Chicken sIgA ELISA Kit

**Chicken sIgA ELISA Kit – For the Quantitative In Vitro Determination of Chicken Secretory Immunoglobulin 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 secretory immunoglobulin A (sIgA) in various biological samples. The assay is based on a sandwich immunoassay principle, where the sIgA in the sample binds to specific antibodies coated on the microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change. The intensity of the color is directly proportional to the concentration of sIgA in the sample. The reaction is stopped, and the optical density (OD) is measured at 450 nm using a microplate reader. To ensure accurate results, calibration standards are provided with known concentrations of sIgA. These standards are run alongside the samples to generate a standard curve, which is used to calculate the sIgA levels in the test 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. Store at -20°C. Avoid repeated freeze-thaw cycles. - **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g for 30 minutes. Store at -20°C. - **Tissue Homogenates, Cell Culture Supernatants, and Other Body Fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs. **Materials Required but Not Supplied:** - Incubator at 37°C - 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 or 12×4 strips) - Standards (6 vials, 0.5 ml/vial) – Concentrations: 8, 4, 2, 1, 0.5, 0.25 ng/mL - Sample Diluent (6.0 ml or 3.0 ml) - HRP-Conjugate Reagent (10.0 ml or 5.0 ml) - 20X Wash Solution (25 ml or 15 ml) - Chromogen Solution A (6.0 ml or 3.0 ml) - Chromogen Solution B (6.0 ml or 3.0 ml) - Stop Solution (6.0 ml or 3.0 ml) - Closure Plate Membrane (2 units) - User Manual (1 copy) - Sealed Bags (1 unit) **Important Notes:** - All reagents should be brought to room temperature (20–25°C) before use. - Do not thaw samples or reagents in a water bath. - Do not use any components beyond their expiration date. - Only use deionized or distilled water for dilutions. - Keep the microtiter plate in its sealed bag until needed. Unused strips must be stored with desiccant. - Use fresh pipette tips for each transfer to avoid cross-contamination. - All biological materials should be handled with care, following proper biosafety protocols. **Wash Solution Preparation:** Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. The 1X solution is stable for one month at 2–8°C. **Assay Procedure:** 1. Prepare all reagents and ensure they reach room temperature. 2. Add 50 µL of standard or sample to appropriate wells (duplicates recommended). 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 plate 4 times (manual or automated method). 5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light. 6. Add 50 µL of Stop Solution to each well. Read OD at 450 nm. **Data Analysis:** - Plot the average OD values of standards vs. their concentrations to create a standard curve. - Calculate the sIgA concentration of unknown samples by comparing their OD values to the standard curve. - Intra-assay and inter-assay CV% are less than 15%. - Sensitivity: <0.1 ng/mL. - Assay range: 0.25 ng/mL to 8 ng/mL. - Cross-reactivity: No significant cross-reaction with other immunoglobulins. **Storage:** - Store at 2–8°C for frequent use; up to 6 months at -20°C. **Safety and Disposal:** - Follow local regulations for disposal of biological waste. - Liquid waste should be treated with 1.0% sodium hypochlorite for at least 30 minutes before disposal. - Substrate B contains 20% acetone; keep away from heat and flame. This kit is intended for research purposes only and should not be used in diagnostic procedures. Always read the entire user manual before performing the assay.

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