SERPINB10 Antibody

What is SERPINB10 and what biological functions does it serve?

SERPINB10 belongs to the serpin (serine protease inhibitor) superfamily, specifically the clade B group. Unlike many other serpins that are secreted, SERPINB10 is an intracellular protein primarily expressed in immune and epithelial cells. Research indicates that SERPINB10 has important regulatory functions in immune responses and inflammatory processes. Genome-wide association studies have identified SERPINB10 as a significant genetic risk locus for cutaneous leishmaniasis caused by Leishmania braziliensis, suggesting its role in infectious disease susceptibility . Additionally, SERPINB10 shows increased expression in type 2 inflammatory conditions, particularly in airway diseases like asthma, indicating its potential involvement in eosinophilic inflammation regulation .

What detection methods are most effective for SERPINB10 research?

For SERPINB10 detection, multiple complementary approaches are recommended:

• ELISA: Provides quantitative measurement of SERPINB10 in biological fluids and tissue homogenates. This method has been successfully employed to detect elevated SERPINB10 levels in induced sputum from asthmatic patients .

• Western Blotting: Allows confirmation of antibody specificity and protein size. Based on serpin family research protocols, use of reducing conditions and proper blocking is essential .

• qRT-PCR: Enables quantification of SERPINB10 mRNA expression. Studies have successfully correlated SERPINB10 mRNA levels with protein expression and clinical parameters in chronic rhinosinusitis with nasal polyps .

• Immunohistochemistry/Immunofluorescence: Provides spatial localization information within tissues. This approach has revealed SERPINB10 expression patterns in airway epithelial cells and infiltrating immune cells .

Each method has specific optimization requirements, and validation using appropriate positive and negative controls is essential for reliable results.

What are the key considerations for antibody selection in SERPINB10 research?

When selecting SERPINB10 antibodies for research applications, consider:

• Antibody specificity: Validate against recombinant SERPINB10 protein and use appropriate knockout/knockdown controls to confirm specificity. This is particularly important because of sequence homology between different serpin family members.

• Application compatibility: Ensure the antibody has been validated for your specific application (WB, IHC, ELISA, flow cytometry). Similar to approaches used with other serpin antibodies, fixation and permeabilization protocols significantly impact antibody performance .

• Clone type: Monoclonal antibodies offer higher specificity but may recognize single epitopes that could be masked in certain applications. Polyclonal antibodies provide broader epitope recognition but potentially lower specificity.

• Species reactivity: Confirm the antibody recognizes SERPINB10 in your species of interest, as sequence variations exist between human and mouse SERPINB10.

• Validation data: Evaluate the manufacturer's validation data and published literature citing specific antibody clones for similar applications.

How can SERPINB10 antibodies be used to study inflammatory airway diseases?

SERPINB10 antibodies have proven valuable in investigating inflammatory airway diseases through several approaches:

• Quantitative assessment: ELISA measurements of SERPINB10 in induced sputum samples have demonstrated significantly increased levels in asthmatic patients compared to healthy controls. This approach enables correlation analysis with clinical parameters (FeNO, eosinophil counts) and therapeutic responses .

• Mechanistic studies: Immunohistochemical staining with SERPINB10 antibodies allows identification of cellular sources and localization patterns within airway tissues. This helps elucidate the relationship between SERPINB10 expression and inflammatory cell infiltration patterns.

• Correlation analysis: Studies have revealed significant positive correlations between SERPINB10 levels and Th2 cytokines IL-4 (r=0.6274, p<0.0001), IL-5 (r=0.5166, p<0.0001), and IL-13 (r=0.5212, p=0.0003) . These findings establish SERPINB10 as a potential signature protein for type 2 high asthma.

• Biomarker development: SERPINB10 expression levels can be measured to predict disease recurrence, as demonstrated in chronic rhinosinusitis with nasal polyps where elevated SERPINB10 mRNA levels correlate with postoperative recurrence (AUC=0.741, p<0.001) .

Researchers should implement appropriate controls, including comparison with other inflammatory markers and correlation with clinical parameters, to validate SERPINB10's role in disease pathogenesis.

What approaches should be used to analyze SERPINB10's role in genetic susceptibility to infectious diseases?

Based on genomic association studies linking SERPINB10 to leishmaniasis susceptibility , researchers investigating SERPINB10's role in genetic susceptibility to infectious diseases should:

• Genotype-phenotype correlation: Combine genotyping of SERPINB10 SNVs with antibody-based measurement of protein expression to determine how genetic variants affect protein levels in patient cohorts.

• Expression quantitative trait locus (eQTL) analysis: Evaluate how SERPINB10 genetic variants influence gene expression in relevant cell types. Research has identified multiple cis-eQTLs across SERPINB10 that map to chromatin interaction regions of transcriptional/enhancer activity in neutrophils, monocytes, B cells, and hematopoietic stem cells .

• Functional validation: Use SERPINB10 antibodies in cell-based assays to examine how different genetic variants affect protein function, cellular localization, or interaction with other immune components.

• Cross-disease analysis: Compare SERPINB10 expression and genetic associations across different infectious and inflammatory conditions to identify shared pathways.

• Chromatin immunoprecipitation: Employ SERPINB10 antibodies in ChIP assays to investigate how genetic variants affect transcription factor binding and chromatin configuration.

This multi-faceted approach can help elucidate how SERPINB10 genetic variations contribute to disease susceptibility mechanisms.

What are the challenges in developing specific antibodies against SERPINB10?

Developing specific antibodies against SERPINB10 presents several challenges:

• Serpin family homology: The serpin superfamily contains multiple members with structural similarities that can lead to cross-reactivity. SERPINB10 shares sequence homology with other clade B serpins, requiring careful epitope selection and extensive cross-reactivity testing.

• Post-translational modifications: SERPINB10 may undergo conformational changes or modifications that affect epitope accessibility. Researchers must consider these variations when developing antibodies for different applications.

• Validation limitations: The relative scarcity of studies specifically focusing on SERPINB10 means fewer validation standards exist compared to more extensively studied proteins.

• Conformational states: Like other serpins that undergo significant conformational changes between active and cleaved forms, SERPINB10 may present different epitopes depending on its functional state .

• Species differences: Sequence variations between human and model organism SERPINB10 may limit cross-species reactivity of antibodies, requiring species-specific antibody development.

Researchers should implement rigorous validation protocols, including recombinant protein controls, knockout/knockdown validation, and side-by-side comparison with multiple antibody clones to ensure specificity.

How should SERPINB10 antibodies be optimized for immunohistochemistry and immunofluorescence?

Optimizing SERPINB10 antibodies for tissue staining requires:

• Fixation optimization: Compare multiple fixation methods (formalin, paraformaldehyde, acetone) to determine which best preserves SERPINB10 epitopes while maintaining tissue morphology. Drawing from serpin research protocols, a 4% paraformaldehyde fixation for 15-20 minutes often provides good results .

• Antigen retrieval: Test both heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0) and enzymatic retrieval methods to optimize epitope accessibility. For SERPINB10 in sinonasal tissues, citrate buffer retrieval has shown effective results .

• Antibody concentration optimization: Perform serial dilutions to determine optimal antibody concentration that maximizes specific signal while minimizing background. Typically starting at 1:100-1:500 dilutions and adjusting based on signal intensity.

• Blocking optimization: Use 5-10% normal serum from the same species as the secondary antibody plus 0.1-0.3% Triton X-100 for permeabilization. Longer blocking times (1-2 hours) may reduce non-specific binding.

• Signal amplification: For low-abundance SERPINB10 detection, consider tyramide signal amplification or high-sensitivity detection systems.

• Controls: Include positive controls (tissues known to express SERPINB10 like airway epithelium or eosinophil-rich tissues), negative controls (antibody diluent only), and isotype controls to validate staining specificity.

When studying inflammatory tissues, counterstaining with cell-type-specific markers can help localize SERPINB10 expression to specific cell populations within the inflammatory infiltrate.

What are the optimal protocols for SERPINB10 western blotting?

For optimal western blot detection of SERPINB10:

• Sample preparation:

  • Tissue samples: Homogenize in RIPA buffer with protease inhibitors

  • Cell lines: Lyse in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitor cocktail

  • Include phosphatase inhibitors if phosphorylation status is relevant

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Load 20-50 μg protein per lane for cell/tissue lysates

  • Include appropriate molecular weight markers (SERPINB10 expected at approximately 45-50 kDa)

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes in 25 mM Tris, 192 mM glycine, 20% methanol

  • PVDF membranes may provide better results than nitrocellulose for SERPINB10

• Blocking:

  • 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature

  • For phospho-specific detection, use 5% BSA instead of milk

• Antibody incubation:

  • Primary: Incubate with anti-SERPINB10 antibody (1:500-1:2000 dilution) overnight at 4°C

  • Secondary: HRP-conjugated secondary antibody (1:2000-1:5000) for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence with exposure times optimized based on signal strength

  • For quantitative analysis, consider fluorescent secondary antibodies

• Controls and validation:

  • Positive control: Recombinant SERPINB10 protein or lysates from cells known to express SERPINB10

  • Loading control: β-actin, GAPDH, or other housekeeping proteins

  • Pre-absorption control: Pre-incubate antibody with recombinant SERPINB10 to confirm specificity

These protocols have been adapted from successful approaches used with other serpin family members .

How can SERPINB10 be accurately quantified in patient samples for biomarker studies?

For quantitative assessment of SERPINB10 as a biomarker:

• ELISA development and validation:

  • Commercial or custom sandwich ELISA using validated antibody pairs

  • Establish standard curves using recombinant SERPINB10 protein

  • Determine assay range, sensitivity, precision, and reproducibility

  • Validate with spike-recovery experiments in matrix-matched samples

  • The approach used for asthma studies demonstrated significant correlation with clinical parameters and disease severity

• Sample collection standardization:

  • For induced sputum: Use standardized induction protocols with 3-5% hypertonic saline

  • For tissue biopsies: Standardize collection site, processing time, and storage conditions

  • For blood: Determine whether serum or plasma is preferable; standardize processing times

• Normalization approaches:

  • For sputum: Normalize to total protein content or use DTT-processed samples

  • For tissue: Normalize to tissue weight, total protein, or housekeeping gene expression

  • For cellular samples: Normalize to cell number or housekeeping proteins

• Correlation with clinical parameters:

  • Establish correlations with established biomarkers (e.g., eosinophil counts, FeNO)

  • Analyze relationship with clinical severity scores

  • Determine cutoff values for diagnostic or prognostic use using ROC analysis

• Statistical analysis:

  • Use appropriate statistical methods for biomarker validation

  • Account for potential confounding factors

  • Consider sensitivity, specificity, positive and negative predictive values

• Longitudinal assessment:

  • Evaluate SERPINB10 stability in samples over time

  • Determine intra-individual variability

  • Assess changes in response to treatment or disease progression

Studies in chronic rhinosinusitis with nasal polyps have successfully employed ROC analysis to establish SERPINB10 as a predictor of postoperative recurrence with an AUC of 0.741 (p<0.001) .

How can SERPINB10 antibodies be used to study its role in type 2 inflammatory diseases?

SERPINB10 antibodies can be employed in multiple ways to investigate type 2 inflammatory diseases:

• Tissue expression profiling:

  • Compare SERPINB10 expression between healthy controls and patients with type 2 inflammatory conditions like asthma and chronic rhinosinusitis

  • Correlate expression levels with disease severity, eosinophilia, and Th2 cytokine levels

  • Research has shown positive correlations between SERPINB10 and IL-4 (r=0.6274, p<0.0001), IL-5 (r=0.5166, p<0.0001), and IL-13 (r=0.5212, p=0.0003) in asthmatic patients

• Cell-specific expression analysis:

  • Use flow cytometry or immunofluorescence co-staining to identify which cell types express SERPINB10

  • Compare expression patterns between different immune cell populations in type 2 vs. non-type 2 inflammation

  • Analyze how SERPINB10 expression changes during cell activation or differentiation

• Functional studies:

  • Employ neutralizing antibodies in ex vivo models to determine SERPINB10's role in eosinophil recruitment or activation

  • Use antibodies to immunoprecipitate SERPINB10 and identify binding partners in inflammatory contexts

  • Develop assays to measure SERPINB10 enzymatic activity and inhibition

• Treatment response monitoring:

  • Evaluate changes in SERPINB10 expression following corticosteroid treatment or biologics targeting type 2 pathways

  • Determine if SERPINB10 levels predict response to specific therapies

• Genetic correlation studies:

  • Correlate genetic variants in the SERPINB10 locus with protein expression levels

  • Investigate how SERPINB10 eQTLs influence inflammatory phenotypes

These approaches have successfully demonstrated SERPINB10's association with type 2 high asthma and its potential as a biomarker for eosinophilic airway inflammation .

What experimental approaches are most effective for studying SERPINB10's relationship with eosinophilic inflammation?

To investigate SERPINB10's relationship with eosinophilic inflammation:

• Co-localization studies:

  • Perform dual immunofluorescence staining for SERPINB10 and eosinophil markers in tissue sections

  • Analyze spatial relationships between SERPINB10-expressing cells and eosinophilic infiltrates

  • Quantify correlation between SERPINB10 expression intensity and eosinophil density

• Correlation analysis:

  • Measure SERPINB10 levels in biological samples and correlate with:

    • Peripheral blood eosinophil counts

    • Tissue eosinophil counts

    • Eosinophil activation markers (ECP, EDN)

  • Studies have demonstrated significant correlations between SERPINB10 expression and both peripheral and tissue eosinophil counts and percentages (p<0.05)

• In vitro models:

  • Study SERPINB10 expression in eosinophils under various stimulation conditions

  • Investigate how recombinant SERPINB10 affects eosinophil survival, migration, and activation

  • Examine interactions between SERPINB10 and eosinophil-derived proteases

• Animal models:

  • Compare eosinophilic inflammation in SERPINB10 knockout versus wild-type mice

  • Evaluate how SERPINB10 modulation affects eosinophil recruitment in allergen challenge models

  • Test anti-SERPINB10 interventions on established eosinophilic inflammation

• Cytokine relationship studies:

  • Analyze how Th2 cytokines regulate SERPINB10 expression

  • Investigate whether SERPINB10 influences IL-5 production or signaling

  • Examine potential feedback loops between SERPINB10 and eosinophilopoietic factors

• Mechanistic investigations:

  • Use SERPINB10 antibodies to immunoprecipitate and identify interacting proteins in eosinophil-rich tissues

  • Develop activity assays to determine which eosinophil-derived proteases are inhibited by SERPINB10

  • Study how SERPINB10 affects eosinophil degranulation and extracellular trap formation

Research has established that SERPINB10 levels in induced sputum are positively correlated with FeNO (r=0.4620, p<0.0001) and peripheral blood eosinophils (r=0.2500, p=0.0218), suggesting an important relationship with eosinophilic inflammation .

What are the future research directions for SERPINB10 in infectious disease applications?

Based on the genome-wide association studies linking SERPINB10 to leishmaniasis susceptibility , several promising research directions emerge:

• Mechanistic studies:

  • Investigate how SERPINB10 influences parasite survival within macrophages

  • Determine whether SERPINB10 inhibits leishmania-derived proteases

  • Examine SERPINB10's role in regulating cell death pathways during infection

• Genetic susceptibility:

  • Expand GWAS findings to additional infectious diseases beyond leishmaniasis

  • Characterize the functional consequences of SERPINB10 polymorphisms

  • Explore population-specific variations in SERPINB10 genetics and their relationship to disease endemicity

• Diagnostic applications:

  • Develop SERPINB10-based assays to identify individuals at higher risk for severe disease

  • Investigate SERPINB10 expression as a biomarker for disease progression or treatment response

  • Explore correlation between SERPINB10 genotype and immunological parameters in infected individuals

• Therapeutic targeting:

  • Design peptide inhibitors or small molecules targeting SERPINB10-mediated pathways

  • Evaluate SERPINB10 modulation as an adjunct to conventional anti-parasitic therapy

  • Investigate SERPINB10's potential as a vaccine target

• Cross-disease applications:

  • Examine SERPINB10's role in other parasitic, bacterial, or viral infections

  • Investigate common mechanisms between infectious and inflammatory SERPINB10 functions

  • Study how SERPINB10 influences the inflammatory-to-resolution transition during infection

Given the identification of multiple cis-eQTLs across SERPINB10 that map to chromatin interaction regions of transcriptional/enhancer activity in immune cells , understanding how these genetic elements influence infection outcomes represents a particularly promising research direction.

What are the common pitfalls in SERPINB10 antibody-based experiments and how can they be addressed?

When working with SERPINB10 antibodies, researchers may encounter several challenges:

• Non-specific binding:

  • Problem: Background staining or multiple bands on western blots

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include additional washing steps, and validate with appropriate controls including recombinant protein controls

• Poor signal intensity:

  • Problem: Weak or absent SERPINB10 signal

  • Solution: Optimize sample preparation to prevent protein degradation, try different epitope retrieval methods for IHC, increase antibody concentration or incubation time, use signal amplification systems

• Inconsistent results between applications:

  • Problem: Antibody works for western blot but not IHC/IF or vice versa

  • Solution: Different applications require different epitope accessibility; try antibodies targeting different regions of SERPINB10

• Epitope masking:

  • Problem: Binding site accessibility issues due to protein conformation or interactions

  • Solution: Test different fixation/denaturation conditions, try antibodies targeting different epitopes

• Cross-reactivity with other serpins:

  • Problem: Antibody recognizes multiple serpin family members

  • Solution: Validate specificity using recombinant proteins of related serpins, use knockout/knockdown controls, consider monoclonal antibodies with confirmed specificity

• Sample processing artifacts:

  • Problem: Variable detection due to sample handling

  • Solution: Standardize collection, fixation, and processing protocols; include processing controls

• Validation challenges:

  • Problem: Limited positive controls for SERPINB10

  • Solution: Generate overexpression systems, use tissues known to express SERPINB10 (e.g., asthmatic airway samples), include recombinant protein controls

Approaches successfully used for other serpin family members, such as those employed with Serpin A5 , can be adapted for SERPINB10 experimental optimization.

How can researchers validate SERPINB10 antibody specificity for their experimental system?

Comprehensive validation of SERPINB10 antibodies should include:

• Recombinant protein controls:

  • Test antibody reactivity against purified recombinant SERPINB10

  • Compare recognition of SERPINB10 versus related serpin family members

  • Perform peptide competition assays to confirm epitope specificity

• Genetic validation:

  • Use SERPINB10 knockout or knockdown systems as negative controls

  • Test samples with known SERPINB10 genetic variants

  • Correlate antibody signal with mRNA expression by parallel qPCR analysis

• Multiple antibody comparison:

  • Use two or more antibodies targeting different SERPINB10 epitopes

  • Compare staining/detection patterns between different antibody clones

  • Verify consistent detection of the target across different applications

• Mass spectrometry validation:

  • Immunoprecipitate SERPINB10 using the antibody

  • Confirm protein identity by mass spectrometry

  • Compare detected peptides with expected SERPINB10 sequence

• Expression system controls:

  • Compare antibody detection in cells with endogenous expression versus overexpression systems

  • Examine signal in cell types known to have differential SERPINB10 expression

  • Use inducible expression systems to confirm antibody sensitivity to expression changes

• Cross-application concordance:

  • Verify that protein detected by western blot corresponds to cells/regions positive by immunostaining

  • Compare flow cytometry results with other protein quantification methods

  • Ensure consistent molecular weight detection across different sample types

• Independent method correlation:

  • Correlate antibody-based detection with SERPINB10 mRNA expression

  • Compare protein levels detected by antibody with activity-based assays if available