SERPINB5 Antibody

What is SERPINB5 and what cellular functions does it serve?

SERPINB5 (Maspin) is a member of the serine protease inhibitor (serpin) superfamily that acts as a tumor suppressor in multiple cancer types. It is a 42 kDa protein encoded by the SERPINB5 gene, with its full amino acid sequence spanning from Met1 to Pro375 in humans . While initially characterized as a tumor suppressor, its complete molecular role remains under investigation .

SERPINB5 is primarily expressed in epithelial cells and can be detected in the extracellular matrix as confirmed by immunohistochemistry and immunofluorescence studies . Recent research has demonstrated that beyond its tumor suppressive role, SERPINB5 may regulate immune function by influencing macrophage phenotype conversion, upregulating M1-related cytokines (TNF-α, IL-12) while suppressing M2-related cytokines (TGF-β, IL-10) .

Which cell lines reliably express SERPINB5 for use as positive controls?

Based on Western blot analysis data, the following cell lines have been confirmed to express detectable levels of SERPINB5:

HeLa parental cell line shows consistent SERPINB5 expression and can serve as a reliable positive control, while SERPINB5 knockout HeLa cells provide an excellent negative control for antibody validation . Gastric cancer cell lines may also express SERPINB5, as it has been identified as a biomarker in gastric high-grade intraepithelial neoplasia (HGIN) .

What are the primary research applications for SERPINB5 antibodies?

SERPINB5 antibodies are utilized across multiple research applications:

• Western Blotting: For protein expression analysis in cell lysates, with detection typically at 40 kDa under reducing conditions .

• Immunohistochemistry (IHC): For tissue localization studies, particularly in gastric, prostate, and cervical tissues .

• Immunofluorescence (IF): For cellular and extracellular localization studies .

• ELISA: For quantitative measurement of SERPINB5 in serum and other biological fluids .

• Prognostic Studies: For correlation analysis between SERPINB5 expression and patient survival in cancer research .

• Biomarker Development: For development and validation of SERPINB5 as a diagnostic biomarker, particularly for gastric HGIN .

How should researchers select an appropriate SERPINB5 antibody for specific applications?

When selecting a SERPINB5 antibody, consider these critical factors:

• Recognition of Native vs. Denatured Forms: Some antibodies, like 16F7, recognize both native and denatured forms of maspin, making them more versatile across applications . This is particularly important as certain commercial antibodies demonstrate specificity limitations.

• Validation Status: Choose antibodies with thorough validation data, especially those tested in knockout systems. For example, MAB2218 has been validated using SERPINB5 knockout HeLa cells .

• Application-Specific Performance:

  • For Western blots: Antibodies tested under reducing conditions with Immunoblot Buffer Group 1 have shown good specificity .

  • For IHC/IF: Antibodies demonstrated to work in formalin-fixed tissues with specific staining patterns in epithelial cells.

  • For quantitative assays: Antibodies validated in ELISA formats with established standard curves.

• Clone Information: Monoclonal antibodies with published clone numbers (e.g., Clone 305339 for MAB2218 or 16F7) provide better reproducibility across experiments .

What validation experiments are essential before using a new SERPINB5 antibody?

A comprehensive validation strategy should include:

• Positive and Negative Controls:

  • Positive controls: HeLa, PC-3, or RT-4 cell lines

  • Negative controls: SERPINB5 knockout cell lines, such as SERPINB5 KO HeLa

• Loading Controls: Use established housekeeping proteins like GAPDH to normalize expression levels .

• Specificity Testing:

  • Western blot should show a single band at approximately 40 kDa

  • Immunostaining should demonstrate the expected cellular localization pattern

  • Knockout/knockdown validation to confirm absence of signal in SERPINB5-deficient samples

• Cross-Reactivity Assessment: Test potential cross-reactivity with other serpin family members, particularly those in the same clade.

• Titration Experiments: Determine optimal dilutions for each application. For Western blots, concentrations around 2 μg/mL have been reported as effective for MAB2218 .

What are the recommended protocols for Western blot detection of SERPINB5?

Based on published successful protocols:

• Sample Preparation:

  • Prepare cell lysates using standard methods

  • Use reducing conditions for optimal detection

  • Utilize Immunoblot Buffer Group 1 for consistent results

• Gel Electrophoresis and Transfer:

  • PVDF membrane shows good results for SERPINB5 detection

  • Transfer using standard protocols for proteins of approximately 40 kDa

• Antibody Probing:

  • Primary antibody: 2 μg/mL of anti-SERPINB5 (e.g., MAB2218)

  • Secondary antibody: HRP-conjugated anti-mouse or anti-rat IgG depending on primary antibody species

  • Include appropriate blocking steps to reduce background

• Detection and Analysis:

  • Look for a specific band at approximately 40 kDa

  • Include positive controls (HeLa cells) and loading controls (GAPDH)

  • For validation, include SERPINB5 knockout cells as negative controls

What methods are recommended for SERPINB5 detection in serum for biomarker studies?

SERPINB5 has shown promise as a serum biomarker, particularly for gastric HGIN. The following protocol has demonstrated high diagnostic efficacy:

• Sample Collection and Processing:

  • Collect serum samples using standard clinical protocols

  • Process immediately or store at -80°C to preserve protein integrity

• ELISA Protocol:

  • Commercial ELISA kits (such as from Cusabio) have been successfully used in clinical studies

  • Follow manufacturer's instructions precisely

  • Read optical density at 450 nm with 570 nm wavelength correction

• Data Analysis:

  • Create standard curves using recombinant SERPINB5

  • Calculate concentrations using appropriate statistical software

  • Compare levels between disease groups and controls

• Performance Characteristics:

  • In gastric HGIN studies, serum SERPINB5 showed excellent diagnostic performance with AUC values of 0.9936 for distinguishing HGIN from chronic gastritis and 0.9750 for distinguishing HGIN from low-grade intraepithelial neoplasia

How does SERPINB5 expression correlate with immune cell infiltration in cancer microenvironments?

Recent research has revealed important connections between SERPINB5 and immune cell infiltration:

• Macrophage Correlation:

  • SERPINB5 expression positively correlates with macrophage infiltration in gastric tissue (GSE130823: r = 0.34, P = 0.044; GSE55696: r = 0.41, P = 0.0051)

  • This suggests SERPINB5 may influence macrophage recruitment to tumor microenvironments

• Macrophage Phenotype Regulation:

  • SERPINB5 expression positively correlates with M1 marker NOS2 expression

  • SERPINB5 expression negatively correlates with M2 marker CSF1R expression

  • In vitro experiments with THP-1-derived macrophages showed that SERPINB5:

    • Upregulates expression of M1-related cytokines (TNF-α, IL-12)

    • Upregulates M1 marker CD86

    • Suppresses production of M2-related cytokines (TGF-β, IL-10)

• Immune Microenvironment:

  • Single sample gene set enrichment analysis (ssGSEA) revealed the immune microenvironment was more active in chronic gastritis compared to gastric precancerous lesions

  • SERPINB5 may play a role in modulating this immune response during disease progression

These findings suggest SERPINB5 may serve as both a biomarker and potential immunomodulatory target in cancer research.

What is the diagnostic efficacy of SERPINB5 as a biomarker for gastric high-grade intraepithelial neoplasia?

SERPINB5 has demonstrated exceptional performance as a serum biomarker for gastric HGIN:

These high AUC values indicate that serum SERPINB5 has excellent discriminatory ability for detecting HGIN compared to less severe conditions . The protein is specifically overexpressed in HGIN but not in LGIN or chronic gastritis, making it particularly valuable for identifying high-risk patients who require further examination.

The correlation between serum SERPINB5 levels and histopathologic lesions suggests this biomarker could potentially supplement traditional histological diagnosis, improving diagnostic accuracy for HGIN . This non-invasive approach could be integrated into surveillance programs for early detection of gastric precancerous lesions.

Why might different anti-SERPINB5 antibodies yield inconsistent results?

Several factors contribute to variability between different SERPINB5 antibodies:

• Epitope Recognition Differences:

  • Some antibodies recognize only denatured forms, while others (like 16F7) recognize both native and denatured forms

  • Epitope masking may occur in certain protein conformations or interactions

• Antibody Quality and Validation:

  • Commercial antibodies vary in their specificity and validation status

  • The 16F7 antibody was specifically noted to outperform several other commercial antibodies in recognizing maspin in both native and denatured forms

• Protocol Optimization:

  • Buffer conditions significantly impact antibody performance

  • Immunoblot Buffer Group 1 has been reported as effective for SERPINB5 detection

• Technical Considerations:

  • Optimal dilutions vary between antibodies and applications

  • For MAB2218, 2 μg/mL has been reported as effective for Western blot detection

To address inconsistencies, researchers should validate multiple antibodies using positive and negative controls (particularly knockout models), standardize protocols, and document the specific clone and conditions used for successful detection.

What are the current challenges in developing SERPINB5 as a diagnostic biomarker?

Despite promising results, several challenges remain in SERPINB5 biomarker development:

• Sample Size Limitations:

  • Larger sample collections and multicenter studies are needed to validate diagnostic efficacy of serum SERPINB5

• Mechanistic Understanding:

  • The precise molecular mechanisms of SERPINB5-mediated effects, particularly regarding macrophage phenotype conversion, are not fully understood

  • More robust evidence supporting phenotype conversion is needed

• Immune Microenvironment Complexity:

  • The components and changes in the immune microenvironment vary across pathological stages

  • The specific role of SERPINB5 in this complex environment requires further elucidation

• Standardization Issues:

  • Assay standardization across laboratories remains challenging

  • Cutoff values for clinical decision-making need to be established

• Tissue vs. Serum Expression:

  • The relationship between tissue expression and serum levels needs further characterization

  • Mechanisms of SERPINB5 secretion into circulation require investigation

Addressing these challenges will require collaborative efforts across multiple research centers with standardized protocols and larger patient cohorts.

What PCR primers and conditions are recommended for SERPINB5 mRNA detection?

Based on published research, the following primers have been successfully used for SERPINB5 amplification:

SERPINB5 primers:

• Sense: 5′-CAGTGGACTAATCCCTGTTC-3′

• Anti-sense: 5′-TCTTCCACTTCTTCCACTGC-3′

• Product size: 107 bp

PCR Conditions:

• Use real-time PCR with SYBR Green or similar detection chemistry

• Results should be presented as Ct values

• Calculate relative changes in gene expression using the ΔΔCt method

• Use appropriate housekeeping genes like GAPDH for normalization

In previous studies, SERPINB5 showed approximately 5.83-fold upregulation in gastric cancer specimens compared to corresponding non-cancerous gastric specimens .

How should researchers interpret contradictory findings regarding SERPINB5's role in different cancer types?

When faced with seemingly contradictory findings about SERPINB5:

• Consider Tissue-Specific Effects:

  • SERPINB5 may function differently in various tissue contexts

  • In gastric tissue, it appears to be upregulated in cancer and precancerous lesions

• Examine Subcellular Localization:

  • The function of SERPINB5 may depend on its cellular localization (nuclear vs. cytoplasmic)

  • IHC studies have shown SERPINB5 in both epithelial cells and extracellular matrix

• Evaluate Methodology Differences:

  • Use of different antibodies may contribute to discrepancies

  • Some antibodies may recognize different forms or epitopes of the protein

• Consider Disease Stage:

  • SERPINB5 expression and function may change during disease progression

  • It appears specifically overexpressed in HGIN but not in LGIN or chronic gastritis

• Analyze Context-Dependent Relationships:

  • SERPINB5's interactions with the immune microenvironment may vary by cancer type

  • Its relationship with macrophage phenotypes adds complexity to its role

By carefully considering these factors, researchers can better interpret seemingly contradictory findings and design experiments to clarify SERPINB5's context-dependent functions.