**Human S-100B ELISA Kit – For the Quantitative In Vitro Determination of Human Soluble Protein-100B Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
This package insert must be read carefully before use. The Human S-100B ELISA Kit is designed for research purposes only and is not intended for diagnostic or therapeutic use. This immunoassay utilizes the enzyme-linked immunosorbent assay (ELISA) principle to quantitatively measure S-100B levels in biological samples.
The reaction involves a colorimetric detection system where the addition of the Stop Solution changes the color from blue to yellow. The intensity of the color is measured at 450 nm using a spectrophotometer. A set of calibration standards is included in the kit to generate a standard curve, which is used to determine the concentration of S-100B in unknown samples by comparing their optical density (OD) values.
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**INTENDED USE AND TEST PRINCIPLE**
This S-100B ELISA Kit is specifically designed for laboratory research use. It is not suitable for clinical diagnostics or therapeutic applications. The test relies on a sandwich ELISA format, where the S-100B protein in the sample binds to specific antibodies coated on the microtiter plate. After incubation with HRP-conjugated secondary antibodies, the substrate is added, producing a color change that correlates with the concentration of S-100B.
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**SAMPLE COLLECTION AND STORAGE**
**Serum:** Use serum separator tubes and allow samples to clot for 2 hours at room temperature or overnight at 4°C. Centrifuge for 20 minutes at approximately 2000×g. Store samples at -20°C and avoid repeated freeze-thaw cycles.
**Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and assay immediately, or aliquot and store at -20°C. Ensure samples are properly centrifuged and free from hemolysis or granules.
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**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
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**REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
| Reagent Name | 96 Determinations | 48 Determinations |
|--------------|-------------------|-------------------|
| MicroELISA Strip Plate | 12×8 strips | 12×4 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 |
**Note:**
- Standard concentrations: 640, 320, 160, 80, 40, 20 pg/mL
- If sample values exceed the highest standard, dilute with Sample Diluent.
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**INSTRUCTIONS FOR USE**
1. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not thaw samples or reagents in a water bath.
2. Do not use any reagent beyond its expiration date.
3. Use only deionized or distilled water for dilutions.
4. Keep unused strip wells in their sealed pouch with desiccant.
5. Use fresh disposable pipette tips for each transfer to prevent contamination.
**Safety Note:**
Disposable gloves must be worn during the entire procedure. All blood-derived products should be treated as potentially infectious. Follow proper biosafety protocols when handling samples.
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**WASTE DISPOSAL**
Liquid waste should be treated with sodium hypochlorite to a final concentration of 1.0% and allowed to stand for at least 30 minutes before disposal.
**SUBSTRATE SOLUTION:**
Avoid contamination. Substrate B contains 20% acetone; keep away from heat or open flame.
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**ASSAY PROCEDURE**
1. Prepare all reagents and ensure standards and samples are run in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition.
3. 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.
4. Wash the plate 4 times with 1X Wash Solution.
- **Manual Washing:** Aspirate and fill each well with 1X Wash Solution, repeating four times.
- **Automated Washing:** Use washer buffer and follow manufacturer instructions.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. Color changes from blue to yellow. Gently tap if uneven color development occurs.
7. Read OD at 450 nm. Generate a standard curve using the average OD values of standards.
**DATA ANALYSIS**
- Calculate mean OD for each standard and sample. Subtract blank OD from all readings.
- Plot OD vs. concentration to create a standard curve.
- Determine unknown concentrations by interpolating OD values on the curve.
**PERFORMANCE CHARACTERISTICS**
- Intra-assay and inter-assay CV <15%
- Assay range: 20–640 pg/mL
- Sensitivity: <20 pg/mL
- Cross-reactivity: No significant cross-reaction with other proteins
**STORAGE**
- Store at 2–8°C for frequent use; up to 6 months at -20°C.
**NOTES:**
- Always follow the manufacturer’s instructions for optimal performance.
- Maintain good lab practices and handle all materials with care.
**For further assistance, contact the manufacturer or refer to the user manual.**
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