S100B Antibody

What is S100B and why is it important as a research target?

S100B is a calcium-binding protein belonging to the S100 family. It is a homodimer composed of two beta chains, distinguishing it from S100A which consists of an alpha and beta chain. S100B is primarily expressed in the brain by astrocytes, oligodendrocytes, and Schwann cells, making it a valuable marker for these cell types .

S100B serves multiple intracellular functions but can also be secreted from cells to exert extracellular effects, some of which may be mediated by RAGE (receptor for advanced glycation end products) . Its importance as a research target stems from its role as a biomarker for blood-brain barrier (BBB) disruption, with elevated levels indicating potential brain injuries or pathologies .

What are the primary applications of S100B antibodies in research?

S100B antibodies are utilized across multiple experimental techniques, including:

These applications allow researchers to investigate S100B expression patterns in various tissues and cell types, supporting studies in neuroscience, oncology, and trauma research .

How do I select the appropriate S100B antibody for my research?

When selecting an S100B antibody, consider:

• Target specificity: Some antibodies are specific to the beta-chain epitope (found in both S100A and S100B), while others may be exclusively specific to S100B . Verify the epitope recognition to ensure appropriate target binding.

• Species reactivity: Confirm reactivity with your experimental species. Most commonly available antibodies react with human, mouse, and rat samples .

• Application compatibility: Ensure the antibody has been validated for your specific application. Some antibodies perform well in multiple applications, while others are optimized for specific techniques .

• Clone type: Consider whether a monoclonal (more specific) or polyclonal (potentially higher sensitivity) antibody better suits your research needs .

• Validation data: Review published data showing the antibody's performance in applications similar to yours .

How can I distinguish between S100B elevation due to brain pathology versus confounding factors?

This represents a significant challenge in S100B research. Based on published findings, several methodological approaches can help:

• Establish appropriate reference values: S100B levels within an athlete can vary depending on the type of physical activity and measurement methodology . The traditional cutoff value of 0.1 μg/L may not always be applicable across different contexts.

• Use combinatorial biomarker approaches: Combining S100B with S100B autoantibody measurements can improve specificity. In a study on brain metastasis detection, using S100B ≥0.058 ng/mL alone had a sensitivity of 89% and specificity of 43%, but when combined with anti-S100B IgG <2.00 AU, specificity improved to 58.2% while maintaining sensitivity .

• Control for extracranial sources: S100B can be released from adipose tissue, muscle, and other non-neural sources. Design experiments that account for or exclude these confounders .

• Temporal assessment: Multiple measurements over time can help distinguish acute elevations (typically seen in injury) from chronic elevations (potentially indicating pathology) .

What factors affect the reliability of S100B immunodetection in experimental models?

Several factors can influence the reliability of S100B detection:

• Sample processing and timing: The timing of sample collection post-intervention (e.g., TBI model) significantly affects results. Standardize collection timepoints based on the kinetics of S100B release in your model .

• Analytical technique variations: Different analytical methods show varied sensitivities. For instance, Simple Western detection identified S100B at approximately 4 kDa in human brain tissue, while traditional Western blot detected it at 10-11 kDa . These discrepancies must be considered when comparing results across studies.

• Buffer conditions and antigen retrieval: For IHC applications, buffer choice impacts detection. Some protocols recommend TE buffer pH 9.0 for antigen retrieval, while others suggest citrate buffer pH 6.0 as an alternative .

• Cross-reactivity considerations: Some antibodies detect both S100A and S100B due to beta-chain recognition. If specific detection of S100B alone is required, careful antibody selection is necessary .

• Detection in complex matrices: When detecting S100B in serum or CSF, matrix effects may influence assay performance. Validate assays specifically for the biological matrix being tested .

How can I interpret contradictory S100B data in neuroinflammation research?

Contradictory S100B findings are not uncommon in neuroinflammation research. To address these contradictions:

What is the optimal protocol for measuring S100B autoantibodies in clinical samples?

Based on published methodologies, the following ELISA protocol has been validated for measuring anti-S100B IgG :

• Plate coating: Coat 96-well plates overnight at 4°C with S100B protein (1 μg/well) in PBS.

• Blocking: After washing three times with PBS, add 100 μL of 1% BSA blocking solution to each well and incubate for 2 hours at room temperature.

• Sample incubation: Wash wells three times with PBS containing 0.05% Tween-20. Add serum samples and standards, then incubate for 1 hour at room temperature.

• Standard curve preparation: Use S100B monoclonal antibody as a standard, with serial dilutions to create a standard curve.

• Secondary antibody incubation: Add 200 μL of HRP-conjugated appropriate secondary antibody (goat anti-mouse IgG for standards, goat anti-human IgG for serum samples) and incubate for 1 hour at room temperature.

• Development: After washing, add 100 μL of OPD solution and incubate for 30 minutes at room temperature. Stop the reaction with 100 μL of 2.5 M sulfuric acid and read at 490 nm .

This method has been shown to reliably detect anti-S100B autoantibodies in human serum samples, with threshold values established for various clinical applications .

How should I optimize Western blot protocols for S100B detection in different tissue types?

Optimizing Western blot protocols for S100B detection requires tissue-specific considerations:

• Sample preparation:

  • Brain tissue: Homogenize in PBS with protease inhibitors. Expect strong signal due to high S100B expression .

  • Non-neural tissues: May require larger sample amounts or more sensitive detection methods due to lower expression levels .

• Protein loading and separation:

  • Load 20-50 μg of total protein per lane.

  • Use 12-15% SDS-PAGE gels for optimal separation of the low molecular weight S100B protein (10-11 kDa) .

• Transfer conditions:

  • Transfer at lower voltage (80-100V) for 60-90 minutes to prevent small proteins like S100B from passing through the membrane.

  • Consider PVDF membranes for better retention of low molecular weight proteins .

• Antibody dilutions:

  • Primary antibody: Use at 1:500-1:1000 dilution for most tissues .

  • For tissues with lower expression, consider longer incubation (overnight at 4°C) rather than higher antibody concentration to improve signal-to-noise ratio.

• Detection system:

  • Enhanced chemiluminescence (ECL) systems work well for most applications.

  • For difficult samples, consider more sensitive detection systems like femto-ECL reagents .

• Expected results:

  • The observed molecular weight should be approximately 10-11 kDa under reducing conditions .

  • Positive controls include A375 cells, human brain (cerebellum) tissue, and mouse brain tissue .

What are the best practices for immunohistochemical detection of S100B in brain tissue sections?

For optimal immunohistochemical detection of S100B in brain tissue:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections typically yield good results at 4-6 μm thickness.

  • Fresh frozen sections can also be used but may show higher background .

• Antigen retrieval:

  • Primary recommendation: TE buffer at pH 9.0.

  • Alternative: Citrate buffer at pH 6.0.

  • Heating in a pressure cooker or microwave for 10-20 minutes generally yields optimal results .

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from the same species as the secondary antibody.

  • Use S100B antibody at dilutions between 1:1000-1:6000, depending on the specific antibody and tissue .

  • Incubate overnight at 4°C for optimal staining with minimal background.

• Detection system:

  • For brightfield microscopy: HRP-DAB systems work well, with hematoxylin counterstaining .

  • For fluorescence: Use appropriate fluorophore-conjugated secondary antibodies; S100B typically shows good results with standard FITC, TRITC, or Alexa Fluor systems .

• Controls:

  • Positive tissue controls: Human malignant melanoma, human gliomas, and mouse brain tissue .

  • Negative controls: Omit primary antibody or use tissues known to lack S100B expression.

• Expected cellular localization:

  • Primarily cytoplasmic and nuclear staining in astrocytes, oligodendrocytes, and Schwann cells.

  • In melanoma tissue, diffuse cytoplasmic staining is typically observed .

How can S100B antibodies be used to distinguish between different brain pathologies?

S100B antibodies can help differentiate various brain pathologies through patterns of expression:

• Traumatic brain injury (TBI): Research using S100B neutralizing antibodies has shown therapeutic potential. Administration of these antibodies significantly reduced TBI-induced lesion volume, improved retention memory function, and attenuated microglial activation in controlled cortical impact models . This suggests a potential diagnostic and therapeutic role in TBI.

• Brain metastasis detection: S100B antibodies are valuable in detecting brain metastases in cancer patients. In lung cancer patients, combining serum S100B levels (≥0.058 ng/mL) with anti-S100B IgG levels (<2.00 AU) achieved 89% sensitivity and 58.2% specificity for detecting brain metastases . This approach could help identify patients requiring brain imaging.

• Alzheimer's disease: S100B is implicated in Alzheimer's pathology through interactions with amyloid-beta, contributing to neuroinflammation and neurotoxicity. Immunohistochemical staining with S100B antibodies can reveal characteristic patterns in Alzheimer's brain tissue .

• Gliomas versus other brain tumors: S100B antibodies are useful in differentiating astrocytic tumors (which typically express S100B) from other CNS neoplasms. The staining pattern and intensity can provide valuable diagnostic information .

• Melanoma metastasis to brain: Almost all malignant melanomas express S100B, making S100B antibodies valuable tools for identifying melanoma metastases in brain tissue .

What are the current challenges in using S100B as a biomarker for sport-related concussion?

Several challenges affect the use of S100B as a biomarker for sport-related concussion:

• Reference value variability: S100B levels within an athlete vary depending on the type of physical activity. Vigorous physical activity can increase peripheral S100B levels beyond the cutoff level of 0.1 μg/L even in the absence of mild traumatic brain injury (mTBI) .

• Methodological inconsistencies: Different studies use varying methodological approaches, including timing of sample withdrawal, sample processing, and analytical techniques, influencing S100B values and making cross-study comparisons difficult .

• Specificity limitations: While S100B has high sensitivity for brain injury, its specificity is limited. At a serum S100B level of 0.058 ng/mL, sensitivity for brain injury reaches 89%, but specificity is only 43% .

• Confounding factors: Extracranial sources of S100B, age-related variations, racial differences, and presence of small vessel disease can all influence S100B levels, complicating interpretation .

• Timing of measurement: Determining the optimal timing for sample collection post-injury remains challenging, as S100B has a relatively short half-life in circulation .

Despite these challenges, S100B measurement has potential as a diagnostic adjunct for concussion in sports settings due to its high sensitivity and excellent negative predictive value . Establishing individualized baseline S100B values for athletes and standardizing measurement protocols could improve its utility.

How can researchers improve the specificity of S100B detection in clinical applications?

To enhance the specificity of S100B detection in clinical applications: