S100A12 Antibody, HRP conjugated

What is S100A12 and why is it important in immunological research?

S100A12 (also known as EN-RAGE or Calgranulin C) is a member of the S100 protein family primarily expressed in myeloid-derived immune cells, particularly CD15-positive neutrophils and CD68-positive macrophages. It functions as a damage-associated molecular pattern (DAMP) molecule and plays important roles in inflammatory responses. Its importance in research stems from its potential as a biomarker for various inflammatory conditions and cancers. For instance, serum S100A12 levels have been found to significantly increase during acute otitis media (AOM) caused by specific bacterial pathogens like Streptococcus pneumoniae (Spn) and Haemophilus influenzae (NTHi) . Additionally, S100A12 expression in tumor tissues has been associated with cancer prognosis, as demonstrated in hepatocellular carcinoma (HCC) .

What does HRP conjugation mean in the context of S100A12 antibodies?

HRP (Horseradish Peroxidase) conjugation refers to the chemical attachment of the enzyme horseradish peroxidase to an antibody specific for S100A12. This conjugation enables direct detection of the antibody-antigen complex through an enzymatic reaction. In immunoassays, the HRP enzyme catalyzes the oxidation of substrates like tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂), producing a colored reaction product that can be measured spectrophotometrically. In the studies reviewed, researchers used "an HRP conjugated polyclonal antibody specific for S100A12" that was added to the wells and incubated at room temperature for 1 hour, followed by washing and reaction with the substrate H₂O₂-tetramethylbenzidine .

What are the primary cellular sources of S100A12 in human tissues?

Based on the research data, S100A12 is exclusively expressed in the cytoplasm of stroma cells, primarily in myeloid-derived immune cells. Specifically, immunofluorescence staining has revealed that S100A12 is predominantly expressed on CD11B-positive myeloid-derived immune cells, particularly on CD15-positive neutrophils and CD68-positive macrophages . Unlike some other markers, S100A12 was not expressed on tumor cells or vascular endothelial cells in studies of hepatocellular carcinoma tissues . This specific cellular expression pattern makes S100A12 a valuable marker for studying neutrophil and macrophage infiltration in disease contexts.

What is the recommended protocol for using S100A12 antibody, HRP conjugated in ELISA assays?

Based on the research methodologies described, a standard protocol for S100A12 ELISA using HRP-conjugated antibodies involves:

• Plate preparation: Coat a 96-well microplate with a monoclonal antibody specific for S100A12.

• Sample addition: Add 100μl of diluted serum (or other biological sample) to the wells, allowing the immobilized antibody to bind any S100A12 present.

• Washing step: Thoroughly wash to remove unbound materials.

• Detection antibody: Add an HRP-conjugated polyclonal antibody specific for S100A12 to the wells and incubate at room temperature for 1 hour.

• Washing step: Perform another thorough wash.

• Substrate reaction: Add the substrate H₂O₂-tetramethylbenzidine to the wells.

• Measurement: Measure the absorbance of the resulting product at 450 nm.

• Analysis: Construct a standard curve by plotting absorbance values versus S100A12 concentrations of calibrators, and determine concentrations of unknown samples using this standard curve .

This protocol has been successfully used to measure S100A12 levels in serum samples from patients with acute otitis media, demonstrating significant differences between disease and healthy states.

How can researchers optimize immunohistochemical detection of S100A12 in tissue samples?

For optimal immunohistochemical detection of S100A12, researchers should:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections of appropriate thickness (typically 4-5 μm).

• Antigen retrieval: Perform heat-induced epitope retrieval to unmask antigenic sites that may be cross-linked during fixation.

• Blocking: Use appropriate blocking solutions to minimize non-specific binding.

• Primary antibody: Apply optimized dilution of anti-S100A12 antibody (determined through titration experiments).

• Detection system: Utilize a sensitive detection system, potentially including HRP-conjugated secondary antibodies.

• Quantification: For analytical studies, consider using total positive staining area (TPSA) measurements. In the HCC study provided, researchers used a threshold of 1,600 μm² to distinguish between high and low S100A12 expression .

• Validation: Confirm specificity through co-localization studies with known markers of myeloid cells (CD11B, CD15, CD68) using immunofluorescence .

This approach allows for both qualitative assessment of S100A12 distribution and quantitative comparison between different tissue samples or conditions.

What are the critical parameters for ensuring reproducible results with S100A12 antibody, HRP conjugated assays?

To ensure reproducible results with S100A12 antibody, HRP conjugated assays, researchers should standardize:

• Antibody quality control: Verify lot-to-lot consistency of the HRP-conjugated anti-S100A12 antibody.

• Incubation conditions: Maintain consistent temperature (room temperature) and duration (1 hour) for antibody incubation .

• Washing procedures: Standardize washing steps to effectively remove unbound antibodies without disrupting specific binding.

• Substrate reaction time: Control the time allowed for color development with HRP substrate.

• Standard curve preparation: Use consistent preparation methods for calibrators to ensure accurate quantification.

• Sample handling: Standardize collection, processing, and storage of biological samples to minimize pre-analytical variables.

• Controls: Include appropriate positive and negative controls in each assay.

• Data analysis: Use consistent methods for standard curve fitting and extrapolation of sample concentrations.

In the studies analyzed, researchers were able to detect significant differences in S100A12 levels between disease states and healthy controls, indicating the reliability of their standardized protocols .

How can researchers differentiate between S100A12 expression patterns in different inflammatory conditions?

Differentiating S100A12 expression patterns across inflammatory conditions requires careful experimental design and analysis:

• Pathogen-specific responses: Research has shown differential S100A12 responses to specific pathogens. For instance, serum S100A12 concentrations were significantly elevated in children with acute otitis media caused by Streptococcus pneumoniae (Spn) and Haemophilus influenzae (NTHi), but not in those with Moraxella catarrhalis (Mcat) infections . This suggests pathogen-specific neutrophil activation patterns.

• Viral vs. bacterial inflammation: S100A12 levels did not significantly increase during viral upper respiratory infections in children, contrasting with the elevated levels seen in bacterial infections. This is consistent with observations that many upper respiratory viral infections decrease neutrophil count .

• Tissue-specific expression: In cancer contexts, intratumoral S100A12 expression was significantly lower (median TPSA, 270 μm²) compared to peritumoral stroma cells (median TPSA, 836 μm²) , suggesting tissue-specific regulation.

• Co-expression analysis: Performing co-localization studies with other immune cell markers can reveal the specific cellular sources of S100A12 in different inflammatory contexts .

By systematically analyzing these patterns, researchers can develop more specific diagnostic or prognostic applications for S100A12 measurements.

What are the potential confounding factors when interpreting S100A12 antibody data in clinical samples?

Several confounding factors should be considered when interpreting S100A12 antibody data:

How does S100A12 detection correlate with other inflammatory biomarkers in research applications?

The correlation between S100A12 and other inflammatory biomarkers varies by context:

These findings highlight that S100A12 may provide unique information not captured by conventional immune cell markers, potentially reflecting specific activation states or functional subsets of myeloid cells.

How can S100A12 antibody, HRP conjugated be used to assess disease progression and treatment efficacy?

S100A12 antibody, HRP conjugated, can be valuable for monitoring disease progression and treatment efficacy through several approaches:

These applications demonstrate the versatility of S100A12 as a biomarker across different disease contexts and treatment modalities.

What is the significance of differential S100A12 expression in bacterial versus viral infections?

The differential expression of S100A12 in bacterial versus viral infections has important implications for both diagnosis and understanding of disease mechanisms:

• Diagnostic potential: Elevated serum S100A12 levels were specifically associated with bacterial infections caused by Streptococcus pneumoniae (Spn) and Haemophilus influenzae (NTHi), but not with viral upper respiratory infections . This suggests S100A12 could help differentiate bacterial from viral infections, addressing a major clinical challenge.

• Pathogen-specific responses: Different bacterial pathogens elicit varying S100A12 responses. Levels were significantly elevated in Spn-induced acute otitis media (AOM), less elevated in NTHi-induced AOM, and not significantly changed in Moraxella catarrhalis (Mcat)-induced AOM . This correlates with the observation that Spn-caused AOM is associated with more symptoms and signs of inflammation than AOM caused by other pathogens.

• Neutrophil dynamics: The lack of S100A12 elevation during viral infections is consistent with observations that many respiratory viral infections, including influenza and parainfluenza, decrease neutrophil count . This highlights the role of S100A12 as a specific marker of neutrophil activation.

• Mechanistic insights: These differential responses suggest distinct immune activation pathways between bacterial and viral infections, which could inform targeted therapeutic approaches.

This distinction makes S100A12 particularly valuable in research on mixed infections or in conditions where distinguishing the underlying cause is critical for appropriate treatment.

What is the relationship between S100A12 expression and tumor microenvironment in cancer research?

Research on S100A12 in cancer contexts has revealed important insights about tumor microenvironment:

These findings suggest that S100A12 may be a marker for specific subtypes of tumor-associated myeloid cells that promote tumor progression, potentially providing new targets for cancer immunotherapy.

Technical Parameters for S100A12 Antibody Detection Methods