**Human WISP1 ELISA Kit – For the Quantitative In Vitro Determination of Human WNT1 Inducible Signaling Pathway Protein 1 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
This kit is designed for the quantitative measurement of Human WISP1 (Wnt-1 Inducible Signaling Protein 1) levels in various biological samples. It employs a sandwich ELISA method, where the target protein is captured by a specific antibody immobilized on the microplate. A horseradish peroxidase (HRP)-conjugated secondary antibody then binds to the captured WISP1, and the enzyme reaction produces a color change that is proportional to the concentration of WISP1 in the sample. The optical density (OD) is measured at 450 nm, and the concentration of WISP1 is determined by comparing the sample OD values with a standard curve.
**INTENDED USE AND TEST PRINCIPLE**
This WISP1 ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic purposes. The test principle involves binding of WISP1 to a capture antibody on the microtiter plate, followed by detection using an HRP-conjugated antibody. A chromogenic substrate is added, and the reaction is stopped to measure the absorbance at 450 nm. The results are interpreted based on a pre-established standard curve.
**SAMPLE COLLECTION AND STORAGE**
- **Serum:** Collect using 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. Store at -20°C after aliquoting. Avoid repeated freeze-thaw cycles.
- **Plasma:** Use heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g, 2–8°C. Store at -20°C.
- **Tissue Homogenate and Cell Culture Supernatants:** Centrifuge to remove particulates. Assay immediately or store at -20°C.
- **Note:** Ensure proper centrifugation, avoid hemolysis, and prevent granules in the samples.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**REAGENTS PROVIDED**
- 96-well microplate (12×8 strips)
- Standard (6 vials, 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 A (6.0 ml / 3.0 ml)
- Chromogen B (6.0 ml / 3.0 ml)
- Stop Solution (6.0 ml / 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**STANDARD CONCENTRATION**
Standard concentrations: 1000, 500, 250, 125, 62.5, 31.2 pg/ml. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**PRECAUTIONS**
- Do not mix reagents from different kits.
- All reagents must reach room temperature (20–25°C) before use.
- Do not use beyond expiration date.
- Keep unused strips in sealed bags with desiccant.
- Wear gloves during handling; treat all samples as potentially infectious.
- Dispose of waste with 1% sodium hypochlorite for 30 minutes before disposal.
- Avoid contact with heat or flame when handling Chromogen A and B (contains 20% acetone).
**REAGENT PREPARATION**
- Wash Solution (1X): Dilute 1 volume of 20X solution with 19 volumes of distilled/deionized water. Store at 2–8°C for 1 month.
**ASSAY PROCEDURE**
1. Prepare all reagents before starting.
2. Add 50 µl of standards or samples in duplicate to the wells.
3. Add 100 µl of HRP-conjugate reagent to each well (except blank), cover, and incubate at 37°C for 60 minutes.
4. Wash 4 times manually or automatically.
5. Add 50 µl of Chromogen A and B to each well, mix gently, and incubate for 15 minutes at 37°C (avoid light).
6. Add 50 µl of Stop Solution. Mix gently if color is uneven.
7. Read OD at 450 nm. Plot standard curve and calculate sample concentrations.
**RESULTS INTERPRETATION**
- Calculate mean OD for each standard and sample. Subtract blank OD.
- Construct a standard curve and determine sample concentration by interpolation.
- Intra-assay and inter-assay CV% < 15%.
- Sensitivity: <10 pg/mL.
- Cross-reactivity: No significant cross-reaction with other proteins.
- Storage: 2–8°C (frequent use); 6 months at -20°C.
**NOTE:** Always read the entire protocol carefully before beginning the assay. Follow all safety guidelines and maintain good lab practices.
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