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

**Rabbit Soluble Leukocyte Differentiation Antigen 40 Ligand (sCD40L) ELISA Kit – Instructions for Use** **Kit Specifications:** - **Configuration:** 48-well or 96-well plate - **Standard Dilution:** 1.5 mL × 1 vial - **Enzyme Standard Reagent:** 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - **Storage Conditions:** 2–8°C - **Validity:** 6 months from the date of receipt **Usage Purpose:** This kit is intended for research purposes only. It is designed to quantitatively determine the concentration of rabbit soluble leukocyte differentiation antigen 40 ligand (sCD40L) in biological samples such as serum, plasma, urine, cell culture supernatants, and tissue homogenates. **Principle of Operation:** The ELISA kit employs a double-antibody sandwich method. A monoclonal antibody specific to sCD40L is pre-coated on the microplate. After incubation with the sample, the sCD40L binds to the immobilized antibody. A HRP-conjugated secondary antibody is then added, forming an immune complex. TMB substrate is used for color development, which changes from blue to yellow upon acid termination. The absorbance at 450 nm is measured, and the concentration of sCD40L is determined by comparing the sample OD values to a standard curve. **Kit Components:** - Sealing Film: 2 pieces (48-well) / 2 pieces (96-well) - Standard: 0.5 mL × 1 vial (2700 ng/L) - Enzyme Standard: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - 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) - Developer B: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Washing Solution: 20 mL × 20 times (48-well) / 20 mL × 30 times (96-well) - Storage: All components should be stored at 2–8°C, except for Developer B, which should be kept at -20°C. **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. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well, centrifuge, and collect the supernatant. 3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. 4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells using repeated freeze-thaw cycles. 5. **Tissue Homogenate:** Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge, and collect the supernatant. 6. **Storage:** Samples should be processed immediately after collection. If not tested right away, store at -20°C. Avoid repeated freezing and thawing. Note: Do not use samples containing NaN3, as it inhibits HRP activity. **Procedure Steps:** 1. **Standard Dilution:** Prepare serial dilutions of the standard according to the instructions. 2. **Sample Addition:** Add 40 µL of sample diluent followed by 10 µL of the sample into each well. 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 50 µL of enzyme conjugate to each well. 6. **Second Incubation:** Incubate again at 37°C for 30 minutes. 7. **Color Development:** Add 50 µL of TMB A and B, incubate for 15 minutes at 37°C. 8. **Stop Reaction:** Add 50 µL of stop solution to each well. 9. **Reading:** Measure the OD at 450 nm within 15 minutes of adding the stop solution. 10. **Data Analysis:** Plot the standard curve using OD values vs. concentration. Calculate the sample concentration using linear regression or reference the standard curve. **Important Notes:** - Equilibrate the kit at room temperature before use. - Avoid cross-contamination; use a new sealing film for each test. - Ensure accurate pipetting and avoid exposure of substrates to light. - Always perform a standard curve in duplicate. If the sample OD exceeds that of the highest standard, dilute the sample and retest. - Follow all safety protocols; treat all waste as biohazardous material. - Do not mix components from different batches. - In case of discrepancy between English and other language manuals, the English version takes precedence. **Performance Characteristics:** - Linear range: 0.2 IU/L – 6 IU/L - Correlation coefficient (R²): ≥ 0.95 - Intra-batch and inter-batch variation: < 9% and < 11%, respectively. **Service Commitment:** - Free technical support during working hours. - Sample testing services available upon request. - Delivery time: From payment to shipment. This ELISA kit provides a reliable and sensitive method for detecting sCD40L levels in various biological matrices, ensuring accurate and reproducible results for your research needs.

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