EGFL7 Antibody, Biotin conjugated

What is EGFL7 and what are its primary biological functions?

EGFL7 is a secreted protein that regulates vascular tubulogenesis in vivo. It plays a dual role by inhibiting platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration while promoting endothelial cell adhesion to the extracellular matrix and angiogenesis . EGFL7 is highly expressed during embryonic vascular development but becomes downregulated in adult endothelium except during tissue repair, regeneration, or pathological conditions such as tumor growth. In cancer contexts, elevated EGFL7 expression has been linked to increased tumor dissemination and reduced survival in various malignancies including glioblastoma .

How does a biotin-conjugated EGFL7 antibody differ from unconjugated versions?

Biotin-conjugated EGFL7 antibodies contain biotin molecules chemically attached to the antibody structure, typically to amino acid residues without affecting the antigen-binding site. This biotin tag enables high-affinity binding to streptavidin or avidin, creating a versatile detection system. Unlike unconjugated antibodies that require labeled secondary antibodies for detection, biotin-conjugated antibodies can be directly detected using streptavidin-conjugated reporter molecules (enzymes, fluorophores, etc.) . For EGFL7 detection, this conjugation improves sensitivity and provides flexibility across multiple applications while maintaining the antibody's binding specificity to the target epitope (such as amino acids 73-198 of human EGFL7) .

What are the primary research applications for biotin-conjugated EGFL7 antibodies?

Biotin-conjugated EGFL7 antibodies are valuable in multiple research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): These antibodies function as detection antibodies in sandwich ELISA systems, working with unconjugated capture antibodies to detect EGFL7 in biological samples .

• Immunohistochemistry (IHC): For visualizing EGFL7 expression patterns in tissue sections, particularly in tumor vasculature studies where approximately 25-40% of intratumoral blood vessels express EGFL7 .

• Western Blotting: When used with streptavidin-HRP detection systems for analyzing EGFL7 expression in cell and tissue lysates. EGFL7 is typically detected in human placenta lysates and HUVEC (human umbilical vein endothelial cell) whole cell lysates, but not in Jurkat cells (human T cell leukemia lymphocytes) .

• Multiplex Immunoassays: For co-localization studies with other vascular markers to understand EGFL7's role in angiogenesis.

• Flow Cytometry: For detecting and quantifying EGFL7 expression in cell populations.

Which cell and tissue types typically express EGFL7?

EGFL7 expression follows specific patterns across different cell and tissue types:

• Primary expression site: Vascular endothelial cells, particularly during developmental angiogenesis and in actively remodeling vessels .

• Normal tissues: High expression during embryonic vascular development with subsequent downregulation in adult quiescent vessels. Expression can be reactivated during wound healing and tissue regeneration .

• Tumor contexts: EGFL7 is predominantly expressed in tumor blood vessels rather than tumor cells themselves. In glioma specimens, large blood vessels with distinct lumens show stronger EGFL7 signal .

• Positive expression models: Human placenta lysates and HUVEC cells serve as reliable positive controls for EGFL7 expression studies .

• Negative expression models: Jurkat cells (human T cell leukemia lymphocytes) have been documented as negative controls for EGFL7 expression .

• Expression regulation: In some cancer cells, the EGFL7 promoter can be epigenetically silenced through methylation. Treatment with DNA methyltransferase inhibitors (5-Aza-dC) and histone deacetylase inhibitors (PBA) can reactivate expression .

What is the optimal protocol for using biotin-conjugated EGFL7 antibodies in sandwich ELISA?

The optimal sandwich ELISA protocol for EGFL7 detection using biotin-conjugated antibodies involves several critical steps:

• Coating: Pre-coat 96-well plates with unconjugated anti-EGFL7 capture antibody (typical concentration: 1-5 μg/ml in carbonate buffer, pH 9.6) overnight at 4°C .

• Blocking: Block non-specific binding sites using appropriate blocking buffer (typically 1-3% BSA or 5% non-fat dry milk in TBST) for 1-2 hours at room temperature .

• Sample addition: Add standards (recombinant EGFL7 protein) and test samples diluted in appropriate buffer, then incubate for 1-2 hours at room temperature or overnight at 4°C for increased sensitivity .

• Detection antibody: Apply biotin-conjugated anti-EGFL7 antibody at the manufacturer's recommended dilution (typically 1:1000 to 1:5000). This antibody binds to EGFL7 captured by the coated antibody on the plate .

• Signal development: Add HRP-Streptavidin conjugate that binds to the biotin on the detection antibody, followed by TMB substrate solution. The enzymatic reaction produces a blue color that turns yellow after adding stop solution .

• Measurement: Read optical density at 450nm using a microplate reader. EGFL7 concentration in samples is calculated using a standard curve, with concentration proportional to OD450 values .

For optimal results, all incubation steps should be followed by thorough washing (typically 3-5 washes with PBST or TBST) to remove unbound reagents.

What are the critical controls for validating EGFL7 antibody specificity?

Validating the specificity of biotin-conjugated EGFL7 antibodies requires multiple control strategies:

• Positive controls:

  • Human placenta lysate and HUVEC (human umbilical vein endothelial cell) whole cell lysate, which reliably express EGFL7 .

  • Recombinant human EGFL7 protein at known concentrations for standard curves.

• Negative controls:

  • Jurkat cells, which have been documented as reliable negative controls for EGFL7 expression .

  • Secondary-only controls (omitting the primary EGFL7 antibody).

  • Isotype controls (using irrelevant biotin-conjugated antibodies of the same isotype).

• Antibody validation approaches:

  • Multiple antibody comparison: Using different anti-EGFL7 antibodies from various sources that should yield 100% overlapping staining patterns in positive tissues .

  • Peptide competition: Pre-incubation with immunizing peptide or recombinant EGFL7 protein should abolish specific staining.

  • Genetic validation: Comparing staining in wild-type versus EGFL7 knockdown/knockout models.

• Application-specific controls:

  • For Western blotting: Molecular weight verification (EGFL7 bands should appear consistent with literature reports) .

  • For IHC: Include both positive (placenta) and negative (Jurkat cells) tissue controls in each experiment .

  • For ELISA: Include complete standard curves and internal quality controls in each assay .

These comprehensive controls ensure that observed signals truly represent EGFL7 rather than non-specific binding or background.

How should samples be prepared for optimal EGFL7 detection in various applications?

Optimal sample preparation varies by application and sample type:

For Immunohistochemistry (IHC)/Immunofluorescence (IF):

• FFPE tissues: After deparaffinization and rehydration, heat-induced epitope retrieval is critical. Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) under pressure cooking conditions typically yields optimal results for EGFL7 .

• Frozen sections: Fix with cold acetone (10 minutes) or 4% paraformaldehyde (10-15 minutes). For paraformaldehyde-fixed samples, a permeabilization step with 0.1-0.3% Triton X-100 may improve antibody accessibility.

• Blocking steps: Critical for both sample types:

  • Block endogenous peroxidase with 0.3% H₂O₂

  • Block endogenous biotin using commercial biotin-blocking kits to prevent false positives

  • Block non-specific protein binding with 5-10% normal serum from the same species as the secondary reagent

For Western Blotting:

• Cell lysates: Lyse cells in RIPA buffer supplemented with protease inhibitors. For endothelial cells (like HUVECs), quick processing is essential as EGFL7 can be rapidly degraded .

• Tissue homogenization: Homogenize tissues in appropriate buffer with protease inhibitors, followed by centrifugation to remove debris.

• Protein quantification: Standardize loading (typically 20 μg per lane for HUVEC and placenta samples) .

• Blocking conditions: 5% non-fat dry milk in TBST has been successfully used for EGFL7 Western blots .

For ELISA:

• Serum/plasma: Collect in appropriate anticoagulant tubes and separate promptly. Multiple freeze-thaw cycles should be avoided.

• Cell culture supernatants: Collect and centrifuge to remove cellular debris. Concentrate if necessary using centrifugal filter units.

• Cell/tissue extracts: Prepare using extraction buffers compatible with the ELISA kit, typically containing protease inhibitors .

Regardless of application, all samples should be processed promptly and stored appropriately (-80°C for long-term storage) to preserve EGFL7 integrity.

What troubleshooting approaches are recommended for high background issues?

High background signal is a common challenge when working with biotin-conjugated antibodies. For EGFL7 detection, several troubleshooting approaches are recommended:

• Endogenous biotin blocking:

  • Implement specific biotin/avidin blocking steps before applying biotin-conjugated EGFL7 antibodies.

  • Use commercial endogenous biotin blocking kits to sequentially block endogenous biotin, avidin, and biotin-binding proteins.

• Optimization of antibody concentration:

  • Titrate the biotin-conjugated EGFL7 antibody to determine the optimal working dilution.

  • For Western blotting, a 1:1000 dilution has been successfully used with 5% non-fat dry milk in TBST as blocking agent .

  • For ELISA, follow the manufacturer's recommended dilution, but consider testing a range above and below this concentration.

• Improve washing procedures:

  • Increase the number and duration of washes between steps.

  • Ensure complete removal of wash buffer between steps.

  • Consider adding low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers.

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, commercial blockers).

  • Increase blocking time and concentration (5% NFDM/TBST has proven effective for EGFL7 Western blots) .

• Reduce streptavidin-conjugate concentration:

  • Dilute streptavidin-HRP more extensively if high background persists.

  • Consider shorter incubation times for the streptavidin detection step.

• Tissue-specific approaches:

  • For tissues with high endogenous biotin (liver, kidney, brain), consider alternative detection methods or implement rigorous biotin blocking.

  • For highly vascularized tissues, ensure proper controls as EGFL7 is naturally expressed in blood vessels.

• Control experiments:

  • Include secondary-only controls to assess non-specific binding.

  • For critical experiments, consider comparing results with non-biotinylated EGFL7 antibodies.

Implementing these approaches systematically can help identify and resolve background issues in EGFL7 detection assays.

How can EGFL7 antibodies be used to study tumor angiogenesis mechanisms?

Biotin-conjugated EGFL7 antibodies provide powerful tools for investigating tumor angiogenesis through several approaches:

• Vascular phenotyping:

  • Multiplex immunohistochemistry combining EGFL7 with endothelial markers (CD31, CD34) and pericyte markers (NG2, PDGFRβ) to assess vessel maturity.

  • Quantitative analysis of EGFL7-positive vessels in relation to total vessel density.

  • Studies have shown that approximately 25-40% of intratumoral blood vessels in glioma specimens express EGFL7, with larger vessels showing stronger signals .

• Functional correlations:

  • Relationship between EGFL7 expression and vessel permeability/leakiness.

  • Research has demonstrated that EGFL7-positive vessels in experimental gliomas showed greater maturity with better pericyte and smooth muscle cell coverage, and reduced leakage of contrast agents during MRI assessment .

• Therapeutic targeting:

  • Monitoring vascular responses to anti-EGFL7 blocking antibodies.

  • Evaluation of combination approaches that pair EGFL7 inhibition with other therapies.

  • Experimental studies have shown that EGFL7-inhibition using specific blocking antibodies reduced vascularization of experimental gliomas and increased survival, particularly when combined with anti-VEGF therapy and temozolomide .

• Molecular mechanism investigations:

  • Using biotin-conjugated antibodies for co-immunoprecipitation to identify EGFL7 binding partners.

  • Studies have identified that EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis and tumor growth .

• Clinical correlations:

  • Association between EGFL7 vascular expression patterns and patient outcomes.

  • Research has linked increased levels of EGFL7 to reduced median survival time in glioblastoma patients .

These approaches help elucidate the role of EGFL7 in tumor angiogenesis and identify potential therapeutic strategies targeting EGFL7-mediated pathways.

What approaches can be used to investigate EGFL7's interaction with integrin α5β1?

The interaction between EGFL7 and integrin α5β1 represents a critical aspect of EGFL7's pro-angiogenic function. Several methodological approaches can be employed to study this interaction:

• Co-immunoprecipitation (Co-IP):

  • Using biotin-conjugated EGFL7 antibodies to pull down protein complexes, followed by immunoblotting for integrin α5β1.

  • Reciprocal approach: immunoprecipitating integrin α5β1 and probing for EGFL7.

  • These assays can determine whether EGFL7 and integrin α5β1 physically interact in endothelial cells.

• Proximity ligation assay (PLA):

  • A technique that detects protein-protein interactions in situ with single-molecule resolution.

  • Combining anti-EGFL7 and anti-integrin α5β1 antibodies with oligonucleotide-conjugated secondary antibodies.

  • When proteins are in close proximity (<40 nm), oligonucleotides can be ligated and amplified, generating fluorescent spots that represent interaction events.

• Functional binding assays:

  • Solid-phase binding assays using purified proteins to determine direct binding.

  • Cell adhesion assays comparing adhesion to EGFL7-coated surfaces with and without integrin α5β1 blocking antibodies.

  • These approaches can demonstrate that EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis .

• Mutational analysis:

  • Generation of EGFL7 variants with modifications in potential integrin-binding domains.

  • Assessment of these variants' ability to bind integrin α5β1 and promote endothelial functions.

  • Correlation with functional outcomes in angiogenesis assays.

• Visualization techniques:

  • Double immunofluorescence staining for EGFL7 and integrin α5β1 in tissue sections.

  • Confocal microscopy to assess co-localization patterns in normal versus tumor vessels.

These experimental approaches provide complementary information about how EGFL7 enhances integrin α5β1 expression and function to promote angiogenesis and tumor growth, as demonstrated in research with glioblastoma models .

How should EGFL7 expression patterns be interpreted in relation to miR-126/126*?

The EGFL7 gene contains the intronic microRNAs miR-126 and miR-126* (its complement), creating a complex regulatory relationship that requires careful interpretation:

Understanding the distinction between EGFL7 protein expression and its intronic miRNAs is essential for correctly interpreting experimental results and developing targeted therapeutic approaches.

What combination therapy approaches show promise when targeting EGFL7?

Research indicates that combining EGFL7 inhibition with other therapeutic modalities offers enhanced efficacy, particularly in highly vascularized tumors:

• EGFL7 inhibition with anti-VEGF therapy:

  • Rationale: Targeting different aspects of the angiogenic process simultaneously.

  • Experimental results: Studies using specific EGFL7 blocking antibodies showed that combining EGFL7 inhibition with anti-VEGF therapy produced greater effects than either treatment alone in reducing vascularization of experimental gliomas .

• Triple combination with chemotherapy:

  • The most promising approach in experimental glioma models involved combining:

    • EGFL7 inhibition (using blocking antibodies)

    • Anti-VEGF therapy

    • Temozolomide (standard chemotherapeutic agent for glioblastoma)

  • This triple combination significantly increased the lifespan of treated animals compared to single or dual therapy approaches .

• Mechanisms of combination effects:

  • Vascular normalization: EGFL7 inhibition led to blood vessels that were more mature as determined by pericyte and smooth muscle cell coverage.

  • Reduced vessel permeability: These normalized vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent.

  • Improved drug delivery: Normalized vasculature may enhance chemotherapeutic penetration into tumors.

• Tumor-specific considerations:

  • Particularly effective in glioblastoma models, where EGFL7 expression in tumor vessels has been well-documented.

  • May be applicable to other highly vascularized tumors where EGFL7 expression has been detected (colon, gastric, breast, kidney, liver cancers) .

• Future directions:

  • Development of humanized anti-EGFL7 antibodies for clinical testing.

  • Exploration of additional combination partners beyond anti-VEGF therapy.

  • Investigation of sequencing effects (concurrent versus sequential administration).

These findings suggest that combinatorial regimens incorporating EGFL7 inhibition may represent novel treatment options for glioblastoma and potentially other EGFL7-expressing malignancies.

How should EGFL7 expression be quantified in immunohistochemical studies?

Quantification of EGFL7 expression in immunohistochemistry requires standardized approaches tailored to its predominantly vascular expression pattern:

• Vessel-based quantification:

  • Count of EGFL7-positive versus total vessels in representative fields.

  • Expression index calculation: percentage of blood vessels staining positive for EGFL7.

  • Research has shown that approximately 25-40% of intratumoral blood vessels stain positive for EGFL7 in glioma specimens .

• Intensity scoring systems:

  • Semi-quantitative scale (0-3): 0 (negative), 1 (weak), 2 (moderate), 3 (strong).

  • Combined scoring: multiply intensity by percentage of positive vessels.

  • Digital image analysis using software to objectively quantify staining intensity.

• Vessel subtype analysis:

  • Large blood vessels with distinct lumens typically show stronger EGFL7 signals compared to smaller vessels .

  • Correlation with vessel maturity markers (pericyte coverage, basement membrane components).

  • Spatial distribution analysis (tumor core versus invasive margin).

• Control-normalized quantification:

  • Express results relative to positive controls (placenta samples) run in parallel.

  • Include negative controls (Jurkat cells) to establish background thresholds .

• Multi-observer validation:

  • Independent scoring by multiple trained observers.

  • Calculation of inter-observer reliability metrics.

  • Consensus scoring for discrepant cases.

• Standardized reporting:

  • Clearly defined methodology including antibody details, dilution, detection system.

  • Consistent threshold criteria for positivity.

  • Representative images showing various expression levels.

These approaches provide robust quantification of EGFL7 expression while accounting for its biological distribution primarily in vascular structures.

What are the expected differences in EGFL7 expression between normal and tumor vasculature?

EGFL7 expression exhibits distinct patterns when comparing normal and tumor vasculature:

• Normal adult vasculature:

  • Generally low to undetectable EGFL7 expression in quiescent adult blood vessels.

  • Expression is physiologically downregulated in the endothelium of adults except during tissue repair or regeneration .

  • When present, expression is primarily restricted to endothelial cells.

• Developmental and regenerative contexts:

  • High expression in embryonic and developing vasculature.

  • Temporary upregulation during wound healing and tissue regeneration processes .

  • Associated with active vascular remodeling phases.

• Tumor vasculature characteristics:

  • Significantly elevated expression compared to normal adult vessels.

  • Heterogeneous pattern with approximately 25-40% of intratumoral blood vessels expressing EGFL7 in glioma specimens .

  • Large blood vessels with distinct lumens typically show stronger EGFL7 signals than smaller vessels .

  • Expression predominantly in vascular endothelial cells rather than tumor cells themselves in glioblastoma .

• Clinical correlations:

  • Increased EGFL7 levels have been linked to increased dissemination of several tumor types .

  • Associated with reduced median patient survival time in glioblastoma patients .

  • Elevated expression documented across multiple tumor types including colon, gastric, breast, kidney, liver, and brain tumors .

• Functional implications:

  • In tumors, EGFL7-positive vessels may show distinct functionality.

  • Research indicates that EGFL7 influences vessel maturity, as determined by pericyte and smooth muscle cell coverage.

  • EGFL7 expression affects vessel permeability, with implications for contrast agent extravasation in imaging studies .

Understanding these expression differences provides valuable context for interpreting EGFL7 staining patterns in normal and pathological specimens.

What methodological approaches can improve detection of low EGFL7 expression levels?

Detecting low levels of EGFL7 expression requires optimized methodological approaches:

• Enhanced sample preparation:

  • Optimal fixation conditions to preserve EGFL7 epitopes.

  • Improved antigen retrieval protocols (extended heat-induced epitope retrieval).

  • Careful blocking of endogenous biotin to reduce background and improve signal-to-noise ratio.

• Signal amplification systems:

  • Tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence.

  • Enhanced chemiluminescence (ECL) substrates for Western blotting.

  • In ELISA applications, consider using poly-HRP streptavidin conjugates and extended substrate development times .

• Antibody optimization:

  • Extended primary antibody incubation (overnight at 4°C).

  • Optimized antibody concentration through careful titration experiments.

  • Use of antibodies targeting highly conserved epitopes (e.g., amino acids 73-198 of EGFL7) .

• Detection system considerations:

  • For Western blotting, PVDF membranes (higher protein binding capacity than nitrocellulose).

  • Longer exposure times for imaging (3-minute exposure has been used successfully for EGFL7 Western blots) .

  • Highly sensitive CCD camera systems for digital imaging.

• Enrichment approaches:

  • Immunoprecipitation to concentrate EGFL7 before detection.

  • For tissue analysis, consider laser capture microdissection to isolate vascular structures.

  • In cell culture studies, concentrate conditioned media for secreted EGFL7