Human S-100B ELISA Kit
**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. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
This ELISA kit is designed to measure the levels of human S-100B protein in various biological samples using a sandwich immunoassay format. The assay relies on specific antibodies that bind to S-100B, followed by an enzymatic reaction that produces a color change. The intensity of the color at 450 nm is directly proportional to the concentration of S-100B in the sample.
To ensure accurate results, a set of calibration standards is included with the kit. These standards are run alongside the samples to generate a standard curve, which allows for precise quantification of S-100B in unknown samples. The optical density (OD) readings from the samples are compared against this curve to determine their respective concentrations.
**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 before centrifugation at 2000×g for 20 minutes. Store at -20°C and avoid repeated freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates, then assay immediately or aliquot and store at -20°C. Ensure no hemolysis or granules are present 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 (stored at 2–8°C):**
| Reagent | 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 |
**Important Notes:**
- Standard concentrations: 640, 320, 160, 80, 40, 20 pg/mL.
- If sample values exceed the highest standard, dilute with Sample Diluent.
- Use only manufacturer-supplied reagents.
- Allow all reagents and materials to reach room temperature (20–25°C) before use.
- Do not thaw samples or reagents in a water bath.
- Do not use reagents beyond their expiration date.
- Only use deionized or distilled water for dilutions.
- Keep microtiter plates in sealed bags until use. Store unused strips with desiccant at 2–8°C.
- Use fresh pipette tips for each transfer to prevent cross-contamination.
- Handle all biological materials as potentially infectious. Follow proper biosafety protocols.
**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 solutions must be handled carefully. Substrate B contains 20% acetone and should be kept away from heat or flame.
**Assay Procedure:**
1. Prepare all reagents and standards before starting.
2. Add 50 µL of standard or sample to appropriate wells (duplicate).
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash plate 4 times with 1X Wash Solution (manual or automated).
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.
7. Measure OD at 450 nm using a microplate reader.
**Data Analysis:**
- Plot average OD values (450 nm) against standard concentrations to create a standard curve.
- Subtract blank OD from all readings before interpretation.
- Locate the sample OD on the Y-axis and draw a line to the curve to determine the corresponding concentration.
**Performance Characteristics:**
- Intra-assay CV <15%, Inter-assay CV <15%
- Assay range: 20 pg/mL – 640 pg/mL
- Sensitivity: <20 pg/mL
- Cross-reactivity: No significant interference observed
- Storage: 2–8°C (frequent use); 6 months at -20°C
**Safety & Handling:**
All biological samples and reagents should be treated with caution. Follow good laboratory practices and dispose of waste according to local regulations. This kit is not intended for diagnostic use. Always read the entire package insert before use.
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