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 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 intended for laboratory research purposes only and should not be used for diagnostic or therapeutic applications. The kit is based on the Enzyme-Linked Immunosorbent Assay (ELISA) principle, which allows for the precise quantification of S-100B protein levels in biological samples. The reaction is terminated by adding a Stop Solution, which changes the color from blue to yellow. The intensity of the yellow color is measured at 450 nm using a spectrophotometer. A standard curve is generated using a set of calibration standards provided in the kit. These standards are run alongside the samples, allowing the operator to determine the concentration of S-100B in unknown samples by comparing their optical density (OD) values to the standard curve. **Sample Collection and Storage** - **Serum**: Collect blood in a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Store at -20°C. Avoid repeated freeze-thaw cycles. - **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Remove particulates by centrifugation. Analyze immediately or aliquot and store at -20°C. Ensure no hemolysis or granules are present in the sample. **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** All reagents are stored at 2–8°C. Check the expiration date on the label. - Microtiter Strip Plate: 12×8 strips or 12×4 strips - Standards (6 vials): 0.5 ml/vial - Sample Diluent: 6.0 ml or 3.0 ml - HRP-Conjugate Reagent: 10.0 ml or 5.0 ml - 20X Wash Solution: 25 ml or 15 ml - Chromogen Solution A: 6.0 ml or 3.0 ml - Chromogen Solution B: 6.0 ml or 3.0 ml - Stop Solution: 6.0 ml or 3.0 ml - Closure Plate Membrane: 2 units - User Manual: 1 unit - Sealed Bags: 1 unit **Notes** - Standard concentrations: 640, 320, 160, 80, 40, 20 pg/mL. - If sample values exceed the highest standard, dilute with Sample Diluent. - Do not use any reagents beyond their expiration date. - Use only deionized or distilled water for dilutions. - Keep microtiter plates in sealed bags until use. Unused strips should be stored at 2–8°C with desiccant. - Use fresh pipette tips for each transfer to prevent cross-contamination. **Safety and Disposal** - Disposable gloves must be treated as potentially hazardous and disposed of accordingly. - All samples and reagents should be considered potentially infectious. Follow good laboratory practices. - Liquid waste should be mixed with sodium hypochlorite to a final concentration of 1.0% and allowed to stand for at least 30 minutes before disposal. - Substrate Solution B contains 20% acetone; keep away from heat and flame. **Reagent Preparation and Storage** - 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. Run standards and samples in duplicate. 2. Add 50 µl of standard or sample to appropriate wells. Blank well: no addition. 3. Add 100 µl of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip. Incubate for 60 minutes at 37°C. 4. Wash the plate 4 times manually or using an automated washer. - Manual: Fill each well with 1X Wash Solution, aspirate, repeat 4 times. Dry by blotting. - Automated: Aspirate and wash four times with 1X Wash Buffer. Adjust brush and fill volume to 350 µL/well. 5. 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, protecting from light. 6. Add 50 µl of Stop Solution to each well. Color changes from blue to yellow. Gently tap if uneven. 7. Read OD at 450 nm. Generate a standard curve by plotting average OD values against known concentrations. **Results Interpretation** - Calculate mean OD for each standard and sample. Subtract blank OD before interpretation. - Plot standard curve and determine sample concentration by interpolation. - Intra-assay and inter-assay CV% < 15%. - Assay range: 20 pg/mL – 640 pg/mL. - Sensitivity: <20 pg/mL. - Cross-reactivity: No significant cross-reactivity observed. **Storage** - Store at 2–8°C for frequent use. Long-term storage: -20°C. Shelf life: 6 months. **Important Notes** - Always allow reagents to reach room temperature (20–25°C) before use. - Avoid using water baths for thawing. - Follow all safety guidelines and dispose of waste properly. This kit is designed for accurate and reliable detection of S-100B in various biological matrices. It is essential to follow all instructions carefully to ensure consistent and reproducible results.

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