EGFR Antibody (PAT2H8AT)

Basic Research Questions

• What is EGFR Antibody (PAT2H8AT) and what is its significance in cancer research?

  EGFR Antibody (PAT2H8AT) is a mouse monoclonal antibody designed to target the human Epidermal Growth Factor Receptor (EGFR), a cell surface protein that regulates cell growth and proliferation. This antibody is derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human EGFR amino acids 424-605 purified from E. coli .

  In cancer research, this antibody serves as a critical tool for studying EGFR-mediated signaling pathways and their role in cancer progression. EGFR dysregulation has been linked to numerous cancers, making it an important target for both research and therapeutic development . By enabling researchers to detect and visualize EGFR expression and localization, this antibody helps advance our understanding of how aberrant EGFR signaling contributes to cancer development and may inform the development of targeted therapies .

• What are the validated applications for EGFR Antibody (PAT2H8AT)?

  EGFR Antibody (PAT2H8AT) has been validated for several research applications:

  • Western Blot (WB): For detecting EGFR protein in cell or tissue lysates

  • Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of EGFR in solution

  • Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing cellular localization of EGFR

  For Western blot applications, researchers should optimize antibody dilutions and blocking conditions to minimize background. For ELISA, the antibody can be used as either a capture or detection antibody depending on assay design. For ICC/IF applications, appropriate fixation and permeabilization protocols should be established to ensure optimal antigen accessibility while preserving cellular structures.

  When designing experiments, include appropriate positive controls (such as A431 cells which overexpress EGFR) and negative controls to validate specificity .

• What are the specific properties of EGFR Antibody (PAT2H8AT)?

  EGFR Antibody (PAT2H8AT) has the following key properties:

  Property	Specification
  Host Species	Mouse
  Clonality	Monoclonal
  Clone ID	PAT2H8AT
  Isotype	IgG2b
  Light Chains	kappa
  Immunogen	Recombinant human EGFR amino acids 424-605
  Reactivity	Human
  Formulation	1mg/ml in PBS, pH 7.4, with 10% Glycerol and 0.02% Sodium Azide
  Purification Method	Protein-G affinity chromatography from mouse ascitic fluids

  This antibody specifically recognizes the extracellular domain of human EGFR, making it suitable for detecting EGFR in its native conformation in live or fixed cells . Understanding these properties is essential for proper experimental design and interpretation of results.

• How should EGFR Antibody (PAT2H8AT) be stored and handled for optimal performance?

  For optimal performance and longevity of EGFR Antibody (PAT2H8AT), follow these storage and handling guidelines:

  Storage Conditions:

  • Short-term storage (up to 1 month): Store at 2-8°C

  • Long-term storage: Store at -20°C

  • Avoid repeated freeze-thaw cycles, which can degrade antibody quality

  Handling Guidelines:

  • The antibody is supplied at 1 mg/ml in a formulation containing PBS (pH 7.4), 10% glycerol, and 0.02% sodium azide

  • Centrifuge the vial briefly before opening to collect all liquid

  • Consider dividing the stock into small working aliquots before freezing

  • When diluting, use high-quality buffers and consider adding carrier proteins to prevent non-specific binding

  • Handle with care as sodium azide in the formulation is toxic if ingested

  Following these guidelines will help maintain antibody integrity and ensure consistent experimental results across different studies.

• What is the relationship between EGFR and cancer development?

  EGFR (Epidermal Growth Factor Receptor) plays a critical role in cancer development through several mechanisms:

  • Oncogenic Signaling: EGFR is a transmembrane protein that acts as a receptor for the extracellular EGF protein family, initiating signaling cascades that regulate cell proliferation, survival, and differentiation . When dysregulated, these pathways can drive uncontrolled cell growth.

  • Mutations and Overexpression: Mutations in the EGFR gene can lead to constitutive activation of the receptor, promoting oncogenic signaling even in the absence of ligand binding . Additionally, EGFR overexpression occurs in various cancer types, including non-small cell lung cancer (NSCLC), providing more receptors for signal transduction.

  • Therapeutic Target: The critical role of EGFR in cancer has made it an important target for cancer therapies. Various approaches have been developed to inhibit EGFR activity, including:

    • Tyrosine kinase inhibitors (TKIs) that target the intracellular domain

    • Monoclonal antibodies that target the extracellular domain

    • Novel inhibitors like furanopyrimidine-based compounds that target specific EGFR mutations such as L858R/T790M

  Research with tools like the EGFR Antibody (PAT2H8AT) helps elucidate these relationships, potentially identifying new therapeutic strategies and improving personalized medicine approaches for patients with EGFR-dependent cancers .

Advanced Research Questions

• How can EGFR Antibody (PAT2H8AT) be optimized for detecting EGFR mutations in clinical samples?

  Optimizing EGFR Antibody (PAT2H8AT) for detection of EGFR mutations in clinical samples requires careful consideration of several technical factors:

  Tissue Processing and Epitope Preservation:

  • Standardize fixation protocols (10% neutral buffered formalin for 24 hours is recommended)

  • Optimize antigen retrieval methods specific to the EGFR extracellular domain

  • Consider using automated immunostaining platforms for consistency across clinical samples

  Mutation-Specific Considerations:

  • The PAT2H8AT antibody targets amino acids 424-605 of EGFR's extracellular domain , while most clinically relevant mutations (like L858R and T790M) occur in the intracellular domain

  • This antibody should detect total EGFR regardless of mutation status, rather than being mutation-specific

  • For comprehensive analysis, combine with mutation-specific antibodies or molecular testing

  Validation Framework:

  • Use cell line controls with known EGFR mutation status

  • Compare antibody staining results with genetic testing (PCR, sequencing) on the same samples

  • Include samples with various EGFR mutations (exon 19 deletions, L858R, T790M) and wild-type controls

  Quantification Strategy:

  • Implement digital pathology tools for standardized scoring

  • Develop an H-score system (intensity × percentage of positive cells)

  • Consider multiplex immunofluorescence to correlate EGFR expression with downstream signaling activation

  This approach ensures reliable detection of EGFR across different mutation contexts, providing valuable complementary information to genetic testing for comprehensive patient assessment.

• What are the best practices for using EGFR Antibody (PAT2H8AT) in combination with EGFR inhibitor research?

  When investigating EGFR inhibitors, especially novel compounds like furanopyrimidine-based inhibitors , the EGFR Antibody (PAT2H8AT) can provide valuable insights into drug mechanisms and efficacy:

  Experimental Design Considerations:

  • Time-course Analysis: Design experiments to capture both immediate (minutes to hours) and long-term (days) effects of inhibitor treatment on:

    • Total EGFR levels

    • EGFR localization (membrane vs. cytoplasmic/endosomal)

    • Receptor degradation rate

  • Dose-response Studies: Test multiple inhibitor concentrations to establish:

    • EC50 values for EGFR inhibition

    • Threshold concentrations for receptor downregulation

    • Selectivity between wild-type and mutant EGFR (especially for inhibitors targeting L858R/T790M mutations)

  Technical Approaches:

  • Western Blot Analysis: To assess total EGFR levels and phosphorylation status

    • Include phospho-EGFR antibodies alongside PAT2H8AT

    • Monitor downstream signaling proteins (ERK, AKT)

    • Validate target modulation similar to studies with compound 52 from Furanopyrimidine research

  • Immunofluorescence: To visualize EGFR trafficking

    • Track receptor internalization following inhibitor treatment

    • Co-stain with endosomal markers to assess receptor fate

    • Quantify membrane-to-cytoplasmic ratio changes

  • Flow Cytometry: For quantitative assessment of surface EGFR

    • Monitor changes in cell-surface receptor density

    • Perform on live cells to avoid fixation artifacts

  Cell Models and Controls:

  • Use both EGFR mutation-positive and wild-type cell lines

    • H1975 cells (EGFR L858R/T790M) as used in inhibitor studies

    • A431 cells (EGFR overexpressing wild-type) as comparison

  • Include treatment-naive controls alongside inhibitor-treated samples

  • Consider resistant cell models to investigate resistance mechanisms

  This systematic approach allows researchers to comprehensively characterize how EGFR inhibitors affect receptor biology and signaling, facilitating the development of more effective targeted therapies.

• How can EGFR Antibody (PAT2H8AT) be incorporated into multiplexed detection systems?

  Integrating EGFR Antibody (PAT2H8AT) into multiplexed detection systems requires careful consideration of compatibility issues and optimization strategies:

  Multiplex Immunofluorescence (mIF) Integration:

  • Panel Design: PAT2H8AT (mouse IgG2b) can be combined with antibodies from different host species or isotypes to minimize cross-reactivity

  • Sequential Staining Protocol:

    1. Begin with heat-mediated antigen retrieval optimized for EGFR

    2. Apply PAT2H8AT as first primary antibody

    3. Detect with isotype-specific secondary antibody

    4. Apply microwave treatment or chemical stripping

    5. Continue with next marker

  • Signal Amplification: Consider tyramide signal amplification (TSA) for detecting low-abundance epitopes alongside EGFR

  Mass Cytometry (CyTOF) Applications:

  • Metal-conjugate PAT2H8AT with lanthanide metals

  • Validate signal specificity after conjugation

  • Develop compensation matrices to account for signal spillover

  Spatial Transcriptomics Integration:

  • Combine PAT2H8AT immunofluorescence with in situ hybridization

  • Correlate protein expression with EGFR mRNA levels

  • Develop protocols for sequential or simultaneous detection

  Cross-Platform Validation:

  • Concordance Testing: Verify that EGFR detection with PAT2H8AT in multiplex systems matches single-plex results

  • Data Integration: Develop computational pipelines to integrate EGFR protein data with other molecular data types

  Multiplex Applications in EGFR Research:

  • Simultaneous detection of EGFR with downstream signaling proteins (pERK, pAKT)

  • Co-detection of EGFR with other ErbB family members

  • Monitoring tumor heterogeneity through single-cell EGFR expression analysis in context of other markers

  By carefully optimizing these protocols, researchers can leverage PAT2H8AT in complex multiplexed systems to gain deeper insights into EGFR biology in cancer and other diseases.

• What methodological approaches are needed for quantitative assessment of EGFR using PAT2H8AT antibody?

  Rigorous quantitative assessment of EGFR using PAT2H8AT antibody requires standardized methodologies across different experimental platforms:

  Western Blot Quantification:

  • Linear Dynamic Range Determination:

    • Perform serial dilutions of lysates to identify linear detection range

    • Avoid saturated signals that prevent accurate quantification

    • Use the 170 kDa band corresponding to full-length EGFR

  • Normalization Strategy:

    • Use housekeeping proteins (β-actin, GAPDH) for loading control

    • Consider total protein normalization methods (Ponceau, REVERT)

    • Include recombinant EGFR standards for absolute quantification

  • Analysis Tools:

    • Use dedicated image analysis software (ImageJ, Image Lab)

    • Apply background subtraction consistently

    • Report results as relative fold change or absolute values

  Flow Cytometry Quantification:

  • Standardization Protocol:

    • Use antibody-binding capacity (ABC) beads to convert fluorescence to molecules per cell

    • Establish consistent voltage settings across experiments

    • Include quantitative fluorescent standards

  • Gating Strategy:

    • Define positive populations based on FMO controls

    • Distinguish membrane from total EGFR through permeabilization comparison

    • Report median fluorescence intensity (MFI) rather than percent positive

  Immunohistochemistry/Immunofluorescence Quantification:

  • Scoring System Development:

    • Implement H-score method (intensity × percentage positive cells)

    • Use digital pathology software for automated quantification

    • Establish regions of interest (ROIs) selection criteria

  • Calibration Approach:

    • Include reference slides with known EGFR expression levels

    • Use cell line microarrays as internal controls

    • Apply batch correction algorithms for multi-slide experiments

  ELISA/Immunoassay Quantification:

  • Standard Curve Optimization:

    • Generate standard curves using recombinant EGFR

    • Ensure curves encompass expected sample concentration range

    • Validate lower limit of quantification (LLOQ)

  • Sample Processing:

    • Standardize protein extraction methods

    • Account for matrix effects in complex samples

    • Run samples in technical triplicates

  These methodological approaches ensure reliable, reproducible quantitative assessment of EGFR across different experimental platforms, facilitating meaningful comparisons between studies.

• How does the epitope specificity of EGFR Antibody (PAT2H8AT) influence its application in studying EGFR-targeted therapies?

  The epitope specificity of EGFR Antibody (PAT2H8AT), which targets amino acids 424-605 in the extracellular domain of human EGFR , has significant implications for studying EGFR-targeted therapies:

  Therapeutic Mechanism Analysis:

  • Monoclonal Antibody Therapeutics: Since therapeutic antibodies like cetuximab also target the extracellular domain, PAT2H8AT can help evaluate:

    • Potential epitope competition or overlap with therapeutic antibodies

    • Receptor conformation changes induced by therapeutic binding

    • Receptor downregulation following therapeutic antibody treatment

  • Tyrosine Kinase Inhibitors (TKIs): For intracellular TKIs targeting the kinase domain (including furanopyrimidine-based compounds ):

    • PAT2H8AT detects total EGFR regardless of phosphorylation status

    • Can monitor receptor levels during treatment without interference from kinase inhibition

    • Allows distinction between inhibition of activity versus receptor degradation

  Resistance Mechanism Investigation:

  • Mutation Analysis: For studying resistance to third-generation inhibitors:

    • The extracellular epitope allows detection of receptors with kinase domain mutations (L858R/T790M)

    • Can monitor whether resistant clones show altered receptor trafficking

    • Enables quantification of total EGFR in models developing resistance to compounds like "52"

  • Bypass Pathway Activation: When investigating alternative signaling:

    • Can determine if EGFR levels change during resistance development

    • Allows co-staining with other receptor tyrosine kinases that might compensate

  Experimental Design Considerations:

  • Pre-clinical Models: For xenograft studies similar to those with compound "52" :

    • Human-specific epitope enables selective detection of tumor EGFR versus host receptors

    • Can be used in ex vivo analysis of tumor samples from drug efficacy studies

    • Allows correlation of EGFR levels with response to treatment

  • Technical Compatibility:

    • The extracellular domain epitope allows live-cell surface EGFR quantification

    • Compatible with non-permeabilized flow cytometry to assess surface receptor modulation

    • Suitable for monitoring receptor internalization kinetics following drug exposure

  Understanding these epitope-related factors allows researchers to design more informative experiments when investigating EGFR-targeted therapies, potentially accelerating the development of effective treatments for EGFR-driven cancers.