Canine transfer growth factor beta 1 (TGF-β1) elisa kit instruction manual

**Canine Transforming Growth Factor β1 (TGF-β1) ELISA Kit – User 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 both 48 and 96 wells) - 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 & Shelf Life:** - Store the kit at 2–8°C. - Shelf life: 6 months from the date of receipt. **Purpose of the Kit:** This kit is designed for the quantitative determination of TGF-β1 in canine serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **Principle of the Assay:** The TGF-β1 ELISA kit uses a double-antibody sandwich method. The microtiter plate is pre-coated with a specific anti-TGF-β1 antibody. After adding the sample, TGF-β1 binds to the immobilized antibody. An HRP-labeled secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, and the reaction is stopped with a stop solution. The color intensity is measured at 450 nm, and the concentration is calculated based on a standard curve. **Sample Preparation Instructions:** 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. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge and collect the supernatant. 3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. 4. **Cell Culture Supernatant:** Centrifuge and collect the supernatant. For intracellular components, lyse cells by freezing and thawing, then centrifuge again. 5. **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, and centrifuge. Collect the supernatant. 6. **Storage:** Keep samples at 2–8°C if testing soon. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles. Do not use samples containing NaN3. **Procedure Summary:** 1. **Standard Dilution:** Prepare a serial dilution of the standard (1800 ng/L to 150 ng/L). 2. **Sample Addition:** Add 40 µL of sample diluent and 10 µL of sample to each well (final dilution: 5×). 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Wash 5 times with diluted washing buffer. 5. **Enzyme Addition:** Add 50 µL of enzyme conjugate to each well. 6. **Incubation:** Incubate again 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. 9. **Reading:** Measure OD at 450 nm within 15 minutes. **Data Analysis:** Plot the standard concentrations against their corresponding OD values to generate a standard curve. Use linear regression to calculate the unknown sample concentrations. Multiply by the dilution factor to obtain the actual concentration. **Notes:** - Equilibrate the kit at room temperature for 15–30 minutes before use. - Avoid cross-contamination; use a new sealing film for each test. - Ensure accurate pipetting and avoid light exposure during color development. - Always prepare a standard curve and perform duplicate measurements. - Follow the manual strictly; results should be confirmed with a microplate reader. **Performance:** - Correlation coefficient (R) ≥ 0.95 - Intra-batch CV < 9%, Inter-batch CV < 11% - Detection range: 0.2 IU/L – 6 IU/L **Service Commitment:** - Free technical support during working hours. - Sample testing services available upon request. - Delivery time: From payment to shipment. **Note:** This product is intended for research use only.

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