**Human MIG/CXCL9 ELISA Kit – For the quantitative in vitro determination of Human monokine induced by interferon-gamma concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
**INTENDED USE AND TEST PRINCIPLE**
This MIG/CXCL9 ELISA kit is specifically designed for laboratory research purposes. It is not intended for diagnostic or therapeutic applications. The assay is based on a sandwich ELISA format, where the colorimetric reaction is detected at 450 nm using a spectrophotometer. The Stop Solution changes the reaction from blue to yellow, and the optical density (OD) is measured to quantify MIG/CXCL9 levels in the samples. A set of calibration standards is included to generate a standard curve, allowing accurate quantification of MIG/CXCL9 concentrations in unknown samples.
**SAMPLE COLLECTION AND STORAGE**
- **Serum**: Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation (20 minutes at ~2000×g). Remove serum and assay immediately, or aliquot and 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. Avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates and assay immediately, or aliquot and store at -20°C. Ensure no hemolysis or contamination occurs during sample preparation.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. Incubator 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**
All reagents are stored at 2–8°C. Check the expiration date on the label.
| Component | 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:**
- 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. Standards, conjugates, and plates are matched for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use components beyond their expiration date.
4. Use only deionized or distilled water for reagent dilutions.
5. Keep microtiter plates in their sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant.
6. Use fresh disposable pipettes. Avoid contact with strong acids or sodium hypochlorite.
7. Handle all blood-derived samples as potentially infectious. Wear gloves during the procedure.
8. Dispose of all samples in a manner that inactivates viruses.
9. Liquid waste: Add 1% sodium hypochlorite, let stand for 30 minutes before disposal.
10. Substrate solution may be contaminated; do not use if it appears bluish.
11. Chromogen B contains 20% acetone; keep away from heat or flame.
12. Allow all reagents to reach 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 before starting. Run standards and samples in duplicate.
2. Add 50 µl of standard or sample to each well. Blank well: 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 plate 4 times (manual or automated).
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. Color changes from blue to yellow. Gently tap if uneven.
7. Read OD at 450 nm. Generate a standard curve by plotting average OD vs. concentration.
8. Calculate mean OD for each standard and sample. Subtract blank OD before interpretation.
9. Locate the OD value on the Y-axis and extend to the standard curve to determine concentration.
10. Variations in technique, timing, or kit age may affect results. Each user must generate their own standard curve.
11. Intra-assay and inter-assay CVs are <15%.
12. Assay range: 156 pg/ml – 5000 pg/ml.
13. Sensitivity: <100 pg/ml.
14. Cross-reactivity: No significant cross-reactivity with other proteins.
15. Storage: 2–8°C (frequent use); -20°C (long-term storage).
**NOTE:** Always read and follow the instructions carefully. This kit is intended for research use only and should not be used in clinical diagnostics.
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