot a standard curve by using the concentration of the standard on the x-axis and the corresponding OD values on the y-axis. Alternatively, calculate the linear regression equation from the standard curve and use it to determine the sample concentration based on its OD value. Multiply the result by the dilution factor to obtain the actual concentration.
**Kit Composition:**
- **Sealing Film:** 2 pieces (48-well) / 2 pieces (96-well)
- **Instructions:** 1 part sealed bag
- **Standard:** 0.5 ml × 1 vial, 2700 ng/L, stored at 2–8°C
- **Enzyme Standard Package:** 1×48 / 1×96, stored at 2–8°C
- **Sample Diluent:** 3 ml × 1 vial (48-well) / 6 ml × 1 vial (96-well), stored at 2–8°C
- **Developer A:** 3 ml × 1 vial (48-well) / 6 ml × 1 vial (96-well), stored at 2–8°C
- **Chromogen B Solution:** 3 ml × 1 vial (48-well) / 6 ml × 1 vial (96-well), stored at -8°C
- **Stop Solution:** 3 ml × 1 vial (48-well) / 6 ml × 1 vial (96-well), stored at 2–8°C
- **Concentrated Washing Solution:** 20 ml × 20 times (48-well) / 20 ml × 30 times (96-well), stored at 2–8°C
**Principle of the Assay:**
The kit uses a double-antibody sandwich ELISA method to detect Human Matrix Lysin (ST3). The microtiter plate is pre-coated with a specific antibody against ST3. After adding the sample, ST3 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, producing a blue color that turns yellow in the presence of acid. The intensity of the color is directly proportional to the ST3 concentration in the sample. Absorbance is measured at 450 nm, and concentrations are calculated using a standard curve.
**Purpose:**
This ELISA kit is intended for the quantitative determination of Human Matrix Lysin (ST3) in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Service Commitment:**
- Fast delivery after payment
- Free technical support during working hours
- Sample testing services available upon request to ensure optimal results
**Storage Conditions & Expiration:**
- **Storage:** 2–8°C
- **Validity:** 6 months
**Sample Preparation and Requirements:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant; if precipitate forms, re-centrifuge.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, 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 freezing and thawing, then centrifuge again.
5. **Tissue Specimen:** Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge at 2000–3000 rpm for 20 minutes. Store the supernatant at 2–8°C or freeze for later use.
6. **General:** Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freezing and thawing.
7. **Note:** Do not use samples containing NaN3, as it inhibits HRP activity.
**Operation Steps:**
1. **Standard Dilution and Loading:** Prepare serial dilutions of the standard and load them into designated wells.
2. **Sample Loading:** Add sample diluent and sample to the test wells. Ensure accurate pipetting.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute the concentrated washing solution and wash the plate 5 times.
5. **Add Enzyme Conjugate:** Add enzyme-labeled reagent to all wells except blank controls.
6. **Incubation:** Repeat incubation step.
7. **Color Development:** Add TMB substrates A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add stop solution to terminate the reaction.
9. **Measurement:** Read absorbance at 450 nm within 15 minutes.
**Notes:**
- Equilibrate the kit to room temperature before use.
- Crystallization of washing solution is normal and does not affect results.
- Use a pipette for accuracy. Load samples within 5 minutes.
- Always prepare a standard curve and run duplicates.
- Discard used sealing films after one use.
- Protect the substrate from light.
- Follow instructions strictly. Results must be confirmed by microplate reader.
- Treat all waste materials as biohazardous.
- Do not mix components from different batches.
- In case of conflict, the English manual takes precedence.
**Kit 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
This manual is designed to guide users through the proper use of the Human Matrix Lysin (ST3) ELISA Kit. Always refer to the latest version of the manual for any updates or changes.
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