ITGAV Recombinant Monoclonal Antibody

What is ITGAV and why is it an important research target?

ITGAV is a transmembrane protein that functions in cell adhesion and signaling pathways. It forms heterodimers with various integrin beta subunits (including β1, β3, β5, β6, and β8) to bind extracellular matrix proteins such as fibronectin and vitronectin. The biological significance of ITGAV stems from its critical roles in regulating cell migration, proliferation, differentiation, and survival. Additionally, ITGAV participates in angiogenesis and wound healing processes. The dysregulation of ITGAV expression and function has been linked to multiple pathological conditions, including cancer, osteoporosis, and autoimmune disorders, making it an important target for both basic and translational research .

How are ITGAV recombinant monoclonal antibodies produced?

The production of ITGAV recombinant monoclonal antibodies follows a sophisticated multi-step biotechnological process. Initially, the gene encoding the ITGAV-specific monoclonal antibody is sequenced from hybridomas that produce the antibody naturally. This gene is then cloned into a plasmid vector that contains appropriate regulatory elements. The recombinant vector is subsequently transfected into a host cell line (commonly mammalian cells) for expression. After expression, the antibody is secreted into the cell culture supernatant, from which it is purified using affinity chromatography techniques, typically involving Protein A and/or Protein G matrices. The purified antibody undergoes extensive testing and characterization to ensure its specificity, affinity, and functionality before being released for research applications .

What are the main differences between standard monoclonal and recombinant monoclonal antibodies against ITGAV?

Standard monoclonal antibodies are traditionally produced via hybridoma technology, where antibody-producing B cells from immunized animals are fused with myeloma cells to create hybridomas that continuously secrete a specific antibody. While effective, this approach can lead to batch-to-batch variability. In contrast, recombinant monoclonal antibodies are produced by cloning the antibody genes and expressing them in controlled expression systems. The recombinant approach offers several advantages: higher consistency between production batches, the ability to engineer the antibody sequence for improved properties, elimination of animal use in production, and potentially superior specificity. For ITGAV research, recombinant antibodies provide more reliable results across experiments, which is particularly important when studying subtle changes in integrin expression or localization during disease progression .

What are the recommended applications for ITGAV recombinant monoclonal antibodies?

ITGAV recombinant monoclonal antibodies have been validated for multiple research applications with specific working dilutions recommended for each technique. The following table summarizes the primary applications and their corresponding dilution ranges based on commercially available antibodies:

These applications enable researchers to investigate ITGAV expression, localization, and interactions in various experimental systems, with each technique providing unique insights into integrin biology .

How should ITGAV antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of ITGAV antibodies are crucial for maintaining their activity and specificity over time. According to manufacturer recommendations, lyophilized antibody preparations should be stored at -20°C until reconstitution. After reconstitution, the antibody can be stored at 4°C for approximately one month for frequent use. For longer storage periods, the reconstituted antibody should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C for up to 24 months (depending on the specific antibody formulation). The buffer composition typically includes stabilizers such as glycerol (often at 50%) and preservatives like sodium azide (NaN₃) or Proclin-300. Thermal stability tests indicate that high-quality antibodies show less than 5% activity loss when incubated at 37°C for 48 hours, suggesting good stability under proper storage conditions. Researchers should always avoid repeated freeze-thaw cycles as these significantly reduce antibody activity and can lead to aggregation and non-specific binding .

How can I optimize Western blot protocols for detecting ITGAV in different sample types?

Optimizing Western blot protocols for ITGAV detection requires careful consideration of several parameters. ITGAV is a large transmembrane protein (observed at 130-140 kDa, calculated at 116 kDa), necessitating specific technical adjustments. For effective separation, use a gradient gel (5-20% SDS-PAGE) with extended running times (2-3 hours) at moderate voltage (70-90V). Sample preparation is critical: use RIPA buffer supplemented with protease inhibitors and ensure complete solubilization of membrane proteins through adequate incubation and occasional vortexing. For cell lines with varying ITGAV expression levels, load 30-50 μg of total protein per well. Use reducing conditions but avoid excessive heating that might cause protein aggregation. During transfer, employ a wet transfer system with methanol-containing buffer for 50-90 minutes at 150mA to ensure efficient transfer of this large protein. Blocking should be performed with 5% non-fat milk in TBS for 1.5 hours at room temperature to minimize background. Optimize primary antibody concentration (typically 0.25-2 μg/mL) and incubate overnight at 4°C for best results. After thorough washing with TBS-0.1% Tween (3 times, 5 minutes each), use an appropriate HRP-conjugated secondary antibody (1:10000 dilution) and develop using enhanced chemiluminescence. If non-specific bands appear, try increasing the blocking time or adjusting the antibody dilution .

What controls should be included when using ITGAV antibodies in immunohistochemistry?

When conducting immunohistochemistry (IHC) experiments with ITGAV antibodies, implementing appropriate controls is essential for result validation and accurate interpretation. Positive controls should include tissues known to express ITGAV, such as human placenta, which shows consistent ITGAV expression. Negative controls should include tissues where ITGAV expression is minimal or absent. For procedural controls, include: (1) a primary antibody omission control (tissue section treated identically but without primary antibody) to assess background from the detection system; (2) an isotype control (using non-specific IgG of the same isotype at the same concentration) to evaluate non-specific binding; (3) a peptide competition assay where the antibody is pre-incubated with the immunizing peptide to confirm specificity. For semi-quantitative analysis, include a dilution series of the primary antibody to establish the optimal signal-to-noise ratio, typically within the 1:50-1:200 range for ITGAV antibodies. Additionally, when studying diseased tissues, always include matched normal tissues as comparative controls. This comprehensive control strategy ensures the specificity of ITGAV detection and allows for confident interpretation of expression patterns in experimental samples .

What is the best approach for validating ITGAV antibody specificity in my experimental system?

Validating ITGAV antibody specificity requires a multi-faceted approach tailored to your experimental system. Begin with Western blot analysis using both positive controls (cell lines known to express ITGAV, such as A549 or A431) and negative controls (cell lines with low or no ITGAV expression). The antibody should detect a specific band at approximately 130-140 kDa. For enhanced validation, use ITGAV knockdown or knockout systems: transfect cells with ITGAV-specific siRNA or generate CRISPR/Cas9 knockout cells and confirm reduced or absent signal. Peptide competition assays provide additional specificity confirmation—pre-incubate the antibody with the immunizing peptide before applying to your samples; a significant reduction in signal indicates specificity. Cross-reactivity testing is crucial when working with multiple species; verify the antibody's reactivity profile matches manufacturer claims by testing on human, mouse, and rat samples if cross-reactivity is indicated. For immunostaining applications, perform dual labeling with a different ITGAV antibody recognizing a distinct epitope; colocalization strongly supports specificity. Finally, correlate protein detection with mRNA expression using qRT-PCR or RNA-seq data from the same samples. This comprehensive validation strategy ensures reliable interpretation of ITGAV expression and localization data in your specific experimental system .

How can flow cytometry protocols be optimized for ITGAV detection?

Optimizing flow cytometry protocols for ITGAV detection requires careful attention to sample preparation, antibody titration, and appropriate controls. Since ITGAV (CD51) is a cell surface protein, begin with gentle cell dissociation methods to preserve surface epitopes—use enzyme-free dissociation buffers when possible, or brief trypsinization followed by recovery in complete medium. Fix cells with 4% paraformaldehyde to maintain membrane integrity. For intracellular detection, include a permeabilization step using 0.1% saponin or 0.1% Triton X-100. Block with 10% normal serum from the same species as the secondary antibody to reduce non-specific binding. The optimal antibody concentration should be determined through titration; start with 1 μg per 1×10⁶ cells and adjust as needed (typical working dilutions range from 1:50 to 1:200). Incubate cells with the primary antibody for 30 minutes at 4-20°C, followed by fluorophore-conjugated secondary antibody (if using an unconjugated primary). Essential controls include an isotype control antibody at the same concentration as the primary antibody and an unstained sample to establish autofluorescence levels. For multicolor analysis, perform compensation using single-color controls. When analyzing results, gate on intact cells using FSC/SSC parameters, exclude doublets, and compare the fluorescence intensity to isotype and unstained controls. This approach enables accurate quantification of ITGAV expression levels and distribution across cell populations .

How can ITGAV antibodies be used to investigate integrin heterodimer formation?

Investigating integrin heterodimer formation with ITGAV antibodies requires sophisticated experimental approaches that preserve protein-protein interactions. Co-immunoprecipitation (Co-IP) serves as a primary method: use ITGAV antibodies conjugated to a solid support to pull down ITGAV along with its beta subunit partners (β1, β3, β5, β6, or β8). The precipitated complexes can then be analyzed by Western blotting using antibodies against specific beta subunits to determine heterodimer composition. For more complex interaction studies, proximity ligation assay (PLA) provides high sensitivity and specificity—pairs of antibodies against ITGAV and various beta subunits generate fluorescent signals only when proteins are in close proximity (<40 nm), allowing visualization of specific heterodimers in situ.

Fluorescence resonance energy transfer (FRET) offers quantitative assessment of heterodimer dynamics in living cells—label ITGAV antibodies with donor fluorophores and beta subunit antibodies with acceptor fluorophores to measure energy transfer as an indicator of protein proximity. For high-throughput screening of multiple integrin combinations, protein array technology can be employed, immobilizing various beta subunits on a chip and probing with fluorescently labeled ITGAV antibodies. Additionally, super-resolution microscopy techniques (STORM, PALM) in combination with dual-labeled antibodies enable visualization of heterodimer distribution at nanometer resolution, revealing spatial organization within adhesion complexes. These methods collectively provide comprehensive insights into the formation, regulation, and functional significance of ITGAV-containing heterodimers in normal and pathological processes .

What methods can be used to study the role of ITGAV in cell migration and adhesion?

Studying ITGAV's role in cell migration and adhesion requires combining antibody-based techniques with functional assays. Wound healing assays provide insights into collective cell migration—create a "wound" in a confluent cell monolayer, treat with ITGAV blocking antibodies (typically at 5-20 μg/mL), and monitor closure rate through time-lapse microscopy. For quantitative single-cell migration analysis, employ Transwell migration assays with ITGAV antibodies to block specific interactions. Adhesion assays directly assess ITGAV-mediated cell attachment—coat surfaces with ITGAV ligands (vitronectin, fibronectin), treat cells with function-blocking ITGAV antibodies, and quantify adherent cells through crystal violet staining or real-time impedance measurements.

For molecular-level studies, integrin activation can be assessed using conformation-specific ITGAV antibodies that recognize active versus inactive states. Immunofluorescence with phospho-specific antibodies targeting focal adhesion proteins (FAK, paxillin) alongside ITGAV staining reveals downstream signaling activation. Advanced techniques include traction force microscopy, where cells are cultured on deformable substrates containing fluorescent beads, and forces generated through ITGAV-mediated adhesions are calculated from substrate deformations. FRAP (Fluorescence Recovery After Photobleaching) with fluorescently tagged ITGAV antibody fragments can measure integrin turnover rates within adhesion sites. Combining these approaches with genetic manipulation (CRISPR/Cas9, siRNA) provides comprehensive understanding of ITGAV's specific contributions to cell migration and adhesion processes in physiological and pathological contexts .

How do ITGAV antibodies contribute to cancer research?

ITGAV antibodies have become instrumental tools in cancer research, enabling investigations across multiple aspects of tumor biology. In tumor tissue analysis, immunohistochemistry with validated ITGAV antibodies (dilutions 1:50-1:200) allows evaluation of expression patterns and correlation with clinical outcomes across various cancer types. ITGAV upregulation has been observed in multiple malignancies, with expression often correlating with invasive potential. For mechanistic studies, ITGAV antibodies facilitate the investigation of angiogenesis pathways—ITGAV/β3 heterodimers play crucial roles in endothelial cell migration and vessel formation, processes targetable with function-blocking antibodies in both in vitro tube formation assays and in vivo models.

In tumor microenvironment research, ITGAV antibodies enable characterization of cancer-associated fibroblasts and immune cell interactions through multicolor flow cytometry and immunofluorescence. For metastasis research, ITGAV antibodies can track changes in adhesion properties during epithelial-mesenchymal transition and identify key interactions with the extracellular matrix components that facilitate invasion. Therapeutically, ITGAV-targeting antibodies serve as both research tools and potential therapeutic agents—function-blocking antibodies can inhibit tumor growth and angiogenesis in preclinical models, while antibody-drug conjugates deliver cytotoxic payloads specifically to ITGAV-expressing cells. Additionally, antibodies recognizing specific conformational states of ITGAV provide insights into integrin activation status during cancer progression. These diverse applications make ITGAV antibodies essential components of the cancer research toolkit, advancing our understanding of tumor biology and identifying new therapeutic strategies .

How do I troubleshoot weak or absent signals when using ITGAV antibodies?

Troubleshooting weak or absent signals when using ITGAV antibodies requires systematic evaluation of each experimental step. First, verify ITGAV expression in your sample type through literature review or preliminary RT-PCR—remember that expression levels vary significantly across tissues and cell lines. For Western blotting, ensure adequate protein loading (40-50 μg total protein) and confirm successful transfer by reversible Ponceau S staining. Try multiple protein extraction methods as ITGAV's membrane localization may require specialized lysis buffers containing mild detergents (1% NP-40 or 0.5% Triton X-100) to efficiently solubilize the protein. Optimize antibody concentration—while manufacturers recommend 0.01-2 μg/mL for Western blotting, signal enhancement may require concentrations at the higher end of this range or extended incubation periods (overnight at 4°C).

For immunohistochemistry/immunofluorescence approaches, test different antigen retrieval methods—ITGAV epitopes may require heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for optimal exposure. When signals remain weak, implement signal amplification systems such as avidin-biotin complex (ABC), tyramide signal amplification (TSA), or polymer-based detection systems. If the problem persists, consider antibody/epitope accessibility issues—try alternative fixation protocols or reduce fixation time, as overfixation can mask epitopes. Finally, verify antibody performance using positive control samples known to express ITGAV at high levels (such as A549 or A431 cell lines) and consider testing alternative ITGAV antibodies that recognize different epitopes, as protein modifications or splice variants may affect epitope availability in your specific samples .

What factors can lead to non-specific binding with ITGAV antibodies and how can they be minimized?

Non-specific binding with ITGAV antibodies can compromise experimental interpretation and can arise from multiple sources. Primary causes include insufficient blocking, inappropriate antibody concentration, and sample-specific factors. To minimize these issues, implement a comprehensive optimization strategy. Begin with thorough blocking—extend blocking time to 1-2 hours using 5% non-fat milk in TBS for Western blotting or 10% normal serum from the same species as the secondary antibody for immunostaining. Consider alternative blocking agents such as 1% BSA or commercial blocking buffers if background persists. Optimize antibody dilutions through systematic titration; excessive antibody concentration is a common cause of non-specific binding—start at the upper end of the recommended range (e.g., 1:500 for Western blotting) and increase dilution if background remains high.

For immunohistochemistry, include additional blocking steps to address tissue-specific factors—incubate sections with avidin/biotin blocking solutions when using biotin-based detection systems to block endogenous biotin, and use hydrogen peroxide treatment to quench endogenous peroxidase activity. To reduce hydrophobic interactions, include 0.1-0.3% Triton X-100 or 0.05% Tween-20 in antibody diluents. When working with tissues containing high levels of endogenous immunoglobulins (such as spleen or lymph nodes), pre-incubate sections with unconjugated Fab fragments to block endogenous Ig. For all applications, increase the number and duration of wash steps using TBS-T (0.1% Tween-20) to remove unbound antibody effectively. Finally, validate results using isotype control antibodies at the same concentration as the primary antibody to distinguish between specific signal and background. This systematic approach will significantly improve signal-to-noise ratio in ITGAV detection across various experimental platforms .

How can I accurately quantify ITGAV expression levels across different experimental conditions?

Accurate quantification of ITGAV expression across experimental conditions requires careful consideration of methodological approaches and appropriate controls. For Western blot quantification, use housekeeping proteins (β-actin, GAPDH) or total protein staining (Ponceau S, SYPRO Ruby) as loading controls. Perform densitometric analysis using software like ImageJ, normalizing ITGAV band intensity to the corresponding loading control. To ensure measurements fall within the linear range of detection, prepare a standard curve using known quantities of recombinant ITGAV protein or serial dilutions of a positive control sample. For more precise quantification, consider capillary-based immunoassays (Wes, Jess systems) which offer higher reproducibility and wider dynamic range than traditional Western blotting.

Flow cytometry provides quantitative analysis of ITGAV surface expression at the single-cell level—use calibration beads with known antibody binding capacity to convert fluorescence intensity to absolute numbers of ITGAV molecules per cell. For immunohistochemical quantification, employ digital image analysis software to measure staining intensity and calculate H-scores or quickscores that incorporate both staining intensity and percentage of positive cells. Real-time qPCR serves as a complementary approach to validate protein-level changes by measuring ITGAV mRNA expression, using validated reference genes for normalization. For absolute quantification, ELISA or bead-based multiplex assays offer high sensitivity—develop a standard curve using recombinant ITGAV protein and ensure samples fall within the linear range of the assay. Regardless of the method chosen, biological replicates (minimum n=3) and technical replicates are essential for statistical validation, along with appropriate statistical tests (t-test, ANOVA with post-hoc tests) to determine significance between experimental conditions .

What are the latest advances in ITGAV antibody-based research for studying disease mechanisms?

Recent advances in ITGAV antibody-based research have expanded our understanding of disease mechanisms across multiple pathological conditions. Single-cell analysis techniques combining ITGAV antibodies with mass cytometry (CyTOF) or single-cell RNA-sequencing have revealed previously unrecognized heterogeneity in ITGAV expression within tumor microenvironments and inflammatory tissues. These approaches have identified distinct cellular subpopulations with unique ITGAV expression signatures that correlate with disease progression and treatment response. Conformation-specific antibodies that selectively recognize active versus inactive ITGAV conformations have enabled researchers to monitor integrin activation states in real-time, providing insights into the dynamic regulation of cell-matrix interactions during disease processes.

In neurodegenerative disease research, ITGAV antibodies have revealed critical roles for this integrin in microglial function and neuroinflammatory responses. Multiplex imaging approaches combining ITGAV antibodies with other markers have mapped the spatial relationships between ITGAV-expressing cells and pathological features in tissues, enhancing our understanding of disease microenvironments. For therapeutic development, antibody engineering has produced ITGAV-targeted bispecific antibodies that simultaneously engage immune effector cells, representing a promising immunotherapeutic strategy for ITGAV-overexpressing tumors. Additionally, antibody-based proximity labeling techniques (BioID, APEX) have identified novel ITGAV-interacting proteins in disease contexts, expanding the network of potential therapeutic targets. These methodological advances collectively drive forward our understanding of ITGAV's contributions to disease pathogenesis and offer new approaches for diagnostic and therapeutic intervention .

How are ITGAV antibodies being used in the development of targeted therapies?

ITGAV antibodies are increasingly central to the development of targeted therapies across multiple disease indications. Function-blocking antibodies that disrupt ITGAV interactions with extracellular matrix components have shown promise in preclinical cancer models by inhibiting tumor angiogenesis, growth, and metastasis. These antibodies typically target the RGD-binding site of ITGAV/β3 or ITGAV/β5 heterodimers, preventing engagement with ligands like vitronectin and fibronectin. Antibody-drug conjugates (ADCs) represent another therapeutic strategy—ITGAV antibodies serve as targeting vehicles to deliver cytotoxic payloads specifically to ITGAV-overexpressing cells, potentially reducing off-target effects compared to conventional chemotherapy.

For immune modulation, ITGAV-targeting bispecific antibodies that simultaneously engage T cells or NK cells can redirect immune responses against ITGAV-expressing tumor cells. Notably, ITGAV antibodies have applications beyond oncology—in fibrotic diseases, antibodies targeting ITGAV/β6 have shown efficacy in reducing TGF-β activation and subsequent fibrosis in lung, liver, and kidney models. In the cardiovascular field, antibodies targeting ITGAV/β3 on platelets have demonstrated antithrombotic effects. Advanced antibody engineering approaches, including site-specific conjugation methods and optimized Fc domains, are enhancing the pharmacokinetic properties and effector functions of ITGAV-targeting therapeutics. Currently, several ITGAV-targeting antibodies and antibody derivatives are in clinical development phases, with preliminary results suggesting both safety and potential efficacy in selected patient populations. This expanding therapeutic landscape highlights the clinical relevance of ITGAV biology and the translational potential of antibody-based approaches targeting this integrin .

What emerging technologies are enhancing the specificity and utility of ITGAV antibodies?

Emerging technologies are significantly enhancing both the specificity and utility of ITGAV antibodies across research and clinical applications. Single B-cell cloning approaches combined with next-generation sequencing have enabled the identification and production of ultra-specific ITGAV antibodies with defined epitope binding characteristics. These technologies allow researchers to map the complete epitope landscape of ITGAV and develop antibodies targeting functionally relevant regions with unprecedented precision. Structural biology techniques including cryo-electron microscopy in combination with antibody fragment analysis have provided atomic-level insights into antibody-ITGAV interactions, guiding rational antibody engineering efforts to enhance specificity and affinity.

Synthetic biology approaches using phage display and yeast display technologies have generated fully human ITGAV antibodies with optimized properties for both research and therapeutic applications. These display platforms, coupled with directed evolution strategies, have yielded antibodies with 10-100 fold improvements in affinity and specificity compared to conventional hybridoma-derived antibodies. For advanced research applications, nanobodies and single-domain antibodies against ITGAV offer superior tissue penetration and epitope access due to their smaller size, enabling visualization of ITGAV in previously inaccessible cellular compartments. Additionally, antibody fragments with site-specific conjugation points for fluorophores, nanoparticles, or other functional moieties allow precise control over labeling stoichiometry and orientation, enhancing signal-to-noise ratios in imaging applications.

Computational approaches including machine learning algorithms are now being employed to predict optimal ITGAV epitopes for antibody generation and to model antibody-antigen interactions before experimental validation. These in silico methods accelerate development timelines and reduce the resources required for antibody optimization. Collectively, these technological advances are transforming ITGAV antibodies from simple detection reagents into sophisticated tools with enhanced specificity, expanded functionality, and increased potential for both basic research and clinical translation .