Mouse leptin (LEP) elisa kit instruction manual

**Mouse Leptin (LEP) ELISA Kit Instruction Manual** **Kit Specifications:** This Mouse Leptin (LEP) ELISA Kit is available in 48-well or 96-well configurations. The kit includes a standard dilution of 1.5 mL × 1 vial, and the enzyme standard reagent is provided as 3 mL × 1 vial (for 48-well) or 6 mL × 1 vial (for 96-well). This reagent is intended for research purposes only. **Standard Curve Preparation:** To determine the concentration of leptin in the samples, a standard curve should be plotted using the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Alternatively, a linear regression equation can be calculated from the standard curve data. The OD value of the sample is then substituted into this equation to calculate the sample concentration, which is subsequently multiplied by the dilution factor to obtain the actual concentration. **Kit Components:** - Sealing film: 2 pieces (48-well) / 2 pieces (96-well) - Standard: 0.5 mL × 1 vial (2700 ng/L) - Enzyme standard: 1×48 / 1×96 - Sample diluent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Developer A: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Chromogen B: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Wash solution: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Concentrated wash solution: 20 mL × 20 times (48-well) / 20 mL × 30 times (96-well) **Principle of the Assay:** The kit employs a sandwich ELISA method to detect mouse leptin (LEP) in the sample. Microtiter plates are pre-coated with anti-mouse LEP antibodies. After incubation with the sample, HRP-conjugated anti-LEP antibodies are added. The resulting antibody-antigen-enzyme complex is detected by adding TMB substrate, which changes color under the action of HRP. The intensity of the color is directly proportional to the amount of LEP in the sample. The absorbance at 450 nm is measured using a microplate reader, and the concentration is determined based on the standard curve. **Purpose:** This kit is designed to quantify leptin levels in serum, plasma, urine, cell culture supernatants, and other biological fluids. **Storage Conditions and Shelf Life:** - Storage: 2–8°C - Shelf life: 6 months **Sample Preparation Guidelines:** 1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant and re-centrifuge if necessary. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge. 3. **Urine:** Collect in a sterile tube and centrifuge. 4. **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freezing and thawing before centrifugation. 5. **Tissue Specimens:** Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C after thawing. 6. **General Notes:** Samples should be processed immediately after collection. If not tested right away, store at -20°C. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN₃, as it inhibits HRP activity. **Procedure Steps:** 1. **Standard Dilution and Loading:** Prepare a serial dilution of the standard and add to the designated wells. 2. **Sample Addition:** Add diluted sample to the test wells. 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Wash the plate 5 times with diluted washing solution. 5. **Enzyme Addition:** Add enzyme-labeled reagent to all wells except blank. 6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes. 7. **Color Development:** Add TMB substrate and incubate for 15 minutes at 37°C. 8. **Stop Reaction:** Add stop solution to terminate the reaction. 9. **Measurement:** Read the absorbance at 450 nm within 15 minutes. **Notes and Warnings:** - Equilibrate the kit to room temperature before use. - Avoid cross-contamination by using a new sealing film for each experiment. - Keep substrates away from light. - Strictly follow the protocol for accurate results. - All waste materials should be treated as biohazardous. - Do not mix reagents from different batches. - In case of conflict between languages, the English manual takes precedence. **Performance Characteristics:** - Linear regression coefficient (R²) ≥ 0.95 - Intra-batch variation < 9%, Inter-batch variation < 11% - Detection range: 0.2 IU/L – 6 IU/L **Service Commitment:** We offer free technical support during working hours and sample testing services upon request to ensure optimal experimental outcomes. Delivery is prompt after payment. **Note:** Always refer to the most recent version of the manual for any updates or additional instructions.

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