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

**Monkey Macrophage Migration Inhibitory Factor (MIF) ELISA Kit Instruction Manual** **Kit Specifications:** - 48-well or 96-well configuration - Standard Diluent: 1.5 mL × 1 bottle - Enzyme Standard Reagent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well) - Storage Conditions: 2–8°C **Calculation Method:** The concentration of the standard is plotted on the x-axis, and the OD value is on the y-axis. A standard curve is drawn on graph paper, and the corresponding sample concentration is determined based on its OD value. The result is then multiplied by the dilution factor. Alternatively, a linear regression equation can be calculated using the standard concentrations and their corresponding OD values. The sample’s OD value is input into this equation to calculate the actual concentration, which is then adjusted by the dilution factor. **Kit Components:** - Sealing Film: 2 pieces (48-well) / 2 pieces (96-well) - Standard: 2700 ng/L, 0.5 mL × 1 bottle - Enzyme Standard: 1×48 or 1×96, stored at 2–8°C - Sample Diluent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at 2–8°C - Developer A: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at 2–8°C - Chromogen B: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at -8°C - Stop Solution: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at 2–8°C - Concentrated Wash Solution: (20 mL × 20 times) × 1 bottle (48-well) / (20 mL × 30 times) × 1 bottle (96-well), stored at 2–8°C **Principle of the Assay:** This kit uses a double-antibody sandwich ELISA method to detect the level of MIF in monkey samples. The microtiter plate is pre-coated with a specific monoclonal antibody against MIF. After incubation with the sample, the captured MIF binds to a HRP-labeled secondary antibody, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, and the color develops under the action of HRP. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm. The intensity of the color is directly proportional to the MIF concentration in the sample, which is then calculated from the standard curve. **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. **Service Commitment:** - Delivery time: From payment to delivery - Free technical support during working hours - Free sample testing service available upon request to ensure optimal experimental results **Storage Conditions & Expiration:** - Kit storage: 2–8°C - Shelf life: 6 months **Sample Preparation & Handling:** 1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. If precipitate forms, re-centrifuge before use. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, centrifuge for 20 minutes, collect supernatant. 3. **Urine:** Collect in sterile tubes, centrifuge at 2000–3000 rpm for 20 minutes. Discard precipitate if present. 4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells by repeated freezing and thawing, then centrifuge again. 5. **Tissue Specimens:** Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge, and collect supernatant. Store at 2–8°C after melting. 6. **General:** Process specimens as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles. 7. **Note:** Avoid using samples containing NaN3, as it inhibits HRP activity. **Operation Steps:** 1. **Standard Dilution and Loading:** Prepare serial dilutions of the standard (1800 ng/L, 1200 ng/L, 600 ng/L, 300 ng/L, 150 ng/L). Load 100 μL into each well. 2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample (final dilution 5×) to each sample well. 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Dilute the concentrated wash solution (30×) with distilled water. Wash 5 times, ensuring complete removal of unbound reagents. 5. **Enzyme Labeling:** Add 50 μL of enzyme conjugate to each well (except blank wells). 6. **Second Incubation:** Incubate at 37°C for 30 minutes. 7. **Color Development:** Add 50 μL of TMB A and B to each well, incubate at 37°C for 15 minutes. 8. **Stop Reaction:** Add 50 μL of stop solution to each well. 9. **Measurement:** Read OD450 using a microplate reader within 15 minutes. **Notes:** - Allow the kit to equilibrate at room temperature for 15–30 minutes before use. - The concentrated wash solution may crystallize; warm it in a water bath before use. - Use a pipette for accuracy, and avoid cross-contamination by using a new sealing film per test. - Always run a standard curve and consider sample dilution if OD exceeds the first standard well. - Keep the substrate away from light. - Follow the manual strictly. Results must be based on instrument readings. - Treat all waste as biohazardous material. - Do not mix reagents from different batches. - In case of conflict, the English version of the manual takes precedence. **Kit Performance:** - Correlation coefficient (R²) of the standard curve ≥ 0.95 - Intra-batch and inter-batch variation < 9% and < 11%, respectively - Detection range: 0.2 IU/L – 6 IU/L

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