Human TGF-β ELISA Kit

**Human TGF-β ELISA Kit – For the quantitative in vitro determination of Human transforming growth factor β concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.** This ELISA kit is designed for research purposes only and should not be used for diagnostic or clinical applications. The assay is based on a sandwich immunoassay principle, where TGF-β in the sample binds to specific antibodies coated on the microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change. The reaction is stopped with a stop solution, and the optical density (OD) is measured at 450 nm using a spectrophotometer. To determine the concentration of TGF-β in the samples, a standard curve is generated using a series of known concentrations. This allows for accurate quantification of unknown samples by comparing their OD values to the standard curve. --- **Sample Collection and Storage:** - **Serum:** Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000 ×g for 20 minutes. Remove serum and assay immediately or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles. - **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000 ×g (2–8°C). Avoid freeze-thaw cycles. - **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or debris is present. --- **Materials Required (Not Supplied):** 1. Incubator set at 37°C 2. Microplate reader capable of measuring absorbance at 450 nm 3. Precision pipettes, disposable tips, and absorbent paper 4. Distilled or deionized water --- **Reagents Provided (Store at 2–8°C):** | Reagent Name | 96 Determinations | 48 Determinations | |-------------------------------|-------------------|-------------------| | MicroELISA Strip Plate | 12 x 8 strips | 12 x 4 strips | | Standard (6 vials) | 0.5 ml/vial | 0.5 ml/vial | | Sample Diluent | 6.0 ml | 3.0 ml | | HRP-Conjugate Reagent | 10.0 ml | 5.0 ml | | 20X Wash Solution | 25 ml | 15 ml | | Chromogen Solution A | 6.0 ml | 3.0 ml | | Chromogen Solution B | 6.0 ml | 3.0 ml | | Stop Solution | 6.0 ml | 3.0 ml | | Closure Plate Membrane | 2 | 2 | | User Manual | 1 | 1 | | Sealed Bags | 1 | 1 | *Standard concentrations: 3200, 1600, 800, 400, 200, 100 pg/mL.* --- **Precautions:** 1. Do not mix reagents from different kit lots. All components are calibrated for optimal performance. 2. Allow all reagents to reach room temperature (20–25°C) before use. Avoid using water baths to thaw. 3. Do not use reagents beyond their expiration date. 4. Use only deionized or distilled water. 5. Store unused strips in the sealed bag with desiccant at 2–8°C. 6. Use fresh pipette tips for each transfer. 7. Handle all biological samples with care. Wear gloves and follow biosafety protocols. 8. Dispose of waste properly after inactivation for at least 30 minutes. 9. Avoid contamination of the substrate solution. Keep Chromogen B away from heat or flame due to its 20% acetone content. --- **Reagent Preparation:** - **Wash Solution (1X):** Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to 1 month. --- **Assay Procedure:** 1. Prepare standards and samples in duplicate. Add 50 µL of each to the appropriate wells. Blank well receives no sample. 2. Add 100 µL of HRP-conjugate reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C. 3. Wash the plate 4 times manually or automatically. 4. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting from light. 5. Add 50 µL of Stop Solution. Color changes from blue to yellow. Gently tap if uneven color appears. 6. Read OD at 450 nm. Subtract blank OD from all readings. --- **Data Interpretation:** - Plot average OD values of standards vs. concentrations. - Determine sample concentration by interpolating OD values on the standard curve. - Each user must generate their own standard curve due to potential variability in technique or conditions. --- **Performance Characteristics:** - Intra-assay CV <15%, Inter-assay CV <15% - Assay range: 100 pg/mL – 3200 pg/mL - Sensitivity: <100 pg/mL - Cross-reactivity: No significant cross-reaction with other factors **Storage:** 2–8°C (for frequent use); -20°C for long-term storage (up to 6 months) **Note:** Always read the entire protocol carefully before starting the assay. Follow all safety guidelines and dispose of materials properly.

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