SERPINB4 Antibody, Biotin conjugated

What is SERPINB4 and how does it differ from SERPINB3?

SERPINB4 (also known as SCCA2, Squamous Cell Carcinoma Antigen 2) is a serine protease inhibitor primarily targeting chymotrypsin-like serine proteases. It shares 92% homology at the amino acid level with SERPINB3, but their catalytic sites show only 54% homology (7 out of 13 identical amino acids). This difference accounts for their distinct inhibitory targets: SERPINB3 inhibits papain-like cysteine proteases, while SERPINB4 mainly inhibits chymotrypsin-like serine proteases. Both are encoded by genes located on chromosome 18 (18q21.3) and consist of 390 amino acids .

What are the biological functions of SERPINB4?

SERPINB4 primarily functions as a protease inhibitor that modulates the host immune response against tumor cells . Originally discovered in squamous cell carcinoma of the cervix in the 1970s, SERPINB4 has been detected in multiple systems including immune, nervous, muscular, secretory, and reproductive systems . Its upregulation has been reported in several cancer types, where it appears to play a role in disease progression and cancer development .

What are the key characteristics of biotin-conjugated SERPINB4 antibodies?

Biotin-conjugated SERPINB4 monoclonal antibodies are specialized research tools with the following specifications:

• Host: Typically mouse-derived for monoclonal variants

• Applications: Primarily Western Blot (WB) analysis

• Reactivity: Human SERPINB4

• Concentration: Generally around 1μg/μl

• Storage: Usually in buffered solution containing TBS (pH 7.4) with BSA, preservatives, and glycerol

• Target location: Cytoplasmic protein

What detection methods are compatible with biotin-conjugated SERPINB4 antibodies?

Biotin-conjugated antibodies offer versatility through the biotin-streptavidin detection system. The primary detection methods include:

What protocols are recommended for optimal antibody performance?

For optimal performance of biotin-conjugated SERPINB4 antibodies:

• Western blot applications typically use dilutions ranging from 1:300-1:5000

• Blocking with 5% skimmed milk or BSA in PBS is recommended to minimize background

• Include multiple washing steps (6× with PBS containing 0.5% Tween 20)

• When working with tissue samples, incorporate an avidin/biotin blocking step to reduce endogenous biotin interference

• Validate antibody specificity against recombinant SERPINB4 and SERPINB3 to confirm selectivity

How can SERPINB4 antibodies be used to study cancer proliferation mechanisms?

SERPINB4 antibodies provide valuable tools for investigating cancer proliferation mechanisms through several approaches:

• Expression analysis in cancer cell lines by Western blot to correlate SERPINB4 levels with proliferation rates

• Immunohistochemical staining of patient tumor samples to establish clinical correlations

• Monitoring changes in SERPINB4 levels following genetic manipulation (knockdown/overexpression) and measuring proliferation using methods such as BrdU incorporation or Ki-67 staining

• Detection of secreted SERPINB4 in cell culture supernatants using ELISA to correlate extracellular levels with cancer cell behavior

Experimental evidence shows that SERPINB4 manipulation significantly affects proliferation, with knockout decreasing proliferation by approximately 12% in HepG2 cells and similar effects demonstrated in melanoma cell lines .

What role does SERPINB4 play in tumor cell invasion and metastasis?

Research demonstrates that SERPINB4 significantly influences tumor invasion capabilities:

• The reactive site loop (RSL) of SERPINB4 appears crucial for invasiveness, as antibodies targeting this region (specifically anti-P#5 antibody) reduced cell invasion by 75% in experimental studies

• SERPINB4 manipulation modulates EMT markers, with overexpression significantly increasing N-cadherin, β-catenin, Snail, and Zeb1 expression, while knockout decreases these markers

• Invasion assays using Matrigel-coated transwell systems can effectively quantify the impact of SERPINB4 on invasive capacity

These findings suggest that targeting the reactive site loop of SERPINB4 could represent a novel approach for inhibiting tumor invasion .

How does SERPINB4 affect immune regulation in the tumor microenvironment?

SERPINB4 appears to modulate immune responses in the tumor microenvironment through:

• Regulation of antigen presentation machinery, as SERPINB4 manipulation significantly modulates expression of MHC Class I molecules (HLA-A, -B, and -C) in melanoma cells

• Potential interference with immune cell activity through its protease inhibitory function

• Creation of an immunosuppressive environment that facilitates tumor immune evasion

These immunomodulatory effects can be studied using biotin-conjugated SERPINB4 antibodies in combination with flow cytometry, immunohistochemistry, and co-culture experiments with immune cells .

How can epitope-specific antibodies against SERPINB4 be developed and validated?

Development of epitope-specific antibodies requires a systematic approach:

• Identification of immunogenic epitopes within SERPINB4, particularly targeting regions with low homology to SERPINB3 such as the catalytic site

• Immunization with synthetic peptides corresponding to these epitopes

• Rigorous validation using multiple methods:

  • ELISA against recombinant SERPINB4, SERPINB3, and murine homologs to confirm specificity

  • Western blot analysis under reducing conditions

  • Testing in cell models with variable SERPINB4 expression levels

  • Functional validation through biological assays

Research has demonstrated that antibodies targeting specific epitopes (like P#5) can have distinct biological effects, reducing cell invasion by 75% while others show trivial results, highlighting the importance of epitope selection .

What techniques can distinguish between SERPINB3 and SERPINB4 in experimental systems?

Distinguishing between these highly homologous proteins requires specialized techniques:

• Use of epitope-specific antibodies targeting regions with minimal homology, particularly the catalytic site region showing only 54% identity

• Validation of antibody specificity through parallel testing against recombinant SERPINB3 and SERPINB4 proteins

• Design of isoform-specific primers for RT-qPCR that target divergent sequences

• Sandwich ELISA configurations with capture and detection antibodies validated for isoform specificity

• Mass spectrometry approaches to identify unique peptide signatures for each isoform

When using these techniques, researchers should implement appropriate controls and validation steps to ensure reliable differentiation between these closely related proteins .

How can researchers monitor SERPINB4 in clinical samples and what are the methodological considerations?

Monitoring SERPINB4 in clinical samples requires careful methodological considerations:

• Sample collection and handling must be standardized as SERPINB4 stability can be affected by processing conditions

• ELISA-based detection in serum/plasma offers quantitative measurement with validated kits showing applicability for human serum, plasma, and other biological fluids

• Immunohistochemical detection in tissue samples requires optimized antigen retrieval protocols and consideration of biotin blocking steps when using biotin-conjugated antibodies

• Western blot analysis of tissue lysates can provide semi-quantitative data on expression levels

• Consideration of pre-analytical variables including:

  • Sample collection method

  • Processing time

  • Storage conditions

  • Number of freeze-thaw cycles

Researchers should validate methods across multiple sample types and include appropriate controls to ensure reliable quantification in clinical specimens .

How might SERPINB4 serve as a therapeutic target in cancer?

The potential of SERPINB4 as a therapeutic target is emerging from several lines of evidence:

• The reactive site loop (RSL) appears critical for SERPINB4's invasion-promoting effects, making it a promising druggable target

• Antibodies specifically targeting the RSL (anti-P#5) demonstrated significant reductions in cell invasion (75%)

• SERPINB4's association with poor prognosis in melanoma suggests potential value as a prognostic biomarker

• Its role in modulating immune responses indicates possible synergistic effects with immunotherapies

• Small molecule inhibitors or peptide mimetics that disrupt SERPINB4's protease inhibitory activity may represent novel therapeutic approaches

Development of therapeutics would require further characterization of structure-function relationships and validation in preclinical models .

What new applications are being developed for biotin-conjugated antibodies in multiplex detection systems?

Emerging applications for biotin-conjugated SERPINB4 antibodies in multiplex systems include:

• Integrated proteogenomic approaches combining protein detection with genetic analysis

• Mass cytometry (CyTOF) incorporating biotin-conjugated antibodies for high-dimensional analysis of cell populations

• Spatial proteomics techniques such as imaging mass cytometry or multiplexed ion beam imaging

• Microfluidic-based single-cell protein analysis systems

• Automated image analysis platforms with machine learning algorithms to quantify SERPINB4 expression in complex tissue architectures

These advanced applications enhance the ability to study SERPINB4 in heterogeneous samples and complex biological contexts .