DEFB4A Antibody

What is DEFB4A and why is it significant in research?

DEFB4A (Defensin β 4A), also known as BD-2, SAP1, DEFB2, DEFB4, HBD-2, DEFB-2 and DEFB102, belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils. These peptides serve important roles in innate immune defense against microbial infections . DEFB4A has gained research significance due to its upregulation in various cancers including colorectal cancer, cutaneous squamous cell carcinoma, and basal cell carcinoma, as well as its potential prognostic value . Its expression pattern correlates with immune markers and potentially influences immunosuppressive activity in tumor microenvironments, making it an attractive target for immunotherapy research .

What antibody formats are available for DEFB4A detection?

Based on current research resources, DEFB4A antibodies are primarily available in polyclonal format derived from rabbit hosts . These antibodies are typically produced using recombinant fusion proteins containing amino acid sequences 1-64 of human DEFB4A as immunogens . Commercial DEFB4A antibodies are generally supplied in liquid form, purified via affinity chromatography to a purity level of ≥95% as determined by SDS-PAGE . While polyclonal antibodies offer broader epitope recognition, researchers should note that the specific literature reviewed does not mention monoclonal options, which might be relevant for experimental design considerations requiring higher specificity.

Which applications are DEFB4A antibodies validated for?

DEFB4A antibodies have been validated for multiple laboratory applications:

These applications allow researchers to detect DEFB4A in various experimental contexts, with optimal dilutions varying by application type . Note that the antibodies may not be validated for other applications such as flow cytometry or chromatin immunoprecipitation, which would require additional validation by researchers.

How does DEFB4A expression correlate with colorectal cancer prognosis?

DEFB4A expression has demonstrated significant prognostic value in colorectal cancer (CRC). Research shows that DEFB4A is consistently upregulated in CRC tumor tissues compared to normal tissues . More specifically, DEFB4A upregulation correlates with:

• Advanced TNM stages (showing progressive increase from stage I through IV)

• Larger tumor sizes

• Presence of lymph node metastasis

• Presence of liver metastasis

• Higher tumor marker levels (CA199, CA 72-4)

What immune mechanisms might explain DEFB4A's role in cancer progression?

Gene Ontology (GO) enrichment and Gene Set Enrichment Analysis (GSEA) reveal that DEFB4A's oncogenic effects may be mediated through immunomodulatory pathways. DEFB4A expression is highly associated with:

• Myeloid leukocyte differentiation

• Leukocyte proliferation

• Positive regulation of leukocyte-mediated immunity

Correlation analyses show positive associations between DEFB4A expression and several immune markers:

• CD11b (myeloid cell marker)

• CD14 (monocyte marker)

• CD45 (leukocyte common antigen)

• CD163 (M2 macrophage marker)

• IL17A (pro-inflammatory cytokine)

These associations suggest DEFB4A may promote tumor growth by fostering an immunosuppressive microenvironment, potentially through recruitment and polarization of myeloid-derived suppressor cells or tumor-associated macrophages. Researchers investigating DEFB4A should consider incorporating these immune markers in their experimental designs to elucidate the complete mechanistic pathway.

How can DEFB4A antibodies be employed in functional studies of cancer cell behavior?

DEFB4A antibodies can be utilized in functional studies to investigate cancer cell behavior through several methodological approaches:

• Knockdown validation studies: Following DEFB4A gene knockdown using siRNA or CRISPR-Cas9, antibodies can confirm protein depletion via Western blot. Research has shown that DEFB4A knockdown affects cell migration properties, suggesting a role in metastatic potential .

• Immunophenotyping of tumor microenvironment: IHC applications of DEFB4A antibodies can help characterize the spatial distribution of DEFB4A expression relative to immune cell infiltrates in tumor sections, helping determine whether DEFB4A correlates with specific immune cell populations in situ.

• Co-immunoprecipitation studies: DEFB4A antibodies can identify binding partners to elucidate signaling pathways, particularly those involving immune receptors given DEFB4A's correlation with immune markers.

• Chromatin immunoprecipitation (ChIP): Though not explicitly validated in the provided sources, optimized DEFB4A antibodies could potentially be employed to investigate transcriptional regulation of DEFB4A in cancer cells.

When designing such functional studies, researchers should include appropriate controls and validate antibody specificity, particularly when studying closely related defensin family members.

What are the optimal storage conditions for DEFB4A antibodies?

DEFB4A antibodies require specific storage conditions to maintain their functionality and prevent degradation:

• Temperature: Store at -20°C for long-term preservation

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting: Divide into small aliquots to avoid repeated freeze/thaw cycles

• Shelf life: Properly stored antibodies maintain activity for approximately 12 months

Researchers should note that repeated freeze-thaw cycles significantly reduce antibody performance. While some sources suggest antibody recycling may be possible, this practice is generally not recommended as buffer conditions change after use, and storage conditions vary between laboratories, potentially compromising experimental reproducibility .

What controls should be included when using DEFB4A antibodies for expression analysis?

Proper experimental controls are essential when working with DEFB4A antibodies:

• Positive tissue controls:

  • For colorectal cancer studies: Include known DEFB4A-expressing tumor tissues

  • For other applications: Epithelial tissues with confirmed DEFB4A expression

• Negative controls:

  • Primary antibody omission control

  • Isotype-matched irrelevant antibody control

  • Tissues known to lack DEFB4A expression

• Knockdown/knockout validation:

  • DEFB4A siRNA or CRISPR-treated samples serve as specificity controls

• Loading and normalization controls:

  • For Western blot: GAPDH (recommended primer sequences: forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′)

  • For qPCR: The 2−ΔΔCq method using GAPDH as reference (DEFB4A primer sequences: forward, 5′-CTCCTCTTCTCGTTCCTCTTCA-3′ and reverse, 5′-GCAGGTAACAGGATCGCCTAT-3′)

These controls ensure experimental validity and help distinguish true DEFB4A signal from background or non-specific binding.

How can researchers address weak or absent signal in DEFB4A immunodetection?

When encountering weak or absent DEFB4A signal in immunodetection experiments, researchers should systematically troubleshoot using this methodology:

• Antibody concentration optimization:

  • For Western blot: Test concentrations ranging from 1:500 to 1:2000

  • For IHC-P: Test concentrations ranging from 1:50 to 1:200

  • For ELISA: Initial concentration of 1 μg/ml with titration as needed

• Antigen retrieval enhancement (for IHC):

  • Test both citrate and EDTA-based antigen retrieval buffers

  • Optimize retrieval time and temperature

• Sample preparation considerations:

  • For Western blot: Ensure proper protein extraction protocol that preserves small proteins (~7 kDa)

  • Consider using gradient gels optimized for low molecular weight proteins

  • Verify transfer efficiency using stained membranes

• Signal amplification strategies:

  • Implement tyramide signal amplification

  • Consider more sensitive detection systems (e.g., chemiluminescence vs. colorimetric)

  • Extend primary antibody incubation time (overnight at 4°C)

• Verify target expression:

  • Run parallel qPCR to confirm DEFB4A mRNA expression in test samples

  • Include known positive controls (e.g., confirmed CRC samples with high DEFB4A expression)

What approaches can overcome cross-reactivity issues with DEFB4A antibodies?

DEFB4A belongs to the defensin family with highly similar protein sequences among members, potentially leading to cross-reactivity issues. Researchers can employ these strategies to ensure specificity:

• Antibody validation using recombinant proteins:

  • Test antibody against recombinant DEFB4A and related defensins

  • Perform competitive binding assays with purified proteins

• Genetic validation approaches:

  • Use DEFB4A-knockout or knockdown samples as negative controls

  • Conduct rescue experiments with DEFB4A overexpression

• Epitope mapping:

  • Determine the specific epitope recognized by the antibody

  • Compare with sequence alignments of related defensins

• Alternative detection methods:

  • Confirm results using orthogonal techniques (e.g., mass spectrometry)

  • Use antibodies targeting different epitopes of DEFB4A

• Pre-absorption controls:

  • Pre-incubate antibody with recombinant DEFB4A protein before application

  • Compare staining patterns with and without pre-absorption

These approaches help ensure that observed signals specifically represent DEFB4A rather than related family members, particularly in tissues expressing multiple defensins.

How might DEFB4A antibodies contribute to understanding its role in immunotherapy response?

Given DEFB4A's association with immune markers and potential immunosuppressive activity, researchers could deploy DEFB4A antibodies to investigate its role in immunotherapy response through:

• Predictive biomarker development:

  • Correlate pre-treatment DEFB4A levels with response to immune checkpoint inhibitors

  • Develop IHC-based scoring systems for DEFB4A expression in tumor microenvironments

• Mechanistic studies:

  • Investigate whether DEFB4A modulates PD-1/PD-L1 or CTLA-4 pathways

  • Examine DEFB4A's effects on tumor-infiltrating lymphocyte activity using co-culture systems

• Therapeutic targeting approaches:

  • Develop function-blocking DEFB4A antibodies to potentially enhance immunotherapy efficacy

  • Explore DEFB4A as a target for antibody-drug conjugates

• Combinatorial treatment assessment:

  • Evaluate whether DEFB4A inhibition synergizes with existing immunotherapies

  • Develop protocols for monitoring DEFB4A expression during treatment response

These research directions could significantly advance our understanding of DEFB4A's role in cancer immunobiology and potentially lead to improved immunotherapeutic approaches, particularly in colorectal cancer where DEFB4A shows prognostic significance .

What technologies might enhance the specificity and sensitivity of DEFB4A detection?

Emerging technologies could significantly improve DEFB4A detection specificity and sensitivity:

• Single-cell analysis approaches:

  • Single-cell Western blotting for heterogeneous samples

  • Imaging mass cytometry for spatial profiling of DEFB4A alongside immune markers

  • Single-cell RNA-seq combined with protein detection (CITE-seq)

• Proximity ligation assays:

  • Detect DEFB4A interactions with potential binding partners

  • Improve specificity through dual antibody recognition requirements

• Nanobody development:

  • Engineer smaller antibody fragments for improved tissue penetration

  • Develop bi-specific antibodies targeting DEFB4A and related immune markers

• Digital pathology integration:

  • Computer-aided quantification of DEFB4A expression in clinical samples

  • Machine learning algorithms to correlate expression patterns with patient outcomes

• Aptamer-based detection:

  • Develop nucleic acid aptamers as alternatives to antibodies

  • Implement aptamer-based biosensors for rapid DEFB4A quantification

These technological advancements could address current limitations in DEFB4A detection and provide more robust tools for both research and potential clinical applications.