**Human Anti-Trypsin (AT) ELISA Kit – For Quantitative In Vitro Determination of Human AT Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Purposes.**
This ELISA kit is designed for the quantitative determination of Human Anti-Trypsin (AT) concentrations in various biological samples. The kit includes all necessary reagents to perform the assay, including calibration standards, a microtiter plate, conjugated reagents, wash solution, chromogen solutions, and a stop solution. The color change from blue to yellow after adding the stop solution allows for accurate measurement of optical density (OD), which correlates with AT concentration.
To determine AT levels, a standard curve is generated using known concentrations of AT. This curve is then used to calculate the unknown sample concentrations based on their OD values at 450 nm. The procedure involves incubation, washing, and color development steps, followed by reading the results on a microplate reader.
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### **Sample Collection and Storage**
- **Serum**: Use serum separator tubes. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g, 2–8°C. Store at -20°C. Avoid repeated freezing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Remove particulates by centrifugation. Assay immediately or store at -20°C. Avoid freeze-thaw cycles.
**Note:** Ensure proper centrifugation and avoid hemolysis or granulation in samples.
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### **Materials Required (Not Supplied)**
1. Incubator set at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
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### **Reagents Provided (Stored at 2–8°C)**
| Reagent | 96 Determinations | 48 Determinations |
|--------|-------------------|-------------------|
| MicroELISA Strip Plate | 12×8 strips | 12×4 strips |
| Standard (6 vials, 0.5 ml/vial) | 0.5 ml/vial | 0.5 ml/vial |
| Sample Diluent | 6.0 ml | 3.0 ml |
| HRP-Conjugate Reagent | 10.0 ml | 5.0 ml |
| 20X Wash Solution | 25 ml | 15 ml |
| Chromogen Solution A | 6.0 ml | 3.0 ml |
| Chromogen Solution B | 6.0 ml | 3.0 ml |
| Stop Solution | 6.0 ml | 3.0 ml |
| Closure Plate Membrane | 2 | 2 |
| User Manual | 1 | 1 |
| Sealed Bags | 1 | 1 |
**Standard Concentrations:** 3200, 1600, 800, 400, 200, 100 pg/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the test.
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### **Precautions**
1. Do not substitute any reagents. All components are matched for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use.
3. Do not use expired reagents.
4. Use only deionized or distilled water for dilution.
5. Keep microtiter plates in sealed bags until use. Unused strips should be stored with desiccant.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Handle all blood-derived products with caution, assuming potential infectious risk.
8. Dispose of all samples and waste properly, following biosafety guidelines.
9. Substrate solutions must be protected from light and contamination.
10. Chromogen B contains 20% acetone; keep away from heat and flame.
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### **Reagent Preparation and Storage**
- **Wash Solution (1X):** Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized water. Store at 2–8°C for up to one month.
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### **Assay Procedure**
1. Prepare all reagents before starting. Add standards and samples in duplicate to the microtiter plate.
2. Add 50 µL of standard or sample to each well. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate at 37°C for 60 minutes.
4. Wash the plate 4 times manually or automatically.
- **Manual Washing:** Aspirate contents, fill with 1X wash buffer, aspirate again. Repeat 4 times. Dry plate by blotting.
- **Automated Washing:** Follow manufacturer instructions.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting from light.
6. Add 50 µL of Stop Solution to each well. Read OD at 450 nm within 15 minutes.
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### **Data Interpretation**
- Generate a standard curve by plotting average OD values (450 nm) against corresponding AT concentrations.
- Subtract blank OD from all readings before interpretation.
- Use graph paper or software to construct the curve.
- Locate the sample OD on the Y-axis, draw a horizontal line to the curve, then a vertical line to the X-axis to find the concentration.
- Intra-assay and inter-assay CVs are less than 15%.
- Assay range: 100 pg/mL – 3200 pg/mL.
- Sensitivity: <10 pg/mL.
- Cross-reactivity: No significant interference observed.
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### **Storage**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C.
- Shelf life: 6 months at -20°C.
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**Important:** Always read the full user manual before beginning the assay. This kit is intended for research purposes only.
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