Human AKA ELISA Kit

**Human Anti-Keratin Antibody (AKA) ELISA Kit – For Quantitative In Vitro Determination of AKA Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids** *For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Purposes.* This ELISA kit is designed for the quantitative measurement of Human Anti-Keratin Antibodies (AKA) in various biological samples. The method is based on the enzyme-linked immunosorbent assay (ELISA) principle, which involves binding of AKA to immobilized antigens on a microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then used to detect bound antibodies, and the reaction is visualized using a chromogenic substrate. The color change is stopped by adding a stop solution, and the optical density (OD) is measured at 450 nm using a microplate reader. The kit includes a set of calibration standards with known concentrations of AKA (160, 80, 40, 20, 10, 5 IU/ml), allowing users to generate a standard curve. By comparing the OD values of unknown samples to this curve, the concentration of AKA in the sample can be accurately determined. --- **Sample Collection and Storage** - **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C before centrifugation 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 at 2000×g (2–8°C) and store at -20°C. Avoid repeated freezing. - **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 contamination occurs. --- **Materials Required but 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 --- **Reagents Provided** | 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 | | 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 | *Note: Standards are prepared at concentrations of 160, 80, 40, 20, 10, and 5 IU/ml. If sample results exceed the highest standard, dilute with Sample Diluent and repeat the test.* --- **Preparation and Handling Instructions** 1. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw using water baths. 2. Do not use reagents beyond their expiration date. 3. Only use deionized or distilled water for dilutions. 4. Keep unused strip wells sealed in the desiccant pouch at 2–8°C until needed. 5. Use fresh pipette tips for each transfer to prevent cross-contamination. 6. Wear disposable gloves throughout the procedure to avoid exposure to potentially infectious materials. 7. Dispose of all biological waste according to local regulations, ensuring proper inactivation of pathogens. 8. Liquid waste should be treated with 1.0% sodium hypochlorite for at least 30 minutes before disposal. 9. Substrate solutions should be checked for discoloration before use. Avoid light exposure during incubation. 10. Chromogen B contains 20% acetone; keep it away from heat and flame. --- **Assay Procedure** 1. Prepare all reagents before starting. Add 100 μL of standards, samples, and blank to the microtiter plate. 2. Cover the plate with an adhesive strip and incubate for 60 minutes at 37°C. 3. Wash the plate 4 times using either manual or automated washing procedures. 4. 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. 5. Add 50 μL of Stop Solution to each well. The color will change from blue to yellow. 6. Read OD at 450 nm within 15 minutes using a microplate reader. --- **Calculation of Results** 1. Plot the average OD values of the standards against their concentrations on a graph. 2. Subtract the blank OD from all measurements before calculating. 3. Determine the AKA concentration of unknown samples by interpolating their OD values on the standard curve. 4. Intra-assay and inter-assay CV% are less than 15%. 5. The detection range is 5–160 IU/ml, with a sensitivity of <1.0 IU/ml. 6. No significant cross-reactivity was observed with other antibodies. --- **Storage and Stability** - Store unopened kit at 2–8°C for frequent use or at -20°C for long-term storage (up to 6 months). - Once opened, store unused strips in the desiccant pouch at 2–8°C. This kit is intended for research purposes only and should not be used for diagnostic or clinical applications. Always follow good laboratory practices and ensure safe handling of all reagents and samples.

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