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

**Canine Transforming Growth Factor Beta 1 (TGF-β1) ELISA Kit – Instructions for Use** **Kit Specifications:** Available in 48-well or 96-well configurations. Standard Diluent: 1.5 mL × 1 vial Enzyme Standard Reagent: 3 mL × 1 vial (for 48-well) / 6 mL × 1 vial (for 96-well) Storage Conditions: 2–8°C **Calculation Method:** Plot the standard curve with concentration on the x-axis and OD values on the y-axis. Determine the sample concentration based on the OD value from the standard curve, then multiply by the dilution factor. Alternatively, calculate the linear regression equation using standard concentrations and corresponding OD values, then input the sample OD into the equation to determine the actual concentration. **Kit Composition:** - 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 Buffer: 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) - Storage: 2–8°C **Principle of Operation:** This ELISA kit uses a double-antibody sandwich method to quantify TGF-β1 in canine samples. The microplate is pre-coated with purified anti-TGF-β1 antibodies. After adding the sample, TGF-β1 binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, producing a blue color that turns yellow in the presence of acid. The intensity of the color is directly proportional to the TGF-β1 concentration in the sample. The absorbance is measured at 450 nm, and the concentration is determined using a standard curve. **Objective:** To quantitatively measure TGF-β1 levels in canine serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **Service Commitment:** - Delivery within the agreed time after payment. - Free technical support during working hours. - Free sample testing service available upon request to ensure optimal experimental results. **Storage & Shelf Life:** - Store the kit at 2–8°C. - Shelf life: 6 months from the date of receipt. **Sample Preparation Guidelines:** 1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing for 10–20 minutes. 3. **Urine:** Collect in sterile tubes, centrifuge at 2000–3000 rpm for 20 minutes. 4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freeze-thaw cycles. 5. **Tissue Specimens:** Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant. 6. **General Note:** Process samples as soon as possible. If not tested immediately, store at -20°C, avoiding repeated freezing and thawing. 7. **Note:** Avoid samples containing NaN₃, as it inhibits HRP activity. **Operating Procedure:** 1. **Standard Dilution & Loading:** Prepare serial dilutions of the standard and load them into designated wells. 2. **Sample Addition:** Add 40 μL of sample diluent and 10 μL of sample (final dilution 5×). 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Dilute concentrated wash solution and wash 5 times. 5. **Enzyme Labeling:** 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 each of Developer A and B, incubate at 37°C for 15 minutes. 8. **Stop Reaction:** Add 50 μL stop solution to each well. 9. **Measurement:** Read OD at 450 nm within 15 minutes. **Notes:** - Equilibrate the kit at room temperature before use. - If the enzyme label is opened, store remaining strips in a sealed bag. - Wash buffer may crystallize; heat gently if needed. - Use accurate pipetting and avoid cross-contamination. - Always include a standard curve and perform duplicate measurements. - Keep substrates away from light. - Follow the manual strictly; results are based on microplate reader readings. - Treat all waste as biohazardous material. - Do not mix reagents from different batches. - In case of conflict, the English manual takes precedence. **Kit Performance:** - Linear regression correlation coefficient R ≥ 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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