Rabbit soluble leukocyte differentiation antigen 40 ligand (sCD40L) elisa kit instruction manual

**Rabbit Soluble Leukocyte Differentiation Antigen 40 Ligand (sCD40L) ELISA Kit – User Manual** **Kit Specifications:** - 48-well or 96-well configuration - Standard dilution: 1.5 ml × 1 bottle - Enzyme standard reagent: 3 ml × 1 bottle (for 48 wells) or 6 ml × 1 bottle (for 96 wells) - This reagent is intended for research use only **Standard Curve Preparation:** To determine the concentration of sCD40L in your samples, prepare a standard curve by plotting the concentration of the standard on the x-axis and the corresponding OD values on the y-axis. Use this curve to find the concentration of the sample based on its OD value. Alternatively, calculate the linear regression equation from the standard curve and substitute the sample’s OD value into the equation. Multiply the result by the dilution factor to obtain the actual sample concentration. **Kit Components:** - Sealing film: 2 pieces (for 48 wells) / 2 pieces (for 96 wells) - Standard: 2700 ng/L, 0.5 ml × 1 bottle - Enzyme standard: 1×48 or 1×96 - Sample diluent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) - Developer A: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) - Chromogen B: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) - Wash solution: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) - Concentrated wash solution: 20 ml × 20 times (for 48 wells) or 20 ml × 30 times (for 96 wells) **Storage Conditions:** - Store all components at 2–8°C unless otherwise specified - Shelf life: 6 months from the date of receipt **Principle of the Assay:** This ELISA kit uses a double-antibody sandwich method to detect soluble CD40 ligand (sCD40L) in rabbit samples. The microplate is pre-coated with a specific antibody against sCD40L. After incubation with the sample, the sCD40L binds to the immobilized antibody. A HRP-conjugated secondary antibody is then added, forming an antibody-antigen-enzyme complex. TMB substrate is used for color development, which changes from blue to yellow upon acid termination. The intensity of the color is directly proportional to the sCD40L concentration in the sample. The absorbance is measured at 450 nm, and the concentration is calculated using a standard curve. **Objective:** This kit is designed to quantify the level of sCD40L in rabbit serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **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 carefully. If precipitate forms, re-centrifuge before use. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge for 20 minutes. Collect the supernatant. 3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Discard any precipitate. 4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freeze-thaw cycles, then centrifuge again. 5. **Tissue:** Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant. Store remaining portions at -20°C. 6. **General Note:** Avoid repeated freezing and thawing. Do not use samples containing NaN3, as it inhibits HRP activity. **Procedure Summary:** 1. **Standard Dilution & Loading:** Prepare a 10-point standard curve with concentrations ranging from 1800 ng/L to 150 ng/L. 2. **Sample Addition:** Add 40 μl of sample diluent followed by 10 μl of sample (final 5-fold dilution). 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Use diluted washing solution (30×) and wash 5 times. 5. **Enzyme Addition:** Add 50 μl of enzyme-labeled reagent to each well (except blank). 6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes. 7. **Color Development:** Add 50 μl of TMB A and B, incubate at 37°C for 15 minutes. 8. **Stop Reaction:** Add 50 μl of stop solution to terminate the reaction. 9. **Reading:** Measure OD at 450 nm within 15 minutes after stopping. **Notes:** - Allow the kit to equilibrate at room temperature for 15–30 minutes before use. - Avoid cross-contamination; use a new sealing film for each experiment. - Ensure accurate pipetting and avoid light exposure during TMB development. - Always prepare a standard curve and run samples in duplicate. - Follow the manual strictly; results should be interpreted using a microplate reader. - Dispose of all waste as biohazardous material. - Do not mix reagents from different batches. **Performance Characteristics:** - Linear correlation coefficient (R²) ≥ 0.95 - Intra-assay CV < 9%, Inter-assay CV < 11% - Detection range: 0.2 IU/L – 6 IU/L **Service Commitment:** - Free technical support during working hours - Free sample testing service available upon request - Delivery within the agreed time frame after payment This kit provides a reliable and sensitive method for detecting sCD40L in various biological samples. Proper handling and adherence to the protocol are essential for accurate and reproducible results.

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