Human FETU-A ELISA Kit

**Human FETU-A ELISA Kit – For the quantitative in vitro determination of Human FETU-A concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.** This ELISA kit is designed to measure Human FETU-A levels using a colorimetric method. The reaction involves an enzyme-linked immunosorbent assay where the intensity of the color developed at 450 nm is directly proportional to the concentration of FETU-A in the sample. A standard curve is generated using a series of calibration standards, allowing accurate quantification of unknown samples by comparing their optical density (OD) values. **INTENDED USE AND TEST PRINCIPLE** The Human FETU-A ELISA Kit is strictly for research purposes and should not be used in diagnostic or clinical settings. The test relies on a competitive binding mechanism, where the FETU-A in the sample competes with a labeled FETU-A for binding sites on the immobilized antibody. After incubation, washing, and addition of chromogenic substrates, the reaction is stopped, and the resulting color is measured at 450 nm. The OD values are then plotted against the standard curve to determine the FETU-A concentration in the sample. **SAMPLE COLLECTION AND STORAGE** - **Serum**: Use a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C before centrifugation 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freezing. - **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs during processing. **MATERIALS REQUIRED BUT 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** All reagents are stored at 2–8°C. Check expiration dates on labels. - Microtiter Strip Plate: 12×8 strips (96 determinations), 12×4 strips (48 determinations) - Standards (6 vials): 0.5 mL/vial - Sample Diluent: 6.0 mL (96), 3.0 mL (48) - HRP-Conjugate Reagent: 10.0 mL (96), 5.0 mL (48) - 20X Wash Solution: 25 mL (96), 15 mL (48) - Chromogen Solution A: 6.0 mL (96), 3.0 mL (48) - Chromogen Solution B: 6.0 mL (96), 3.0 mL (48) - Stop Solution: 6.0 mL (96), 3.0 mL (48) - Closure Plate Membrane: 2 pieces - User Manual: 1 copy - Sealed Bags: 1 bag **STANDARD CONCENTRATIONS** Standards: 1000, 500, 250, 125, 62.5, 31.2 μg/mL. If sample values exceed the highest standard, dilute with sample diluent and repeat the assay. **PRECAUTIONS** 1. Only use reagents provided by the manufacturer. 2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw in a water bath. 3. Do not use expired components. 4. Use only deionized or distilled water for dilutions. 5. Keep microtiter plates in sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant. 6. Use fresh pipette tips for each transfer to prevent cross-contamination. 7. Treat all blood-derived materials as potentially infectious. Follow proper biosafety protocols. 8. Dispose of all samples according to local regulations. Allow liquid to stand for 30 minutes to inactivate viruses before disposal. 9. Substrate solution may be easily contaminated. Handle with care. 10. Avoid exposure to heat or flame when handling Chromogen B, which contains 20% acetone. **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 all reagents before starting. 2. Add 50 µL of standard or sample to appropriate wells (excluding blank). Cover with adhesive strip and incubate for 60 minutes at 37°C. 3. Wash microtiter plate 4 times. - *Manual Washing*: Aspirate, fill with 1X Wash Solution, aspirate again. Repeat 4 times. Invert and blot dry. - *Automated Washing*: Aspirate and wash 4 times. Adjust brush to remove maximum liquid. Fill 350 µL/well. Invert and blot dry. 4. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Mix gently and incubate for 15 minutes at 37°C, protected from light. 5. Add 50 µL of Stop Solution to each well. Read OD at 450 nm using a microplate reader. **DATA ANALYSIS** - Plot average OD values of standards vs. concentrations on a graph. - Subtract blank OD from all readings. - Locate the OD value of the sample on the Y-axis and draw a horizontal line to intersect the standard curve. Draw a vertical line to the X-axis to determine the concentration. - Each user should generate their own standard curve due to possible variations in technique. - Intra-assay CV <15%, Inter-assay CV <15%. - Assay range: 31.2–1000 µg/mL. - Sensitivity: <10 µg/mL. - Cross-reactivity: No significant cross-reactivity observed. **STORAGE** - Store at 2–8°C for frequent use; for long-term storage, keep at -20°C. Shelf life: 6 months at -20°C. **NOTES** Always read the entire package insert before use. Follow all safety and handling instructions carefully. This kit is intended for research purposes only.

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