**Chicken SP-A ELISA Kit – For the Quantitative In Vitro Determination of Chicken Pulmonary Surfactant-Associated Protein A (SP-A) in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Purposes.*
This ELISA kit is designed to quantitatively measure the concentration of SP-A in various biological samples using a colorimetric method. The Stop Solution changes the reaction from blue to yellow, and the optical density (OD) is measured at 450 nm with a microplate reader. A set of standard solutions is included to generate a standard curve, which allows for accurate calculation of SP-A levels in unknown samples.
**Sample Collection and Storage:**
- **Serum:** Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Remove serum and assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g and 2–8°C. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and assay immediately or store at -20°C. Ensure no hemolysis or debris is present.
**Materials Required but Not Supplied:**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- Microtiter Strip Plate (12×8 strips / 12×4 strips)
- Standards (6 vials, 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 Solutions A & B (6.0 ml / 3.0 ml each)
- Stop Solution (6.0 ml / 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Note:** Standard concentrations are 64, 32, 16, 8, 4, and 2 pg/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions:**
- Do not mix reagents from different kit lots.
- Allow all reagents to reach room temperature (20–25°C) before use.
- Do not use reagents beyond their expiration date.
- Use only deionized or distilled water for dilution.
- Store unused strips in sealed bags with desiccant at 2–8°C.
- Wear gloves during handling; treat all samples as potentially infectious.
- Discard waste after 30 minutes to inactivate viruses.
- Avoid contamination by using fresh pipette tips for each transfer.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents before starting.
2. Add 50 µL of standards or samples to appropriate wells (duplicate).
3. Add 100 µL of HRP-Conjugate reagent to all wells except the blank.
4. Incubate for 60 minutes at 37°C.
5. Wash the plate 4 times manually or automatically.
6. Add 50 µL of Chromogen A and 50 µL of Chromogen B. Incubate for 15 minutes at 37°C.
7. Add 50 µL of Stop Solution. The color should turn yellow.
8. Measure OD at 450 nm and plot a standard curve.
**Interpretation:**
- Plot average OD values against standard concentrations.
- Subtract blank OD from all readings.
- Calculate SP-A concentration based on the standard curve.
- Intra-assay and inter-assay CV% <15%.
**Assay Range:** 2 pg/mL – 64 pg/mL
**Sensitivity:** <1.0 pg/mL
**Cross-Reactivity:** No significant cross-reactivity observed.
**Storage:** 2–8°C (frequent use); 6 months at -20°C.
**Important Note:** This kit is for research purposes only. It is not intended for diagnostic or clinical use. Always follow safety protocols when handling biological materials.
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