Monkey macrophage migration inhibitory factor (MIF) elisa kit instruction manual

**Monkey Macrophage Migration Inhibitory Factor (MIF) ELISA Kit Instruction Manual** **Kit Specifications:** This ELISA kit is available in 48-well or 96-well configurations. It includes: - Standard Diluent: 1.5 ml × 1 vial - Enzyme Standard Reagent: 3 ml × 1 vial (for 48-well) / 6 ml × 1 vial (for 96-well) - Sealing Film: 2 pieces (for 48-well) / 2 pieces (for 96-well) - Standard: 0.5 ml × 1 vial, 2700 ng/L - Sample Diluent: 3 ml × 1 vial (for 48-well) / 6 ml × 1 vial (for 96-well) - Developer A: 3 ml × 1 vial (for 48-well) / 6 ml × 1 vial (for 96-well) - Chromogen B: 3 ml × 1 vial (for 48-well) / 6 ml × 1 vial (for 96-well) - Stop Solution: 3 ml × 1 vial (for 48-well) / 6 ml × 1 vial (for 96-well) - Concentrated Washing Solution: 20 ml × 20 times (for 48-well) / 20 ml × 30 times (for 96-well) **Storage Conditions and Expiration:** - Kit Storage: 2–8°C - Shelf Life: 6 months from the date of manufacture **Purpose:** This ELISA kit is designed for the quantitative determination of Macrophage Migration Inhibitory Factor (MIF) in monkey serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **Principle of Operation:** The MIF ELISA kit uses a double-antibody sandwich method. The microtiter plate is pre-coated with a specific monoclonal antibody against MIF. After incubation with the sample, the MIF present in the sample binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, which changes color under the action of HRP. The intensity of the color is directly proportional to the concentration of MIF in the sample. The absorbance is measured at 450 nm using a microplate reader, and the MIF concentration is calculated based on a standard curve. **Sample Preparation and Handling:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. If precipitate forms, re-centrifuge before use. - **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. - **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells by repeated freezing and thawing, then centrifuge again. - **Tissue Homogenate:** Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. - **Storage:** Samples should be processed as soon as possible. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN₃, as it may inhibit HRP activity. **Operation Steps:** 1. **Standard Dilution and Loading:** Prepare a serial dilution of the standard solution. Load 100 μL into the first two wells, then perform a 1:1 dilution for each subsequent well. 2. **Sample Loading:** Add 40 μL of sample diluent, followed by 10 μL of the sample. Ensure the final dilution is 5-fold. 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Dilute the concentrated washing solution as instructed. Wash the plate 5 times, ensuring thorough removal of unbound substances. 5. **Enzyme Conjugate Addition:** Add 50 μL of enzyme-labeled reagent to each well, except blank wells. 6. **Second Incubation:** Repeat the incubation step at 37°C for 30 minutes. 7. **Color Development:** Add 50 μL of TMB A and B solutions sequentially. Incubate at 37°C for 15 minutes in the dark. 8. **Stop Reaction:** Add 50 μL of stop solution to each well. 9. **Measurement:** Read the OD values at 450 nm within 15 minutes after stopping the reaction. **Notes and Recommendations:** - Allow the kit to equilibrate to room temperature for 15–30 minutes before use. - The concentrated washing solution may crystallize; heat gently if necessary. - Use separate pipettes for each step to avoid contamination. - Always run a standard curve in duplicate. If the sample OD exceeds the highest standard, dilute the sample and retest. - Discard all waste materials as biohazardous. - Do not mix reagents from different batches. - Follow the manual strictly. Results must be confirmed with a microplate reader. **Performance Characteristics:** - Linear regression correlation coefficient (R²) ≥ 0.95 - Intra-batch and inter-batch variation < 9% and < 11%, respectively - Detection range: 0.2 IU/L – 6 IU/L **Service Commitment:** We offer free technical support during working hours. Our team can assist with sample testing to ensure accurate and reliable results. **Note:** This manual is for research use only.

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