EMB3003 Antibody

What is EMab-300 antibody and what targets does it recognize?

EMab-300 is a monoclonal antibody (rat IgG1, kappa) specifically developed to detect mouse Epidermal Growth Factor Receptor (mEGFR). It was established using the Cell-Based Immunization and Screening (CBIS) method, which involves immunizing rats with LN229/mEGFR cells and screening the resulting hybridomas for mEGFR-specific binding . This antibody recognizes mEGFR in both overexpressed systems (such as CHO/mEGFR cells) and endogenously expressing cell lines (including NMuMG and Lewis lung carcinoma cells) . Unlike many commercially available antibodies, EMab-300 was specifically designed for recognizing mouse EGFR, making it valuable for preclinical studies in mouse models.

How does the specificity of EMab-300 compare to other anti-EGFR antibodies?

EMab-300 demonstrates high specificity for mouse EGFR, as evidenced by flow cytometry analysis showing dose-dependent recognition of mEGFR-expressing cells while showing no reactivity with parental CHO-K1 cells that do not express mEGFR . This specificity is particularly important for distinguishing between true signal and background in experimental settings. Unlike some antibodies that may cross-react with multiple species or related receptors, EMab-300 appears to maintain its specificity even at higher concentrations (10 μg/mL), where non-specific binding often becomes problematic with less specific antibodies . When selecting an antibody for mEGFR detection, researchers should consider this specificity profile, particularly when working with complex tissue samples where multiple related receptors may be present.

What is the binding affinity of EMab-300 and how is it determined?

The binding affinity of EMab-300 to mEGFR has been determined through kinetic analysis using flow cytometry. The dissociation constant (KD) values were established as:

These moderate affinity values were calculated using serial dilutions of EMab-300 (from 656 to 0.08 nM) incubated with target cells, followed by detection with Alexa Fluor 488-conjugated anti-rat IgG and analysis using a cell analyzer . The KD was subsequently calculated using GraphPad PRISM 8. This methodological approach provides a quantitative measure of antibody-antigen interaction strength, which is crucial for predicting antibody performance in various applications and for comparing different antibodies targeting the same antigen.

How should EMab-300 be optimally used for flow cytometry applications?

For optimal flow cytometry applications with EMab-300, the following methodological approach is recommended:

• Cell preparation: Harvest cells using gentle dissociation methods (e.g., 1 mM EDTA) to preserve surface epitopes. Avoid using trypsin which may cleave EGFR.

• Antibody concentration titration: Test a range of concentrations (e.g., 10, 1, 0.1, and 0.01 μg/mL) to determine optimal signal-to-noise ratio for your specific cell line. EMab-300 has shown dose-dependent detection in multiple cell lines .

• Incubation conditions: Incubate cells with EMab-300 for 30 minutes at 4°C in blocking buffer (0.1% BSA in PBS) .

• Secondary antibody: Use an appropriate fluorophore-conjugated secondary antibody (e.g., Alexa-Fluor-488-conjugated anti-rat IgG) at a dilution of approximately 1:200 .

• Controls: Always include isotype control (rat IgG1) and unstained controls. For cell lines where EGFR expression is uncertain, include both known positive and negative controls.

This methodological approach ensures consistent and reliable detection of mEGFR while minimizing background signal and non-specific binding issues that could complicate data interpretation.

What purification methods are recommended for EMab-300?

For the purification of EMab-300 from hybridoma culture supernatants, the following methodology has proven effective:

• Initial capture: Apply the hybridoma supernatant to an appropriate affinity matrix, such as Ab-Capcher (which contains the alkali-resistant antibody-binding protein Protein A-R28 immobilized at high density) .

• Washing: Thoroughly wash the matrix with phosphate-buffered saline (PBS) to remove non-specifically bound proteins and contaminants .

• Elution: Elute the bound antibodies using an IgG elution buffer under controlled pH conditions that disrupt the antibody-matrix interaction without denaturing the antibody .

• Buffer exchange and concentration: Concentrate the eluate and replace the elution buffer with PBS using an appropriate molecular weight cut-off filter such as Amicon Ultra .

• Quality control: Verify the purity of the antibody using SDS-PAGE and confirm activity using a functional assay such as flow cytometry.

This purification approach yields highly pure antibody preparations while maintaining functional integrity. For researchers requiring larger quantities, scaling up this protocol with larger columns is feasible, though additional optimization may be necessary.

How can researchers validate the epitope specificity of EMab-300?

Validating the epitope specificity of EMab-300 requires a systematic approach:

• Competitive binding assays: Test whether pre-incubation with recombinant mEGFR protein or known epitope-containing peptides blocks antibody binding to cells.

• Domain deletion/mutation analysis: Express variants of mEGFR with specific domains deleted or mutated to identify the region containing the epitope.

• REMAP (RIEDL insertion for epitope mapping) method: This technique, mentioned in the literature for mapping conformational epitopes, involves creating a library of antigen variants with small peptide insertions and testing antibody binding to identify epitope regions .

• Cross-reactivity testing: Examine binding to related receptors (e.g., ErbB2, ErbB3) to determine epitope uniqueness.

• Western blot under different conditions: Compare binding under reducing and non-reducing conditions to determine if the epitope is conformational.

While specific epitope data for EMab-300 is not provided in the search results, researchers working with EMab-300 should consider these methodological approaches as they were successfully applied to other antibodies developed with similar techniques (including anti-human EGFR mAbs EMab-51 and EMab-134) .

How can EMab-300 be applied in preclinical mouse models for cancer research?

EMab-300 offers several methodological approaches for preclinical cancer research in mouse models:

• Pharmacokinetic and biodistribution studies: EMab-300 can be labeled (e.g., with radioisotopes or near-infrared dyes) to track EGFR targeting in vivo, allowing for quantitative assessment of tumor accumulation versus normal tissue distribution.

• Syngeneic mouse models: EMab-300 can be particularly valuable in studies using Lewis lung carcinoma or other murine cancer cell lines that express endogenous mEGFR, enabling investigation in immunocompetent mice with intact immune systems .

• Epithelial-to-mesenchymal transition (EMT) studies: Since EMab-300 recognizes mEGFR in NMuMG cells (commonly used for EMT research), it can be employed to study EGFR's role in this process, particularly in sphere formation assays where EGF signaling is critical .

• Antibody engineering for therapeutic assessment: Converting EMab-300 to mouse IgG format (particularly mouse IgG2a subclass) could enable evaluation of potential therapeutic effects through enhanced antibody-dependent cellular cytotoxicity in syngeneic models .

• Combination therapy models: EMab-300 derivatives could be used in combination with immune checkpoint inhibitors in syngeneic models to evaluate potential synergistic effects .

These approaches can provide valuable insights into EGFR-targeted therapies before advancing to human studies, potentially improving the predictive value of preclinical research and reducing late-stage clinical failures.

What considerations are important when adapting EMab-300 for antibody-drug conjugate (ADC) development?

When adapting EMab-300 for antibody-drug conjugate development, researchers should consider several methodological factors:

• Isotype switching: Converting EMab-300 (rat IgG1) to mouse IgG (particularly IgG2a) is essential for appropriate effector functions in mouse models. This involves cloning the variable regions and expressing them with mouse constant regions .

• Glycoengineering: Consider using fucosyltransferase 8-deficient expression systems to produce defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity, as demonstrated with other antibodies .

• Conjugation site selection: Analyze the EMab-300 sequence for appropriate conjugation sites that won't interfere with antigen binding. Options include:

  • Random lysine conjugation (simpler but less controlled)

  • Site-specific conjugation via engineered cysteines

  • Enzymatic approaches (e.g., transglutaminase)

• Drug-to-antibody ratio (DAR) optimization: Systematically test different DARs to balance potency with pharmacokinetic properties.

• Linker selection: Choose appropriate linkers (cleavable vs. non-cleavable) based on the target cell's endosomal properties and the payload's mechanism of action.

• In vitro validation: Prior to in vivo studies, confirm that conjugation doesn't impair antigen binding using the same flow cytometry methods used to characterize the original EMab-300 .

This methodological framework can guide researchers in developing EMab-300-based ADCs with optimal efficacy and specificity profiles.

How does EMab-300 perform in detecting EGFR in different cellular contexts and activation states?

The performance of EMab-300 across different cellular contexts and EGFR activation states is a complex consideration:

What are common technical issues when using EMab-300 in flow cytometry and how can they be resolved?

When using EMab-300 in flow cytometry, researchers may encounter several technical challenges, each with specific methodological solutions:

• High background signal:

  • Problem: Non-specific binding to Fc receptors on cells.

  • Solution: Include an Fc blocking step before antibody incubation using commercial Fc block or normal rat serum (2-5%) .

  • Validation: Compare blocking with isotype control to confirm improvement.

• Weak or absent signal despite known EGFR expression:

  • Problem: Epitope masking or destruction during cell preparation.

  • Solution: Use gentler cell dissociation methods (EDTA rather than enzymatic methods) .

  • Validation: Compare different dissociation methods with the same cell line.

• Inconsistent results between experiments:

  • Problem: Variability in antibody concentration or cellular EGFR expression.

  • Solution: Establish standard curves using calibration beads and carefully control cell culture conditions that might affect EGFR expression.

  • Validation: Include a standard cell line with known EGFR expression in each experiment.

• False positives in certain cell populations:

  • Problem: Autofluorescence or non-specific binding.

  • Solution: Use appropriate fluorescence-minus-one (FMO) controls and consider alternative fluorophores if autofluorescence occurs in the same channel.

  • Validation: Confirm results using an alternative detection method (e.g., immunoblotting).

• Poor discrimination between positive and negative populations:

  • Problem: Suboptimal antibody concentration.

  • Solution: Perform careful titration series (as mentioned in the EMab-300 study, which tested 10, 1, 0.1, and 0.01 μg/mL) .

  • Validation: Calculate the staining index at each concentration to identify optimal signal-to-noise ratio.

These methodological approaches systematically address common technical challenges while providing validation steps to ensure reliable results.

How should researchers interpret contradictory results between EMab-300 binding and other EGFR detection methods?

When faced with contradictory results between EMab-300 binding and other EGFR detection methods, researchers should follow this methodological framework for resolution:

• Consider epitope accessibility differences:

  • Different detection methods may access different epitopes

  • EMab-300 likely recognizes a conformational epitope based on development methodology

  • Some methods (e.g., certain immunohistochemistry protocols) may denature EGFR, affecting epitope recognition

• Evaluate method-specific factors:

  • Flow cytometry (EMab-300's validated application): Detects surface expression primarily

  • Western blotting: Detects denatured protein, may miss conformational epitopes

  • RT-PCR: Measures mRNA, not protein expression

  • Mass spectrometry: Provides total protein without spatial information

• Systematic reconciliation approach:

  • Verify antibody functionality using appropriate positive controls

  • Perform parallel analyses with multiple antibodies targeting different EGFR epitopes

  • Consider receptor internalization or shedding affecting surface detection

  • Examine post-translational modifications that might affect epitope recognition

• Quantitative comparison matrix:

  Detection Method	What It Measures	Potential Discrepancy Causes
  EMab-300 Flow Cytometry	Surface mEGFR with specific conformation	Receptor internalization, epitope masking
  Western Blot	Total denatured EGFR protein	Conformational epitope loss, sample preparation differences
  IHC/IF	Spatially localized EGFR	Fixation effects on epitope, accessibility issues
  ELISA	Soluble or extracted EGFR	Extraction efficiency, conformational changes
  RT-PCR	EGFR mRNA expression	Post-transcriptional regulation

• Biological interpretations:

  • Different results may reflect actual biological states rather than technical errors

  • Consider whether discrepancies reveal meaningful insights about receptor dynamics

This methodological framework helps researchers systematically evaluate contradictory results to determine whether they represent technical limitations or biologically significant phenomena.

How can researchers determine if decreased EMab-300 binding is due to reduced EGFR expression or epitope masking?

Distinguishing between reduced EGFR expression and epitope masking requires a systematic methodological approach:

• Multiple epitope targeting strategy:

  • Use additional antibodies targeting different EGFR epitopes in parallel experiments

  • If all antibodies show reduced binding, expression is likely decreased

  • If only EMab-300 shows reduced binding, epitope masking is more likely

• Total vs. surface EGFR comparison:

  • Compare flow cytometry with EMab-300 (surface detection) to permeabilized cell staining or Western blot (total protein)

  • Significant discrepancy suggests epitope masking or internalization rather than expression changes

• mRNA expression analysis:

  • Quantify EGFR mRNA using RT-qPCR

  • Reduced mRNA with reduced EMab-300 binding suggests true expression changes

  • Normal mRNA with reduced EMab-300 binding suggests post-transcriptional regulation or epitope issues

• Competition assay with potential masking agents:

  • Pre-treat cells with agents that might displace masking factors (mild detergents, high salt, pH changes)

  • Restored binding after treatment suggests epitope masking

• Ligand-induced changes assessment:

  • Measure EMab-300 binding before and after EGF stimulation

  • Rapid decrease after stimulation suggests internalization or conformational change rather than expression changes

  • Sustained decrease over hours suggests actual downregulation

• Quantitative flow cytometry approach:

  • Use calibration beads to convert fluorescence intensity to absolute antibody binding capacity

  • This enables more precise quantification of binding site number changes

This methodological framework provides multiple, complementary approaches to distinguish between these mechanistically distinct phenomena, leading to more accurate biological interpretations.

What modifications to EMab-300 could enhance its utility in advanced imaging applications?

Several methodological approaches could enhance EMab-300's utility for advanced imaging applications:

• Direct fluorophore conjugation:

  • Site-specific conjugation of bright, photostable fluorophores (e.g., Alexa Fluor 647, Janelia Fluor dyes) to EMab-300

  • Validation through comparison with secondary detection methods to ensure epitope recognition is preserved

  • Benefit: Reduced background and enhanced signal-to-noise ratio for super-resolution microscopy

• Fragment generation:

  • Development of Fab or single-chain variable fragment (scFv) derivatives of EMab-300

  • Methodological approach: Enzymatic digestion (papain for Fab) or recombinant expression of variable regions

  • Benefit: Improved tissue penetration and reduced steric hindrance in dense samples

• Bispecific formats for proximity detection:

  • Engineer bispecific antibodies combining EMab-300 binding specificity with another relevant target

  • Applications include proximity ligation assays or FRET-based interaction studies

  • Enables investigation of EGFR heterodimers or EGFR interaction with downstream signaling molecules

• Click chemistry-compatible derivatives:

  • Introduce bio-orthogonal functional groups for post-labeling via click chemistry

  • Methodological advantage: Flexibility to attach various imaging agents (fluorophores, MRI contrast agents, PET tracers)

  • Applications in multimodal imaging across different resolution scales

• Integration with emerging microscopy techniques:

  • Optimize EMab-300 for DNA-PAINT or STORM super-resolution microscopy

  • Develop EMab-300 derivatives compatible with expansion microscopy protocols

  • Engineer derivatives suitable for light-sheet microscopy in whole tissue samples

Each of these methodological approaches requires validation to ensure that modifications don't compromise the binding specificity and affinity that make EMab-300 valuable for mEGFR detection , while extending its applications to cutting-edge imaging techniques.

How might EMab-300 be adapted for studying EGFR biology in models of cancer resistance?

Adapting EMab-300 for studying EGFR biology in models of cancer resistance involves several methodological strategies:

• Development of EMab-300 derivatives for mutation-specific detection:

  • Engineer variant antibodies that distinguish between wild-type EGFR and resistance-associated mutations

  • Validation through parallel testing on cell lines expressing defined EGFR variants

  • Applications in tracking the emergence of resistant clones in heterogeneous populations

• Combination with phospho-specific detection systems:

  • Develop protocols for co-detection of total EGFR (using EMab-300) and phosphorylated EGFR

  • Methodological approach: Sequential or simultaneous immunostaining with phospho-specific antibodies

  • Enables assessment of EGFR activation status in resistant models

• Analysis of EGFR dynamics in resistant models:

  • Employ EMab-300 in pulse-chase experiments to study receptor internalization and recycling

  • Compare trafficking patterns between sensitive and resistant cells

  • Potential for revealing altered EGFR localization as a resistance mechanism

• Integration with functional readouts:

  • Develop assays linking EMab-300 binding to downstream functional consequences

  • Example methodology: Flow cytometry combining EMab-300 detection with simultaneous assessment of key signaling nodes (e.g., phospho-ERK, phospho-AKT)

  • Applications in identifying bypass mechanisms in resistant models

• EMab-300-based analysis of heterogeneity:

  • Apply EMab-300 in single-cell analysis platforms to quantify EGFR expression heterogeneity

  • Methodological approach: Index sorting or mass cytometry combined with single-cell sequencing

  • Potential for identifying resistant subpopulations before clinical resistance emerges

These methodological approaches leverage EMab-300's specificity for mEGFR while extending its applications to address key questions in cancer resistance biology, particularly in murine models where matching the species specificity of the antibody is critical.

What are the methodological considerations for using EMab-300 in multiplexed protein detection systems?

Integrating EMab-300 into multiplexed protein detection systems requires careful methodological consideration:

• Antibody panel design and validation:

  • Confirm EMab-300 compatibility with fixation and permeabilization protocols used for other targets

  • Test for potential cross-reactivity with other primary antibodies in the panel

  • Validate signal specificity in positive and negative control samples for each target

  • Methodological approach: Sequential staining with careful blocking between steps may be necessary

• Spectral considerations for fluorescence-based multiplexing:

  • Select secondary antibody fluorophores with minimal spectral overlap

  • For direct conjugation, choose fluorophores compatible with instrumentation and other panel components

  • Consider brightness hierarchy: assign brightest fluorophores to least expressed targets

  • Validation: Perform fluorescence-minus-one controls for each channel to confirm specificity

• Mass cytometry (CyTOF) adaptation:

  • Metal conjugation of EMab-300 using appropriate chelating polymers

  • Titration to determine optimal concentration after conjugation

  • Integration into existing panels with attention to potential signal spillover

  • Validation: Compare pre- and post-conjugation binding patterns to ensure epitope recognition is preserved

• Sequential multiplexing approaches:

  • Optimize EMab-300 for cyclic immunofluorescence or iterative bleaching protocols

  • Determine antibody removal efficiency between cycles

  • Develop appropriate imaging registration protocols for precise overlay of multiple rounds

  • Application: This approach has been cited for use with SYCP3 antibodies in iterative bleaching extends multiplexity applications

• Computational analysis frameworks:

  • Develop appropriate compensation matrices for fluorescence spillover

  • Implement clustering algorithms appropriate for the biological question

  • Include visualization methods that effectively communicate multidimensional relationships

Following these methodological considerations ensures that EMab-300 can be effectively integrated into multiplexed systems while maintaining its demonstrated specificity for mEGFR , enabling complex analyses of receptor biology in relation to other cellular components.