Human AKA ELISA Kit

**Human AKA ELISA Kit – For the Quantitative In Vitro Determination of Human Anti-Keratin Antibody Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids** **FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE.** This ELISA kit is specifically designed for the quantitative determination of Human Anti-Keratin Antibodies (AKA) in various biological samples. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, which involves binding the target antibody to a microtiter plate coated with antigens. After incubation, a horseradish peroxidase (HRP)-conjugated secondary antibody is added, followed by a chromogenic substrate that produces a color change. The reaction is stopped, and the optical density (OD) is measured at 450 nm using a microplate reader. To ensure accurate results, a set of calibration standards is included in the kit. These standards are run alongside the samples to generate a standard curve, allowing the operator to calculate the concentration of AKA in unknown samples by comparing their OD values to the standard curve. **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. Assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles. - **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. - **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 granules are present. **Materials Required (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 (12x8 or 12x4 strips) - Standards (6 vials, 0.5 ml/vial) - 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 Solutions A & B (6.0 ml or 3.0 ml each) - Stop Solution (6.0 ml or 3.0 ml) - Closure Plate Membrane (2 pieces) - User Manual and Sealed Bags **Important Notes:** - Standard concentrations: 160, 80, 40, 20, 10, 5 IU/ml. - If sample values exceed the highest standard, dilute with Sample Diluent and repeat. - All reagents should be brought to room temperature (20–25°C) before use. Do not thaw using water baths. - Never use reagents past their expiration date. - Only use deionized or distilled water for dilutions. - Keep unused strips in sealed bags with desiccant at 2–8°C. - Always use fresh pipette tips to prevent cross-contamination. - Handle all biological materials with caution, assuming potential infectious risk. - Dispose of all samples and waste according to local regulations. - Liquid waste must be treated with 1.0% sodium hypochlorite for 30 minutes before disposal. - Substrate solutions are sensitive; discard if discolored. - Chromogen B contains 20% acetone—keep away from heat and flame. - Allow all reagents to reach room temperature before starting the assay. **Assay Procedure:** 1. Prepare all reagents and add them to the microtiter plate. 2. Incubate for 60 minutes at 37°C. 3. Wash the plate four times manually or automatically. 4. Add 50 μL of Chromogen A and 50 μL of Chromogen B to each well. 5. Incubate for 15 minutes at 37°C, protecting from light. 6. Add 50 μL of Stop Solution to each well. 7. Measure OD at 450 nm within 15 minutes. **Data Interpretation:** - Plot average OD values of standards against their concentrations to create a standard curve. - Subtract blank OD values from all measurements before interpretation. - Each user should generate their own standard curve due to possible variations in technique. - Intra-assay and inter-assay CV% are less than 15%. - Assay range: 5–160 IU/ml. - Sensitivity: <1.0 IU/ml. - Cross-reactivity: No significant interference observed. **Storage:** - Store at 2–8°C for frequent use; up to six months at -20°C. **Note:** This kit is intended for research purposes only and not for diagnostic procedures. Always follow proper safety protocols when handling biological samples.

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