Human MIG/CXCL9 ELISA Kit

**Human MIG/CXCL9 ELISA Kit – For Quantitative In Vitro Determination of Human Monokine Induced by Interferon-Gamma in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids** *For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Procedures.* --- ### **Intended Use and Test Principle** This Human MIG/CXCL9 ELISA Kit is designed for the quantitative measurement of Human monokine induced by interferon-gamma (MIG/CXCL9) concentrations in various biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. The assay is based on a sandwich ELISA format, where specific antibodies are used to capture MIG/CXCL9 from the sample. A horseradish peroxidase (HRP)-conjugated secondary antibody then binds to the captured antigen, and the reaction is developed with a chromogenic substrate. The addition of a stop solution changes the color from blue to yellow, and the optical density (OD) is measured at 450 nm using a spectrophotometer. To determine the concentration of MIG/CXCL9 in unknown samples, a standard curve is generated using a set of calibration standards provided in the kit. The OD values of the samples are compared to this curve to calculate their respective MIG/CXCL9 concentrations. --- ### **Sample Collection and Storage** - **Serum**: Collect blood in 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 aliquot and store at -20°C. Avoid repeated freeze-thaw cycles. - **Plasma**: Use heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freeze-thaw cycles. - **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles. Ensure samples are properly centrifuged and free from hemolysis or granules. --- ### **Materials Required but Not Supplied** 1. 37°C incubator 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** All reagents should be stored at 2–8°C and used before the expiration date indicated on the label. | Component | 96 Determinations | 48 Determinations | |----------|-------------------|-------------------| | MicroELISA Strip Plate | 12 x 8 strips | 12 x 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 | *Standard concentrations: 5000, 2500, 1250, 625, 312, 156 pg/ml.* *If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.* --- ### **Precautions** 1. Do not mix reagents from different kit lots. All components are optimized for use together. 2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Avoid using water baths for thawing. 3. Do not use reagents past their expiration date. 4. Use only deionized or distilled water for dilutions. 5. Keep microtiter plates in sealed bags until use. Unused strips should be stored at 2–8°C with desiccant. 6. Use fresh disposable pipettes and avoid contact with strong acids or sodium hypochlorite. 7. Handle all biological materials as potentially infectious. Wear gloves during the procedure. 8. Dispose of all samples and waste according to local biosafety regulations. 9. Liquid waste must be treated with 1% sodium hypochlorite for at least 30 minutes before disposal. 10. Substrate solutions must be used before they turn bluish. Chromogen B contains 20% acetone—keep away from heat and flame. 11. Ensure all reagents are at room temperature before starting the assay. --- ### **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 one month. --- ### **Assay Procedure** 1. Prepare all reagents and standards before beginning. Run standards and samples in duplicate. 2. Add 50 µl of standard or sample to each well. The blank well receives no sample. 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 Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat four times. - **Automated Washing**: Aspirate and wash four times, adjusting washer settings for optimal performance. 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. The color should change from blue to 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 standard concentrations. --- ### **Data Interpretation** 1. Calculate the mean OD for each standard and sample. 2. Subtract the blank OD from all values before interpretation. 3. Locate the sample OD on the Y-axis and draw a horizontal line to intersect the standard curve. Read the corresponding concentration. 4. Variability due to operator technique, incubation time, or reagent age may occur. Each user should generate their own standard curve. 5. Intra-assay and inter-assay CV% are less than 15%. 6. Assay range: 156 pg/ml to 5000 pg/ml. 7. Sensitivity: <100 pg/ml. 8. Cross-reactivity: No significant cross-reaction with other proteins. 9. Storage: 2–8°C for frequent use; -20°C for long-term storage. --- **Note:** This product is intended for research purposes only and should not be used in clinical diagnostics. Always follow proper safety protocols when handling biological samples.

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