ITGB7 Antibody

What is ITGB7 and why is it important in immunological research?

ITGB7 (Integrin Beta 7) is a single-pass type I membrane protein belonging to the integrin beta chain family. It plays critical roles in leukocyte adhesion, signaling, proliferation, and migration by forming heterodimers with specific alpha integrin subunits. Most notably, ITGB7 associates with integrin alpha 4 (CD49d) to form the alpha 4 beta 7 integrin (LPAM-1) expressed on intraepithelial lymphocytes, and with alpha E (CD103) to form the alpha E beta 7 integrin (HML-1) expressed on T cells adjacent to mucosal epithelium .

The importance of ITGB7 in immunological research stems from its role in mediating the trafficking of immune cells to the gut mucosa. The alpha 4 beta 7 integrin binds to ligands including VCAM-1 (CD106), MAdCAM-1, and fibronectin, while alpha E beta 7 primarily binds to E-cadherin (CD324) . These interactions facilitate leukocyte adhesion to endothelial cells and promote transmigration to extravascular spaces during inflammatory responses, making ITGB7 a key target in studies of inflammatory bowel diseases, mucosal immunity, and targeted therapeutics .

What applications are ITGB7 antibodies commonly used for in research?

ITGB7 antibodies are utilized across multiple experimental applications:

The appropriate application depends on experimental goals, with flow cytometry being particularly valuable for analyzing expression on immune cell subsets and Western blotting for confirming antibody specificity and protein molecular weight .

What are the key differences between polyclonal and monoclonal ITGB7 antibodies?

For initial characterization studies, polyclonal antibodies may offer advantages in detecting native protein under various conditions, while monoclonal antibodies are preferred for consistent results in quantitative analyses and therapeutic applications .

How should I optimize ITGB7 antibody concentration for Western blot applications?

Optimizing ITGB7 antibody concentration for Western blot requires systematic titration to balance signal strength with background:

• Initial titration: Test a range of dilutions (e.g., 1:200, 1:500, 1:1000, 1:2000) using positive control samples like THP-1 cells, K-562 cells, or mouse spleen tissue, which are known to express ITGB7 .

• Loading control: Include 40-50 μg of total protein per lane with appropriate molecular weight markers to identify the expected 87-100 kDa ITGB7 band .

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST buffer; for phospho-specific detection, BSA is preferred over milk proteins.

• Incubation conditions: Test both overnight incubation at 4°C and 1-2 hours at room temperature to determine optimal signal-to-noise ratio.

• Secondary antibody selection: Use HRP-conjugated anti-rabbit IgG for polyclonal antibodies (typically at 1:5000-1:10000 dilution) .

• Exposure time adjustment: Begin with 10-minute exposure and adjust based on signal intensity .

• Validation: Confirm specificity by including negative control samples and/or using reducing vs. non-reducing conditions to verify expected molecular weight.

For challenging samples, membrane stripping and reprobing may be necessary, though this can reduce signal intensity with each cycle. In quantitative studies, consider using digital imaging systems rather than film for more accurate densitometric analysis .

What sample preparation methods yield optimal results for ITGB7 immunohistochemistry?

Effective ITGB7 immunohistochemistry requires careful sample preparation:

• Fixation protocol:

  • For formalin-fixed paraffin-embedded (FFPE) tissues: Fix in 10% neutral buffered formalin for 24-48 hours

  • For frozen sections: Flash freeze in OCT compound and prepare 5-8 μm sections

• Antigen retrieval methods:

  • Primary recommendation: TE buffer at pH 9.0 for heat-induced epitope retrieval

  • Alternative: Citrate buffer at pH 6.0 if TE buffer yields suboptimal results

  • Heat source: Pressure cooker or microwave heating to 95-100°C for 15-20 minutes

• Blocking procedure:

  • Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes

  • Block non-specific binding with 5-10% normal serum (species of secondary antibody) in PBS containing 0.1% Triton X-100

• Antibody incubation:

  • Dilution range: 1:50-1:500, with 1:100 as a recommended starting point

  • Incubation time: 1 hour at room temperature or overnight at 4°C in a humidified chamber

  • Wash steps: 3-5 washes with PBS-T (PBS + 0.05% Tween-20)

• Detection system:

  • For brightfield microscopy: HRP-polymer detection systems with DAB substrate

  • For fluorescence: Secondary antibodies conjugated to fluorophores like NorthernLights™ 557

Positive control tissues should include human tonsillitis tissue, which has been validated for ITGB7 detection . When working with mucosal tissue samples, special attention should be paid to mucin interference by extending washing steps .

What are the most reliable positive controls for validating ITGB7 antibodies across different applications?

Reliable positive controls for ITGB7 antibody validation vary by application:

For negative controls, consider using tissues known to have low ITGB7 expression or cells treated with ITGB7-specific siRNA. Additionally, isotype controls (e.g., rabbit IgG or mouse IgG matched to the primary antibody) should be included in experimental design to assess non-specific binding, particularly in flow cytometry applications .

How can I effectively use ITGB7 antibodies to study gut-homing lymphocyte trafficking in inflammatory bowel disease models?

Studying gut-homing lymphocyte trafficking in IBD models using ITGB7 antibodies requires a multi-faceted approach:

• Flow cytometric profiling:

  • Panel design: Combine anti-ITGB7 (clone FIB504 at ≤0.125 μg per test) with markers for T cell subsets (CD4, CD8), memory phenotype (CD45RA, CD45RO), and gut-homing receptors (CCR9)

  • Sample preparation: Isolate lymphocytes from peripheral blood, mesenteric lymph nodes, and intestinal lamina propria

  • Analysis: Quantify the percentage of α4β7⁺ cells within each subset and monitor changes during disease progression

• In vivo trafficking studies:

  • Adoptive transfer: Label lymphocytes with fluorescent dyes (CFSE) before transfer into recipient animals

  • Tracking: Use anti-ITGB7 antibodies to identify transferred cells in gut tissues

  • Functional blockade: Administer blocking ITGB7 antibodies (such as vedolizumab analogues) to assess impact on lymphocyte homing

• Ex vivo adhesion assays:

  • Endothelial binding: Culture primary intestinal endothelial cells or MAdCAM-1 expressing cell lines

  • Quantification: Measure adhesion of lymphocytes pre-treated with anti-ITGB7 blocking antibodies versus controls

  • Shear stress conditions: Perform assays under static and flow conditions to mimic physiological environments

• Tissue analysis:

  • Sequential immunohistochemistry: Perform ITGB7 staining followed by markers of inflammation

  • Confocal microscopy: Co-localize ITGB7⁺ cells with adhesion molecules (MAdCAM-1, VCAM-1)

  • Computational analysis: Quantify the spatial distribution of ITGB7⁺ cells relative to inflammatory foci

• Therapeutic intervention assessment:

  • Time-course studies: Monitor ITGB7 expression before, during, and after therapeutic interventions

  • Dose-response: Evaluate the relationship between anti-integrin antibody dosage and trafficking inhibition

  • Combination approaches: Analyze synergistic effects of targeting multiple adhesion pathways

This approach allows for comprehensive assessment of how ITGB7-mediated trafficking contributes to disease pathogenesis and response to therapy .

What are the key considerations when using ITGB7 antibodies for analyzing the α4β7 vs. αEβ7 integrin complexes?

Analyzing distinct ITGB7-containing integrin complexes requires careful experimental design:

• Antibody selection strategies:

  • ITGB7-specific antibodies: Detect both α4β7 and αEβ7 complexes

  • Complex-specific antibodies: Use antibodies against conformational epitopes formed by specific αβ pairs

  • Paired detection: Combine anti-ITGB7 with either anti-CD49d (α4) or anti-CD103 (αE) antibodies

  • Humanized therapeutic antibodies: Some, like vedolizumab (TAB-H75), specifically target the α4β7 heterodimer

• Flow cytometry panel design:

  • Three-color approach: Anti-ITGB7 + anti-CD49d + anti-CD103 to distinguish populations

  • Gating strategy: Identify CD49d⁺ITGB7⁺ (α4β7) vs. CD103⁺ITGB7⁺ (αEβ7) populations

  • Controls: Include FMO (fluorescence minus one) controls for accurate gate placement

• Functional discrimination:

  • Ligand binding assays: α4β7 binds MAdCAM-1 and VCAM-1, while αEβ7 binds E-cadherin

  • Differential blocking: Use ligand-specific blocking antibodies to distinguish functions

  • Cell type correlation: αEβ7 is predominantly expressed on intraepithelial lymphocytes, while α4β7 is more broadly expressed on circulating lymphocytes

• Tissue distribution analysis:

  • Sequential IHC: Stain consecutive sections with ITGB7, CD49d, and CD103 antibodies

  • Multiplex IF: Use differently labeled antibodies against ITGB7, CD49d, and CD103

  • Quantitative analysis: Calculate co-localization coefficients to determine heterodimer distribution

• Technical challenges and solutions:

  • Epitope masking: Some complex conformations may mask epitopes; test multiple antibody clones

  • Activation-dependent expression: Use both resting and activated lymphocytes in analyses

  • Detergent sensitivity: Some epitopes are sensitive to detergent solubilization; optimize lysis conditions

These approaches allow researchers to distinguish between the gut-homing α4β7 integrin and the tissue-retention αEβ7 integrin, which have distinct functions in mucosal immunology .

How can I use ITGB7 antibodies to evaluate the efficacy of anti-integrin therapeutics in experimental models?

Evaluating anti-integrin therapeutic efficacy using ITGB7 antibodies involves multiple analytical approaches:

• Target engagement assessment:

  • Receptor occupancy assay: Measure binding competition between therapeutic antibody and detection antibody

  • Flow cytometry: Quantify percentage of α4β7⁺ cells with accessible (unoccupied) epitopes

  • Experimental design: Compare pre- and post-treatment samples to determine occupancy kinetics

• Functional blocking validation:

  • In vitro adhesion assays: Measure reduction in lymphocyte adhesion to MAdCAM-1 or VCAM-1

  • Competitive binding ELISA: Use recombinant α4β7 protein to determine IC50 values of therapeutic antibodies

  • Dot blot analysis: Direct comparison of binding affinity between different anti-integrin antibodies

• Pharmacodynamic biomarkers:

  • Circulating lymphocytes: Monitor changes in peripheral blood α4β7⁺ lymphocyte counts

  • Tissue infiltration: Quantify reduction in ITGB7⁺ cells in intestinal biopsies using IHC

  • Inflammation markers: Correlate ITGB7⁺ cell changes with inflammatory cytokine levels

• Treatment regimen optimization:

  • Dose-response studies: Establish relationship between antibody dose and receptor occupancy

  • Dosing interval assessment: Determine duration of target engagement to inform dosing frequency

  • Combination strategy: Evaluate synergistic effects with other immunomodulatory approaches

• Resistance mechanism investigation:

  • Epitope analysis: Detect mutations or conformational changes affecting therapeutic binding

  • Alternative pathway upregulation: Monitor compensatory increases in other adhesion molecules

  • Sequential therapy response: Assess cross-resistance between different anti-integrin therapeutics

This methodological framework allows for comprehensive assessment of anti-integrin therapeutic candidates, facilitating translational research toward clinical applications in inflammatory bowel diseases and other immune-mediated conditions .

What are common sources of false positives/negatives when using ITGB7 antibodies, and how can they be mitigated?

For quantitative applications, always include concentration curves of positive control samples to establish assay linearity and dynamic range .

How should I interpret discrepancies in ITGB7 detection between different antibodies or experimental approaches?

When faced with discrepancies in ITGB7 detection across antibodies or methods, consider this systematic approach:

• Epitope mapping analysis:

  • Different antibodies target distinct epitopes on ITGB7 (N-terminal, middle region, C-terminal)

  • Some epitopes may be inaccessible in certain experimental conditions

  • Compare immunogen sequences: CAB5873 targets amino acids 1-140, while others target different regions

• Methodological differences:

  • Native vs. denatured protein detection: Western blot detects denatured protein, while flow cytometry and IF detect native conformation

  • Sample preparation effects: The ITGB7 conformation in FFPE tissues differs from frozen sections

  • Buffer compatibility: Some antibodies perform better in specific buffer systems

• Validation hierarchy:

  • Establish a "gold standard" method (often WB) to confirm specificity

  • Compare observed molecular weights (87-100 kDa) across antibodies

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to verify specificity

• Quantitative considerations:

  • Different antibodies may have varying affinities affecting detection threshold

  • Establish standard curves with recombinant protein for absolute quantification

  • When comparing results, normalize to common positive controls

• Biological variables:

  • Heterodimer formation affects epitope accessibility (α4β7 vs. αEβ7)

  • Activation state alters integrin conformation and antibody binding

  • Post-translational modifications (glycosylation) may mask epitopes

• Reporting guidelines:

  • Document complete antibody information (clone, catalog number, lot)

  • Specify exact experimental conditions and image acquisition parameters

  • Present data from multiple antibodies when discrepancies exist

When publishing, acknowledge limitations and potential causes of discrepancies rather than selectively reporting concordant results .

What are the critical quality control parameters for validating new lots of ITGB7 antibodies before experimental use?

Implementing rigorous quality control for ITGB7 antibody validation ensures experimental reliability:

• Physicochemical characterization:

  • Concentration verification: Measure protein concentration (expected ~2.2 mg/ml for many preparations)

  • Purity assessment: Run SDS-PAGE to confirm >90% purity of antibody preparation

  • Aggregation analysis: Verify <10% aggregation by HPLC or native PAGE

• Immunoreactivity testing:

  • Titration curve: Perform serial dilutions on standard positive controls (e.g., THP-1 cells)

  • EC50 determination: Calculate half-maximal effective concentration and compare to reference lot

  • Sensitivity threshold: Determine minimum detectable ITGB7 concentration

• Specificity validation:

  • Western blot: Confirm single band at expected 87-100 kDa molecular weight

  • Cross-reactivity panel: Test against related integrins (β1, β2) to confirm specificity

  • Species cross-reactivity: Verify reactivity with human, mouse, and rat samples as claimed

• Application-specific controls:

  • For IHC: Test on reference tissue sections (human tonsillitis tissue) with established staining pattern

  • For flow cytometry: Compare percentage of positive cells in standard lymphocyte preparations

  • For IF: Verify membrane/cytoplasmic localization pattern in PBMCs

• Stability assessment:

  • Freeze-thaw stability: Test activity after multiple freeze-thaw cycles

  • Temperature sensitivity: Compare performance after storage at different temperatures

  • Long-term stability: Monitor activity over time following storage recommendations

• Documentation requirements:

  • Certificate of analysis: Maintain records showing lot-specific QC results

  • Validation report: Document all testing parameters, acceptance criteria, and results

  • Reference standards: Maintain aliquots of reference lots for future comparisons

Implementing these quality control measures reduces experimental variability and increases confidence in research findings using ITGB7 antibodies .

How can ITGB7 antibodies be utilized in single-cell analysis of mucosal immune populations?

ITGB7 antibodies offer powerful capabilities for dissecting mucosal immune heterogeneity at single-cell resolution:

• Single-cell flow cytometry applications:

  • High-dimensional phenotyping: Incorporate anti-ITGB7 (e.g., FIB504 clone) in 15+ color panels

  • CITE-seq integration: Combine ITGB7 antibody-derived tags with single-cell transcriptomics

  • Isolation strategies: Use ITGB7 as a sorting parameter to enrich gut-homing populations for downstream analysis

• Tissue-based spatial analysis:

  • Multiplex immunofluorescence: Combine ITGB7 with lineage markers, activation markers, and adhesion molecules

  • Imaging mass cytometry: Incorporate metal-conjugated ITGB7 antibodies for high-parameter tissue imaging

  • Digital spatial profiling: Analyze ITGB7⁺ cell distribution relative to tissue niches

• Function-phenotype correlation:

  • Live cell imaging: Track ITGB7⁺ lymphocyte migration in tissue explants

  • Calcium flux assays: Measure signaling in ITGB7⁺ vs. ITGB7⁻ cells upon activation

  • Cytokine secretion analysis: Correlate ITGB7 expression with functional output at single-cell level

• Technical considerations:

  • Antibody panel design: Place ITGB7 in appropriate fluorochrome channel based on expression level

  • Dissociation protocols: Optimize tissue digestion to preserve ITGB7 epitopes

  • Fixation compatibility: Validate ITGB7 antibody performance with fixation needed for intracellular markers

• Computational analysis approaches:

  • Dimensionality reduction: Visualize ITGB7⁺ populations using tSNE or UMAP

  • Trajectory inference: Map developmental relationships of ITGB7-expressing cells

  • Spatial statistics: Quantify clustering and interaction patterns of ITGB7⁺ cells in tissues

These approaches enable researchers to uncover previously unappreciated heterogeneity within mucosal immune populations and link ITGB7 expression to functional specialization .

What role can ITGB7 antibodies play in developing organoid-based models for studying intestinal immunity?

ITGB7 antibodies provide valuable tools for enhancing intestinal organoid models of mucosal immunity:

• Organoid-immune cell co-culture optimization:

  • Selection of lymphocytes: Isolate ITGB7⁺ lymphocytes by flow cytometry for co-culture with intestinal organoids

  • Migration assessment: Quantify ITGB7-dependent homing of lymphocytes to organoids

  • Adhesion blockade: Use blocking antibodies to modulate immune cell-organoid interactions

• Functional readouts:

  • Live imaging: Track labeled ITGB7⁺ cells interacting with organoid structures

  • Barrier function: Measure how ITGB7⁺ cell incorporation affects epithelial integrity

  • Cytokine production: Analyze secretory profiles of organoids with integrated ITGB7⁺ populations

• Disease modeling applications:

  • Inflammatory conditions: Recreate IBD microenvironments by combining patient-derived organoids with ITGB7⁺ cells

  • Therapeutic testing: Evaluate anti-integrin therapies in organoid-immune cell systems

  • Host-microbe interactions: Study how ITGB7-mediated immune positioning affects response to microbial stimuli

• Analytical approaches:

  • Flow cytometry: Dissociate co-cultures and analyze changes in ITGB7 expression

  • Confocal microscopy: Visualize ITGB7⁺ cell distribution within 3D organoid structures

  • Transcriptional profiling: Compare gene expression in organoids with vs. without ITGB7⁺ cell incorporation

• Technical optimizations:

  • Antibody penetration: Optimize staining protocols for intact 3D structures

  • Fixation compatibility: Test fixatives that preserve both organoid architecture and ITGB7 epitopes

  • Long-term monitoring: Develop strategies for repeated imaging of the same organoid-immune cell cultures

This emerging research area bridges the gap between traditional 2D culture systems and in vivo models, offering new insights into ITGB7-mediated immune-epithelial interactions .

How can researchers effectively incorporate ITGB7 antibodies in multi-omic studies of tissue-resident immune cells?

Integrating ITGB7 antibodies into multi-omic approaches enables comprehensive profiling of tissue-resident immune populations:

• Combined protein-transcriptome approaches:

  • CITE-seq workflow: Conjugate ITGB7 antibodies to oligonucleotide barcodes for combined protein-RNA profiling

  • Cell isolation strategy: Sort ITGB7⁺ vs. ITGB7⁻ populations before single-cell RNA sequencing

  • Computational integration: Correlate ITGB7 protein levels with gene expression patterns

• Spatial multi-omics integration:

  • Sequential workflows: Perform imaging with ITGB7 antibodies followed by spatial transcriptomics

  • Registration algorithms: Align ITGB7 protein data with spatial gene expression maps

  • Cell type deconvolution: Use ITGB7 as a marker for identifying specific immune subsets in spatial data

• Epigenome-proteome correlation:

  • ATAC-seq with protein detection: Profile chromatin accessibility in ITGB7-defined populations

  • CyTOF followed by ChIP-seq: Isolate ITGB7⁺ subsets for histone modification analysis

  • Multi-modal data integration: Develop analytical frameworks to connect ITGB7 expression with chromatin states

• Methodological considerations:

  • Sample processing compatibility: Optimize protocols to preserve both protein epitopes and nucleic acid integrity

  • Antibody performance: Validate ITGB7 antibody specificity in multi-omic workflows

  • Batch effect management: Implement controls to align data across different analytical platforms

• Computational analysis frameworks:

  • Multi-modal clustering: Identify cell states based on integrated ITGB7 protein and gene expression data

  • Trajectory reconstruction: Map developmental relationships with ITGB7 as a key feature

  • Systems biology modeling: Incorporate ITGB7 data into network analyses of mucosal immunity

This multi-omic approach provides unprecedented resolution of the biological context in which ITGB7 functions, connecting molecular profiles to cellular identities and tissue localization .