**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 Procedures.*
This ELISA kit is designed for the quantitative determination of SP-A levels in various biological samples. The assay is based on a sandwich ELISA format, utilizing specific antibodies to detect and quantify SP-A. The color change from blue to yellow occurs upon addition of the Stop Solution, and the optical density (OD) is measured at 450 nm using a spectrophotometer. A standard curve is generated using a set of calibration standards, allowing accurate quantification of SP-A concentrations in unknown samples.
**Sample Collection and Storage:**
- **Serum:** Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge 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 (2–8°C). Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are 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, 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. All components are matched for optimal performance.
- Allow all reagents to reach room temperature (20–25°C) before use. Avoid water baths for thawing.
- Do not use expired reagents.
- Use only deionized or distilled water for dilutions.
- Keep unused microtiter plate strips in sealed bags with desiccant.
- Use fresh pipette tips for each transfer to prevent contamination.
- Handle disposable plasma as potentially hazardous. Wear gloves during the procedure.
- Waste must be inactivated for at least 30 minutes before disposal.
- Substrate solutions may be contaminated; discard if discolored.
- Substrate B contains 20% acetone; keep away from heat or flame.
- Allow all reagents to reach room temperature before use.
**Reagent Preparation and Storage:**
- **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 1 month.
**Assay Procedure:**
1. Prepare all reagents before starting the assay. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-Conjugate reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the microtiter plate 4 times. Manual washing involves filling and aspirating each well with 1X Wash Solution. Automated washing requires setting the washer to aspirate and fill 350 µL per well.
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. The color should turn yellow. If green or uneven, gently tap the plate.
7. Measure OD at 450 nm. Generate a standard curve by plotting average OD values against SP-A concentrations. Calculate mean OD for each standard and sample, subtracting the blank value. Use graph paper or software for curve fitting.
**Additional Notes:**
- Intra-assay and inter-assay CVs are less than 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); -20°C for up to six months.
This kit is ideal for researchers studying pulmonary surfactant proteins in avian models. Always follow safety protocols and ensure proper handling of biological materials.
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