**Human MIG/CXCL9 ELISA Kit – For Quantitative In Vitro Determination of Human Monokine Induced by Interferon-Gamma Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Purposes.*
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### **Intended Use and Test Principle**
This Human MIG/CXCL9 ELISA Kit is specifically designed for the quantitative measurement of monokine induced by interferon-gamma (MIG/CXCL9) in various biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. The kit is intended solely for laboratory research purposes and should not be used in diagnostic or clinical procedures.
The assay is based on a sandwich ELISA format. A color change occurs when the stop solution is added, shifting the color from blue to yellow. The intensity of this color is measured at 450 nm using a spectrophotometer. Calibration standards are included in the kit to generate a standard curve, which allows for accurate quantification of MIG/CXCL9 concentrations in the test samples.
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### **Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Remove the 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 and 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 granules are present in the samples.
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### **Materials Required (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
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### **Reagents Provided**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
| Reagent Name | 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 |
**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.
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### **Precautions**
1. Do not substitute reagents from different kit lots. All components are matched for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents past their expiration date.
4. Use only deionized or distilled water for dilution.
5. Keep microtiter plates in their sealed bags until ready to use. Store unused strips at 2–8°C with desiccant.
6. Use fresh disposable pipettes.
7. Handle all blood-derived materials as potentially infectious. Wear gloves during the procedure.
8. Dispose of all samples properly, following biohazard protocols.
9. Liquid waste must be treated with sodium hypochlorite (final concentration 1.0%) and left for at least 30 minutes before disposal.
10. Substrate solutions may be easily contaminated. If the solution appears bluish, discard it.
11. Chromogen B contains 20% acetone; keep away from heat and flame.
12. Bring all reagents to room temperature before starting the assay.
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### **Reagent Preparation and Storage**
- **Wash Solution (1X):** Mix 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
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### **Assay Procedure**
1. Prepare all reagents before starting. It is recommended to run standards and samples in duplicate.
2. Add 50 µl of standard or sample to appropriate wells. Blank well receives no addition.
3. Add 100 µl of HRP-conjugate reagent to each well except the blank. Cover with an 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 4 times.
- **Automated Washing:** Aspirate and wash 4 times with 350 µL/well. Ensure maximum aspiration.
5. After final wash, invert the plate and blot dry.
6. 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.
7. Add 50 µl of Stop Solution. The color changes from blue to yellow. If uneven or green, gently tap the plate.
8. Read OD at 450 nm. Plot the average OD of each standard against its concentration to create a standard curve.
9. Subtract blank OD from all sample OD values.
10. Determine sample concentrations by comparing OD to the standard curve.
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### **Performance Characteristics**
- **Intra-assay CV (%) and Inter-assay CV (%):** <15%
- **Assay Range:** 156 pg/ml – 5000 pg/ml
- **Sensitivity:** <100 pg/ml
- **Cross-reactivity:** No significant cross-reactivity observed
- **Storage:** 2–8°C (for frequent use); -20°C (long-term storage)
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**Important Note:** This kit is intended for research use only. Always follow proper safety and handling procedures. Consult the user manual for full instructions and details.
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