**Human MIP-1δ ELISA Kit – For the quantitative in vitro determination of Human Macrophage Inflammatory Protein 1δ 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.**
This ELISA kit is designed to measure the levels of Human MIP-1δ in various biological samples. The test works based on the principle of competitive immunoassay. The color change occurs when the Stop Solution is added, turning the reaction from blue to yellow. The optical density (OD) at 450 nm is then measured using a spectrophotometer. A standard curve is generated using a set of calibration standards, allowing the user to determine the concentration of MIP-1δ in the samples by comparing their OD values with the standard curve.
**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. Centrifuge for 20 minutes at ~2000×g. 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 at 2000×g, 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other body fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
**Materials Required but 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):**
- MicroELISA Stripplate (12×8 or 12×4 strips)
- Standard (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 Solution A (6.0 ml or 3.0 ml)
- Chromogen Solution B (6.0 ml or 3.0 ml)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Note:** Standard concentrations are 480, 240, 120, 60, 30, and 15 pg/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions:**
- Do not mix reagents from different kit lots. All components are matched for optimal performance.
- Bring all reagents to room temperature (20–25°C) before use. Do not use water baths to thaw samples.
- Do not use expired reagents.
- Only use the provided reagents. Keep microtiter plates in their storage bags until needed. Unused strips should be stored at 2–8°C with desiccant.
- Use fresh pipette tips for each step. Handle all blood-derived samples as potentially infectious. Wear gloves during the procedure.
- Dispose of all waste properly. Add sodium hypochlorite to a final concentration of 1.0% to inactivate viruses before disposal.
- Chromogen Solutions A and B contain 20% acetone. Keep away from heat or flame.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Store at 2–8°C for up to 1 month.
**Assay Procedure:**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
2. Add 50 µl of standard or sample to appropriate wells. Leave blank well empty.
3. Add 100 µl of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash plate 4 times manually or automatically. Ensure thorough washing.
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 to each well. Gently mix if color is uneven.
7. Read OD at 450 nm. Generate standard curve by plotting average OD values against concentrations.
**Interpretation:**
- Calculate mean OD for each standard and sample. Subtract blank OD from all readings.
- Plot the standard curve and determine sample concentrations by matching OD values to the curve.
- Intra-assay and inter-assay CVs are <15%. Assay range: 15–480 pg/mL. Sensitivity: <10 pg/mL.
- Cross-reactivity is minimal. No significant interference observed.
**Storage:**
- Store at 2–8°C for frequent use; for long-term storage, keep at -20°C for up to 6 months.
**Important Notes:**
- Always follow good lab practices and safety protocols.
- This kit is not intended for diagnostic use. It is strictly for research purposes.
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