FZD7 Antibody, HRP conjugated

What is FZD7 and why is it an important research target?

FZD7 (Frizzled-7) is a seven-transmembrane receptor that plays a critical role in the Wnt signaling pathway. It has emerged as a significant research target due to its overexpression in multiple cancer types, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival . FZD7 shows increased protein expression in ovarian tumors compared to normal ovarian tissue, suggesting its potential as a tumor-specific antigen . Additionally, FZD7 has been implicated in the regulation of pluripotency in human pluripotent stem cells, with its downregulation accompanying differentiation and exit from the pluripotent stem cell state .

What are the key specifications of commercially available FZD7 antibody, HRP conjugated?

Commercial FZD7 antibody, HRP conjugated products typically have the following specifications:

Table based on product information from search result .

How does the specificity of FZD7 antibodies compare to other FZD family receptors?

The specificity of FZD7 antibodies is a critical concern given the high conservation among FZD proteins. Advanced FZD7 antibodies have been developed to specifically target the 'neck' region between the cysteine-rich domain (CRD) and the first transmembrane domain of FZD7, which contains distinguishing sequences .

Research has confirmed that well-characterized FZD7 antibodies, such as F7-Ab, do not cross-react with the other nine human FZD receptors (FZD1-6, 8-10) . The epitope specificity has been mapped to an eight amino acid stretch, with a key leucine residue at position 188 in human FZD7 (hFZD7) being critical for recognition . Notably, a single amino acid change from leucine to proline at position 188 renders hFZD7 non-reactive to some antibodies, while the corresponding amino acid change in mouse Fzd7 (mFzd7) from proline to leucine can restore reactivity .

What are the optimal conditions for using FZD7 antibody, HRP conjugated in Western blot analyses?

For optimal Western blot analysis using FZD7 antibody, HRP conjugated:

• Sample Preparation:

  • Use either reducing or non-reducing conditions depending on the specific antibody recommendations. Some FZD7 antibodies perform better under non-reducing conditions for detecting native protein conformations .

  • Include appropriate positive controls, such as lysates from cells known to express FZD7 (e.g., HepG2 cell line) .

• Gel Electrophoresis:

  • Use a 10-12% SDS-PAGE gel for optimal separation.

  • Load 20-50 μg of total protein per lane.

• Transfer and Blocking:

  • Transfer to PVDF membrane (preferred over nitrocellulose for some FZD7 antibodies).

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Antibody Incubation:

  • Dilute primary FZD7-HRP antibody to 0.25-0.5 μg/mL in blocking buffer.

  • Incubate overnight at 4°C for optimal results.

  • Since the antibody is HRP-conjugated, no secondary antibody is required.

• Detection:

  • Wash membrane thoroughly with TBST (3-5 times, 5 minutes each).

  • Develop using ECL substrate.

  • FZD7 typically appears as a band at approximately 65 kDa .

How should researchers optimize FZD7 antibody, HRP conjugated for ELISA applications?

For ELISA applications using FZD7 antibody, HRP conjugated:

• Plate Coating:

  • For direct ELISA: Coat microplate wells with recombinant FZD7 protein at 1-10 μg/mL in carbonate buffer (pH 9.6) overnight at 4°C.

  • For sandwich ELISA: Coat with a capture antibody recognizing a different epitope of FZD7 than the HRP-conjugated detection antibody.

• Blocking:

  • Block with 1-5% BSA or casein in PBS for 1-2 hours at room temperature.

• Sample and Antibody Incubation:

  • For direct ELISA: Add serially diluted FZD7-HRP antibody (starting from 1:1000 dilution).

  • For sandwich ELISA: Add samples containing FZD7, followed by FZD7-HRP conjugated antibody.

  • Incubate for 1-2 hours at room temperature or overnight at 4°C.

• Optimization Parameters:

  • Test various antibody dilutions to determine optimal signal-to-noise ratio.

  • Optimize incubation times and temperatures.

  • Consider adding 0.05% Tween-20 to reduce non-specific binding.

• Detection:

  • Add TMB substrate and measure absorbance at 450 nm after stopping the reaction with 2N H₂SO₄.

  • Include a standard curve using recombinant FZD7 protein for quantification.

What controls should be included when using FZD7 antibody, HRP conjugated in experiments?

Proper controls are essential for validating results with FZD7 antibody, HRP conjugated:

• Positive Controls:

  • Cell lines with verified FZD7 expression (e.g., HepG2, MDA-MB-231 for TNBC studies) .

  • Recombinant human FZD7 protein.

  • Tissue samples known to express FZD7 (e.g., embryonic mouse gastrointestinal tract) .

• Negative Controls:

  • FZD7 knockout cell lines (e.g., HEK293T cells with FZD7 loss-of-function mutations) .

  • Cells with minimal FZD7 expression.

  • Isotype control antibody (rabbit IgG-HRP) to assess non-specific binding.

• Specificity Controls:

  • Pre-absorption with recombinant FZD7 protein to confirm antibody specificity.

  • Testing cross-reactivity with other FZD family members, especially FZD1, FZD2, and FZD5 which share higher sequence homology .

• Technical Controls:

  • No primary antibody control (for direct HRP detection systems).

  • Gradient of antigen concentration to verify dose-dependent response.

• Genetic Validation:

  • siRNA or shRNA knockdown of FZD7 to demonstrate antibody specificity through reduced signal intensity .

How can FZD7 antibodies be used to investigate Wnt signaling pathways in cancer research?

FZD7 antibodies are valuable tools for investigating Wnt signaling in cancer research through multiple approaches:

• Signaling Inhibition Studies:

  • FZD7 antibodies can competitively bind FZD7 receptors to block extracellular Wnt activation, leading to the destabilization of β-catenin and decreased Axin2 expression . This provides insight into the dependency of cancer cells on FZD7-mediated Wnt signaling.

  • Monitoring changes in downstream Wnt targets (β-catenin, Axin2, SP5) after antibody treatment can elucidate pathway dynamics .

• Receptor-Specific Signaling:

  • Using highly specific FZD7 antibodies allows researchers to dissect the contribution of FZD7 versus other FZD receptors in Wnt signaling contexts .

  • Engineered antibody derivatives like F7L6 (a bispecific binder to FZD7 and LRP6) can be used to study the selective activation of FZD7 signaling and its effects on cell differentiation .

• Multivalent Binding Effects:

  • FZD7 antibody-nanoshell conjugates (FZD7-NS) have been shown to inhibit Wnt signaling more effectively than freely delivered antibodies due to multivalent binding effects .

  • These studies have revealed that FZD7-NS have approximately 100-fold increased binding affinity to FZD7 cell surface receptors relative to freely delivered antibodies (effective dissociation constants of 4.83E-10 M versus 1.38E-8 M) .

• Cancer Subtype Characterization:

  • FZD7 antibodies can be used to identify cancer subtypes with elevated FZD7 expression, which correlate with more aggressive phenotypes and poorer patient outcomes, particularly in ovarian cancer .

What are the considerations for developing FZD7 antibody-drug conjugates (ADCs) for targeted cancer therapy?

Developing FZD7 antibody-drug conjugates requires careful consideration of several factors:

• Target Expression and Specificity:

  • FZD7 is overexpressed in multiple solid cancers, particularly in mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma .

  • High specificity antibodies that don't cross-react with other FZD family members are essential to minimize off-target effects .

• Antibody Selection and Engineering:

  • Chimeric human-mouse IgG1 antibodies against FZD7 have been successfully used in ADCs (e.g., septuximab vedotin/F7-ADC) .

  • Key characteristics include:

    • High affinity for human FZD7

    • Minimal cross-reactivity with mouse Fzd7 to enable preclinical safety studies

    • Strategic selection of binding epitopes that facilitate internalization

• Payload Selection:

  • Monomethyl auristatin E (MMAE) has been successfully used as a cytotoxic payload in FZD7-ADCs .

  • This microtubule-inhibiting drug effectively kills ovarian cancer cells when delivered via FZD7-targeting ADCs.

• Conjugation Chemistry:

  • The method of conjugating the drug to the antibody affects:

    • Drug-to-antibody ratio (DAR)

    • Stability in circulation

    • Release kinetics in target cells

• Efficacy and Safety Profile:

  • FZD7-ADCs like septuximab vedotin have demonstrated:

    • Selective binding to human FZD7

    • Potent killing of ovarian cancer cells in vitro

    • Regression of ovarian tumor xenografts in murine models

    • Favorable safety profile in mice expressing humanized Fzd7

• Resistance Mechanisms:

  • Consider potential resistance mechanisms such as:

    • Downregulation of FZD7 expression

    • Alterations in internalization or trafficking pathways

    • Upregulation of drug efflux pumps

How can FZD7 antibodies be incorporated into nanoparticle-based delivery systems to enhance cancer treatments?

FZD7 antibodies can be effectively incorporated into nanoparticle-based delivery systems to enhance cancer treatments through several strategies:

• Antibody-Nanoparticle Conjugation Methods:

  • Direct conjugation via chemical coupling (e.g., maleimide-thiol chemistry, EDC/NHS coupling)

  • Biotin-streptavidin interactions for non-covalent attachment

  • Site-specific conjugation strategies to preserve antibody functionality

• Advantages of FZD7 Antibody-Nanoparticle Systems:

  • Enhanced multivalent binding: FZD7 antibody-nanoshell conjugates (FZD7-NS) demonstrate approximately 100-fold increased binding affinity compared to free antibodies

  • Improved pharmacokinetics and tumor accumulation

  • Dual functionality: simultaneous targeting and Wnt signaling inhibition

• Polymer Nanoparticle Applications:

  • FZD7-targeted polymer nanoparticles encapsulating doxorubicin (DOX) have shown enhanced efficacy against triple-negative breast cancer (TNBC) cells

  • At concentrations of 5-10 μM, these particles inhibit cellular metabolic activity to a greater extent than free DOX

  • The nanoparticles simultaneously deliver chemotherapy and inhibit Wnt signaling by triggering β-catenin phosphorylation

• Gold Nanoshell Applications:

  • FZD7 antibody-functionalized gold nanoshells (FZD7-NS) demonstrate cell-selective binding to TNBC cells but not to healthy cells

  • These conjugates decrease β-catenin stabilization and Axin2 mRNA expression in TNBC cells at much lower antibody doses than freely delivered antibodies

• Design Considerations:

  • Nanoparticle size optimization (typically 50-200 nm) for enhanced permeability and retention effect

  • Surface density of FZD7 antibodies to maximize binding while maintaining colloidal stability

  • Selection of appropriate nanoparticle core material based on the intended application (therapy, imaging, or combination)

What are common challenges when working with FZD7 antibody, HRP conjugated, and how can they be addressed?

Researchers often encounter several challenges when working with FZD7 antibody, HRP conjugated:

• Non-specific Binding:

  • Challenge: High background signal in Western blots or immunostaining.

  • Solution:

    • Increase blocking time or concentration (5% BSA or milk)

    • Add 0.1-0.3% Triton X-100 or Tween-20 to wash buffers

    • Pre-absorb antibody with cell/tissue lysates lacking FZD7 expression

    • Use more stringent washing conditions (increased salt concentration)

• Variable Detection Sensitivity:

  • Challenge: Inconsistent detection of FZD7 across different samples.

  • Solution:

    • Optimize protein extraction methods to preserve membrane proteins (use non-ionic detergents)

    • Consider non-reducing conditions for Western blot to maintain epitope conformation

    • Increase antibody concentration or incubation time

    • Use signal amplification systems compatible with HRP (e.g., tyramide signal amplification)

• Cross-reactivity Issues:

  • Challenge: Potential cross-reactivity with other Frizzled family members.

  • Solution:

    • Validate results with knock-down/knock-out controls

    • Use antibodies specifically validated against all FZD family members (such as F7-Ab)

    • Perform pre-absorption controls with recombinant FZD proteins

• HRP Conjugate Stability Issues:

  • Challenge: Loss of enzymatic activity during storage.

  • Solution:

    • Avoid repeated freeze-thaw cycles

    • Store in smaller aliquots with 50% glycerol at -20°C

    • Add stabilizers like BSA (0.1-1%)

    • Check activity periodically with simple dot blot assays

• Epitope Masking:

  • Challenge: Reduced antibody binding due to post-translational modifications or protein-protein interactions.

  • Solution:

    • Try different protein extraction methods

    • Consider mild denaturation protocols

    • Test alternative FZD7 antibodies targeting different epitopes

How can researchers differentiate between FZD7-mediated canonical and non-canonical Wnt signaling using antibody-based approaches?

Differentiating between FZD7-mediated canonical and non-canonical Wnt signaling requires sophisticated antibody-based approaches:

• Canonical Wnt Signaling Detection:

  • β-catenin Stabilization: Monitor cytoplasmic and nuclear β-catenin accumulation using Western blot after FZD7 activation or inhibition .

  • TCF/LEF Reporter Assays: Use in combination with FZD7 antibodies to measure canonical pathway activation.

  • Downstream Target Expression: Measure canonical Wnt target genes like Axin2, SP5, or c-Myc using RT-qPCR following FZD7 antibody treatment .

• Non-canonical Wnt Signaling Detection:

  • JNK Phosphorylation: Use phospho-specific antibodies to detect JNK activation in the PCP pathway.

  • Calcium Flux: Combine FZD7 antibody treatments with calcium indicators to monitor Wnt/Ca²⁺ pathway activation.

  • RhoA/Rac1 Activation: Use pull-down assays to assess small GTPase activation in non-canonical pathways.

• Pathway-Specific Co-receptor Analysis:

  • Canonical Co-receptors: Evaluate FZD7 co-localization with LRP5/6 using co-immunoprecipitation or proximity ligation assays.

  • Non-canonical Co-receptors: Assess interaction with Ror1/2 or Ryk for non-canonical signaling.

• Specific Antibody Tools:

  • Bispecific Antibodies: Use tools like F7L6 (targeting both FZD7 and LRP6) to specifically activate canonical signaling .

  • Epitope-Specific Antibodies: Different epitope-targeting antibodies may preferentially interfere with canonical versus non-canonical signaling.

• Combinatorial Approaches:

  • Pathway Inhibitors: Combine FZD7 antibodies with canonical (e.g., tankyrase inhibitors) or non-canonical (e.g., JNK inhibitors) pathway blockers.

  • Genetic Tools: Use siRNAs targeting specific downstream effectors in combination with antibody treatments.

What advanced validation techniques ensure the specificity and functionality of FZD7 antibody, HRP conjugated in complex biological systems?

Ensuring specificity and functionality of FZD7 antibody, HRP conjugated in complex biological systems requires rigorous validation approaches:

• Genetic Validation Strategies:

  • CRISPR/Cas9 Knockout Controls: Generate FZD7 knockout cell lines as negative controls for antibody validation .

  • siRNA/shRNA Knockdown: Demonstrate reduced antibody signal corresponding to reduced FZD7 expression levels .

  • Rescue Experiments: Restore FZD7 expression in knockout cells to confirm antibody specificity.

• Epitope Mapping and Mutagenesis:

  • Alanine Scanning Mutagenesis: Identify critical residues for antibody binding, such as the L188 position in human FZD7 .

  • Domain Swapping: Create chimeric constructs between FZD7 and other FZD family members to confirm epitope specificity.

  • Truncation Constructs: Test antibody binding to different FZD7 fragments (e.g., neck region between CRD and first transmembrane domain) .

• Species Cross-reactivity Analysis:

  • Multi-species Testing: Evaluate reactivity against FZD7 orthologs from different species (e.g., human vs. mouse) .

  • Humanized Mouse Models: Test antibody functionality in mice expressing humanized FZD7 .

• Functional Validation Approaches:

  • Signaling Readouts: Confirm antibody's ability to modulate Wnt signaling through β-catenin stabilization assays .

  • Receptor Internalization: Assess antibody-induced receptor internalization using imaging techniques.

  • Therapeutic Efficacy: Evaluate tumor growth inhibition in relevant cancer models .

• Advanced Imaging Techniques:

  • Super-resolution Microscopy: Visualize FZD7 distribution and clustering at nanoscale resolution.

  • Proximity Ligation Assay (PLA): Detect protein-protein interactions involving FZD7 in situ.

  • Live-cell Imaging: Monitor antibody binding dynamics and subsequent cellular responses in real-time.

How is FZD7 antibody research evolving in the development of cancer immunotherapies?

FZD7 antibody research is advancing cancer immunotherapy development through several innovative approaches:

• Dual-Function Antibodies:

  • Development of antibodies that simultaneously block FZD7 signaling and recruit immune effectors through their Fc regions

  • Bispecific antibodies targeting both FZD7 and immune checkpoint receptors (PD-1, CTLA-4) to enhance anti-tumor immunity

• Immune-Engaging Strategies:

  • FZD7 antibody-based bispecific T-cell engagers (BiTEs) that bring T cells in proximity to FZD7-expressing tumor cells

  • CAR-T cell therapies utilizing FZD7-binding domains derived from highly specific antibodies

• Combination Therapies:

  • Synergistic approaches combining FZD7 antibodies with:

    • Immune checkpoint inhibitors

    • Conventional chemotherapies

    • Targeted therapies against complementary oncogenic pathways

• Antibody-Drug Conjugates (ADCs):

  • Advanced FZD7-ADCs like septuximab vedotin that deliver cytotoxic payloads directly to tumor cells

  • Development of ADCs with immunomodulatory payloads that can alter the tumor microenvironment

• Nanoparticle-Based Immunotherapies:

  • FZD7 antibody-functionalized nanoparticles carrying immune-stimulating agents (TLR agonists, cytokines)

  • Combined delivery systems incorporating both tumor-targeting and immune-activating capabilities

• Personalized Approaches:

  • Stratification of patients based on FZD7 expression levels in tumors

  • Development of companion diagnostics to identify patients most likely to respond to FZD7-targeted immunotherapies

What are the newest techniques for characterizing FZD7 antibody binding kinetics and their implications for research applications?

Cutting-edge techniques for characterizing FZD7 antibody binding kinetics are providing deeper insights with significant research implications:

• Surface Plasmon Resonance (SPR) Advancements:

  • Multi-parameter SPR allowing simultaneous measurement of binding to multiple FZD family members

  • Kinetic titration approaches that preserve sample and increase throughput

  • SPR imaging for antibody epitope mapping with higher resolution

• Bio-Layer Interferometry (BLI):

  • Real-time, label-free detection of antibody-FZD7 binding

  • High-throughput screening capabilities for antibody variant libraries

  • Evaluation of binding in complex biological matrices

• Single-Molecule Techniques:

  • Fluorescence Correlation Spectroscopy (FCS) to measure binding in solution at single-molecule resolution

  • Single-molecule FRET to analyze conformational changes upon antibody binding

  • Optical tweezers to measure binding forces at the single-molecule level

• Cell-Based Binding Assays:

  • Advanced flow cytometry with Quantum calibration beads for absolute quantification of binding sites

  • Real-time cell analysis systems measuring binding kinetics on living cells

  • Modified Langmuir isotherm models for measuring effective dissociation constants in cellular contexts

• Computational Approaches:

  • Molecular dynamics simulations to predict antibody-FZD7 interactions

  • Machine learning algorithms to optimize antibody design based on binding kinetics data

  • In silico epitope mapping to guide antibody engineering

• Research Implications:

  • Correlation between binding kinetics and functional outcomes (pathway inhibition, receptor internalization)

  • Identification of optimal antibody properties for specific applications (detection vs. therapeutic)

  • Understanding the relationship between multivalent binding effects and enhanced signaling inhibition

How can researchers leverage FZD7 antibodies to study stem cell differentiation and developmental processes?

Researchers can leverage FZD7 antibodies to investigate stem cell differentiation and developmental processes through several sophisticated approaches:

• Lineage-Specific Differentiation Control:

  • FZD7 plays a prominent role in regulating pluripotency of human pluripotent stem cells (hPS cells) .

  • Selective activation of FZD7 signaling using engineered antibody constructs like F7L6 promotes mesendodermal differentiation .

  • FZD7 antibodies can be used to block specific differentiation pathways, as demonstrated by the inhibition of hematopoietic differentiation when FZD7 signaling is activated .

• Temporal Regulation Studies:

  • Pulse-chase experiments with FZD7 antibodies at different developmental timepoints to determine critical windows for FZD7 signaling.

  • Inducible antibody-based systems to temporarily block or activate FZD7 signaling during specific developmental stages.

• Spatial Organization Analysis:

  • Immunofluorescence with FZD7 antibodies to map receptor distribution during organoid development.

  • Co-localization studies to identify FZD7 interactions with different co-receptors during fate specification.

• Mechanistic Investigations:

  • Combining FZD7 antibodies with downstream signaling analyses to distinguish between canonical and non-canonical pathway contributions to differentiation.

  • Structure-function studies using domain-specific antibodies to identify critical regions of FZD7 in developmental signaling.

• Developmental Biology Applications:

  • FZD7 antibodies can be used to study gastrulation-like processes, as FZD7 activation initiates transcriptional programs similar to those observed during primitive streak formation .

  • Ex vivo embryonic tissue culture with FZD7 antibody treatments to examine effects on morphogenesis.

• Regenerative Medicine Implications:

  • Directed differentiation protocols utilizing stage-specific manipulation of FZD7 signaling.

  • Optimization of definitive endoderm induction for generating insulin-producing cells or hepatocytes.

  • Development of FZD7-based strategies to maintain stemness or drive differentiation depending on therapeutic goals.