Met Antibody, Biotin conjugated

What is the Met receptor and why is it an important research target?

Met (c-Met) is a 145 kDa receptor tyrosine kinase (RTK) widely expressed in epithelial cells of the brain, kidney, liver, and other tissues. It serves as the receptor for Hepatocyte Growth Factor (HGF), also known as scatter factor. Upon ligand binding, Met undergoes autophosphorylation, activating several downstream effectors including ERK-1, ERK-2, and PLC gamma .

Met plays critical roles in:

• Embryonic development including gastrulation

• Development of muscles and neurons

• Angiogenesis and kidney formation

• Adult processes such as wound healing and organ regeneration

The importance of Met as a research target stems from its dysregulation being linked to numerous human malignancies including gastric, renal, and breast cancers . After activation, Met interacts with multiple signaling components:

• PI3-kinase subunit PIK3R1

• PLCG1

• SRC

• GRB2

• STAT3

These interactions activate several signaling cascades including RAS-ERK, PI3-kinase-AKT, and PLCgamma-PKC pathways , making Met an attractive target for cancer therapeutics and diagnostic testing.

What are the advantages of using biotin-conjugated antibodies for Met detection?

Biotin-conjugated anti-Met antibodies offer several methodological advantages in research settings:

• Enhanced detection sensitivity: The strong non-covalent interaction between biotin and streptavidin allows for signal amplification in various detection systems .

• Versatile detection options: Biotin-conjugated antibodies can be detected with different streptavidin-conjugated reporters (fluorophores, enzymes, quantum dots) without needing multiple secondary antibodies .

• Reduced background in multi-labeling experiments: Using biotin-conjugated primary antibodies eliminates cross-reactivity issues sometimes encountered with species-specific secondary antibodies .

• Specific plasma membrane detection: When used on live cells, biotin-conjugated anti-Met antibodies can specifically detect plasma membrane-associated Met with high signal-to-noise ratio .

• Biological activity: Unlike some tags that might interfere with protein function, biotin-conjugated anti-Met antibodies can effectively activate Met signaling, making them useful for functional studies of receptor dynamics .

The utility of these conjugates is further enhanced by their ability to detect both total and phosphorylated forms of Met, depending on the epitope recognized by the specific antibody .

How does the biotinylation process affect antibody functionality?

The biotinylation process can significantly impact antibody functionality, with several important considerations for researchers:

• Challenge ratio impact: The level of biotin incorporation is directly influenced by the challenge ratio (CR) of biotin to protein. Higher challenge ratios generally result in greater biotin incorporation, but excessive biotinylation can potentially interfere with antigen recognition .

• Incorporation variability: The median incorporation of biotin across different antibodies using a standard protocol (Sulfo-NHS-LC-Biotin at 2 mg/ml and CR 10) is approximately 46%, but varies significantly between antibodies . This variability is protein-dependent and may be due to differences in surface-accessible lysine residues.

• Functional effects: Evidence shows that biotin-conjugated anti-Met antibodies can induce clustering of Met receptors on the plasma membrane of live cells, promoting receptor activation similar to that induced by its natural ligand HGF . This indicates that biotinylation preserves and may even enhance certain functional properties of the antibody.

• Detection consistency: At standardized challenge ratios, the coefficient of variation (CV) for biotinylation measurements typically does not exceed 4.2%, except at very low challenge ratios (CR 1), suggesting good reproducibility of the conjugation process .

When optimizing biotinylation protocols, researchers should consider testing multiple challenge ratios and confirming that the conjugated antibody maintains its desired binding and functional properties through appropriate validation experiments.

What applications are biotin-conjugated Met antibodies most suitable for?

Biotin-conjugated Met antibodies demonstrate utility across multiple research applications:

• Flow cytometry: These antibodies are particularly effective for flow cytometric analysis of Met expression on cell surfaces. The recommended usage is ≤1 μg per test, where a test is defined as the amount of antibody needed to stain a cell sample in a final volume of 100 μL (cell counts typically range from 10^5 to 10^8 cells/test) .

• Sandwich ELISA: Biotin-conjugated anti-Met antibodies can function as detection antibodies in enzyme-linked immunosorbent assays, particularly in competition analysis where they are mixed with MET928 before addition to microplates coated with HGF/SF or fragment NK1 .

• Live cell imaging: When conjugated with fluorophores like Allophycocyanin (APC), these antibodies can specifically detect plasma membrane-associated Met in live cells with high signal-to-noise ratio, revealing receptor clustering patterns .

• Western blotting: Biotinylated phospho-Met antibodies are effective for detecting activated Met (phosphorylated at Tyr1234/1235) in western blot applications at a recommended dilution of 1:1000 .

• Complex formation analysis: These antibodies can be used in studies examining Met protein interactions, where the Fab and MET proteins are co-incubated at specific molar ratios and analyzed by size exclusion chromatography .

For optimal results, researchers should carefully titrate the antibody for each specific application and validate its performance in their experimental system.

How do biotin-conjugated anti-Met antibodies influence receptor activation and signaling?

Biotin-conjugated anti-Met antibodies exert profound effects on receptor activity through specific mechanisms:

• Receptor clustering induction: These antibodies cause a rapid and dramatic redistribution of Met proteins on the plasma membrane, inducing a "clustered" or "patched" pattern that resembles activation by the natural ligand HGF .

• Temporal dynamics: The antibody-activated clustering of Met occurs very rapidly and transiently. Following administration of biotin-conjugated anti-Met antibodies, clustering is observable within 2-5 minutes, reaches peak intensity at 7-10 minutes, and then the Met staining disappears from the cell surface within 15-30 minutes as the antibody-Met complexes are internalized .

• Signaling pathway activation: Biochemical analyses demonstrate that biotin-conjugated anti-Met antibodies induce phosphorylation of Y1234/Y1235 on Met within 7 minutes, comparable to activation by HGF. This leads to phosphorylation and activation of downstream kinases such as AKT .

• Cell-type versatility: The ability of biotin-conjugated anti-Met antibodies to activate Met signaling has been demonstrated across multiple human glioblastoma cell lines, including U373-MG, T98G, LN229, and LN18, suggesting broad applicability in cancer research models .

This ability to mimic ligand-induced activation makes biotin-conjugated anti-Met antibodies valuable tools for studying receptor dynamics and signaling processes in the absence of HGF, providing controlled experimental conditions for investigating Met-dependent cellular responses.

What methodological approaches can effectively characterize the degree of antibody biotinylation?

Several methodological approaches can accurately characterize the degree of antibody biotinylation:

• Modified Quant*Tag method: This is a widely used approach that measures the concentration of bound biotin. When combined with protein concentration measurements (determined by absorbance at UV280), this allows calculation of the average amount of biotin residues per antibody (B/P) .

• Background subtraction considerations: Proteins may interact with the QuantTag reagent even in the absence of biotinylation, producing background color changes. It's therefore recommended to use unbiotinylated material for background subtraction in the QuantTag assay to improve accuracy .

• Capillary isoelectric focusing (cIEF): This technique can detect the acidic shift that occurs in antibodies following biotinylation. cIEF graphs can reveal the presence of multiple peaks, allowing estimation of the proportion of unconjugated antibody remaining in the preparation. The technique has shown sensitivity to detect approximately 10% unconjugated material in a biotinylated preparation .

The data from these analyses can be presented as follows:

This characterization is crucial for ensuring batch-to-batch consistency and for optimizing antibody performance in various experimental applications.

How can researchers mitigate biotin interference in assays using biotin-conjugated Met antibodies?

Biotin interference presents a significant challenge in streptavidin-based assays using biotin-conjugated antibodies. Researchers can implement several strategies to mitigate this interference:

These approaches allow researchers to optimize their experimental protocols based on anticipated biotin levels and required assay sensitivity.

What mechanistic insights have been gained from studying Met receptor clustering using biotin-conjugated antibodies?

Research with biotin-conjugated anti-Met antibodies has revealed significant mechanistic insights into Met receptor biology:

These insights have important implications for understanding Met-mediated signaling in both normal and pathological contexts, particularly in cancer where Met dysregulation contributes to malignant progression.

How do biotinylated phospho-specific Met antibodies contribute to studying receptor activation in cancer research?

Biotinylated phospho-specific Met antibodies have become valuable tools in cancer research, offering unique advantages for studying receptor activation:

• Sensitive detection of activation state: Phospho-Met (Tyr1234/1235) biotinylated antibodies specifically detect the activated form of Met, recognizing the autophosphorylation sites that are critical indicators of receptor activity. These antibodies detect endogenous levels of Met only when phosphorylated at Tyr1234/1235, providing a sensitive measure of receptor activation status .

• Applications in signaling pathway analysis: In western blotting applications (typical dilution 1:1000), these antibodies allow researchers to monitor Met activation in response to various stimuli and to evaluate the effects of potential therapeutic agents on Met signaling. The biotinylation enables signal amplification through streptavidin-based detection systems .

• Cross-reaction considerations: An important methodological consideration is that these antibodies may cross-react with overexpressed tyrosine phosphorylated Src proteins in western blot analysis. This potential cross-reactivity necessitates appropriate controls when studying complex signaling environments .

• Temporal signaling dynamics: Studies using biotin-conjugated anti-Met antibodies revealed that addition of these antibodies to serum-starved cells induced phosphorylation of Y1234/Y1235 on Met within 7 minutes, comparable to activation by HGF. This provides valuable insights into the kinetics of Met activation .

• Comparative activation analysis: Research has demonstrated that biotin-conjugated anti-Met antibodies strongly induced MET and AKT phosphorylation compared to HGF control in multiple glioblastoma cell lines (U373-MG, T98G, LN229, and LN18), suggesting these antibodies can effectively activate Met in various cancer models .

The ability to specifically detect phosphorylated Met makes these antibodies crucial for evaluating the efficacy of Met-targeting therapeutics and for exploring the role of Met activation in cancer progression and resistance mechanisms.

What considerations are important when developing Met-targeting antibody-drug conjugates with biotin components?

Developing Met-targeting antibody-drug conjugates (ADCs) with biotin components requires careful consideration of several factors:

• Conjugation strategy optimization: Site-specific conjugation approaches tend to produce more homogeneous ADCs with improved therapeutic windows compared to random conjugation methods. BYON3521, a novel site-specifically conjugated duocarmycin-based ADC targeting Met, demonstrates this approach's promise in creating effective therapeutics .

• Binding affinity considerations: High-affinity antibodies against Met are critical for effective targeting. BYON3521 comprises a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus Met, highlighting the importance of strong target binding .

• Efficacy across expression levels: Met-targeting ADCs should demonstrate efficacy across varying levels of target expression. BYON3521 showed good efficacy in cell lines and in vivo tumor models with low, moderate, and high Met expression, suggesting broader applicability than therapeutics dependent on Met signaling addiction .

• Safety profile assessment: The therapeutic window of Met-targeting ADCs is critical. BYON3521 exhibited an encouraging safety profile in a 4-cycle Good Laboratory Practice toxicity study in cynomolgus monkeys, predicting a therapeutic window that would allow efficacious and safe treatment of patients with Met-positive cancers .

• Advantage over signaling-dependent approaches: Met-targeting ADCs operate through a different mechanism than kinase inhibitors or antibodies designed to block signaling. Their efficacy depends on Met expression on the cell surface to mediate binding and cellular uptake of conjugated toxins, rather than on downstream signaling dependency. This makes them promising for a broader range of Met-expressing cancers beyond those with MET amplification or mutation .

These considerations highlight the potential of biotinylated components in developing the next generation of Met-targeting therapeutics with improved efficacy and safety profiles.

How can researchers optimize protocols for analyzing Met receptor internalization using biotin-conjugated antibodies?

Optimizing protocols for analyzing Met receptor internalization with biotin-conjugated antibodies requires careful attention to several methodological aspects:

• Temporal sampling strategy: Based on research findings, Met internalization following antibody binding follows specific kinetics that should guide experimental design:

  • Initial clustering occurs within 2-5 minutes

  • Formation of strong patched areas at 7-10 minutes

  • Internalization begins at approximately 10 minutes

  • Complete internalization with strong intracellular staining occurs within 15-30 minutes

  Researchers should include multiple time points in this critical 0-30 minute window to capture the full internalization process.

• Live-cell imaging optimization: For live-cell tracking of Met internalization:

  • Use serum-starved cells to establish baseline receptor distribution

  • Apply biotin-conjugated anti-Met antibodies at 1 μg per test for approximately 10^5 to 10^8 cells

  • Maintain physiological conditions (37°C, appropriate pH and CO2) during imaging

  • Capture images at 2-3 minute intervals to track internalization dynamics

• Multi-channel detection approach: Implement a three-channel detection strategy to simultaneously visualize:

  • Biotinylated antibodies (using streptavidin-conjugated fluorophores like Brilliant Violet 421)

  • Activated Met (using phospho-specific antibodies with distinct fluorophores such as Alexa Fluor 647)

  • Total Met proteins (using antibodies against different epitopes conjugated to fluorophores like Alexa Fluor 488)

• Complementary biochemical analysis: Combine imaging with western blotting to quantify changes in surface versus internalized Met:

  • Collect samples at matched time points (0, 7, 15, and 30 minutes)

  • Analyze phosphorylation status of Met (Y1234/Y1235) and downstream targets like AKT

  • Compare activation patterns induced by biotin-conjugated antibodies versus HGF as a control

• Cell-type considerations: Validate protocols across multiple cell types, as internalization kinetics may vary. Studies have demonstrated antibody-induced Met internalization in multiple glioblastoma cell lines including U373-MG and T98G .

This methodological approach allows researchers to comprehensively characterize Met internalization dynamics while distinguishing between antibody-induced and ligand-induced receptor trafficking.

What are the challenges and solutions in using biotin-conjugated Met antibodies for reproducible research?

Researchers face several challenges when using biotin-conjugated Met antibodies, but methodological solutions can enhance reproducibility:

• Variation in biotinylation levels

  • Challenge: The degree of biotinylation can vary significantly between antibody preparations, with median incorporation around 46% but substantial variation between antibodies .

  • Solution: Implement consistent characterization methods for each batch, including modified Quant*Tag assays to determine biotin-to-protein ratios, and maintain detailed records of conjugation conditions (challenge ratio, protein concentration) .

• Presence of unconjugated antibody

  • Challenge: Low challenge ratios (CR≤5) often result in significant amounts of unconjugated antibody remaining in the preparation, which can interfere with experimental outcomes .

  • Solution: Use capillary isoelectric focusing (cIEF) to assess the proportion of unconjugated antibody. The technique can detect approximately 10% unconjugated material in a preparation and guide purification needs .

• Biotin interference in streptavidin-based assays

  • Challenge: Endogenous or exogenous biotin can compete with biotinylated antibodies in streptavidin-based detection systems.

  • Solution: Increase streptavidin concentration in the assay. Research shows that using 3X standard concentration of streptavidin-coated microparticles (M3) enables resistance to biotin concentrations up to 500 ng/mL .

• Receptor activation by antibody binding

  • Challenge: Biotin-conjugated anti-Met antibodies can induce receptor clustering and activation, potentially confounding experiments designed to measure baseline Met status .

  • Solution: Include appropriate controls (non-targeting biotinylated antibodies) and be aware of the temporal dynamics of antibody-induced activation (peaks at 7-10 minutes) .

• Cross-reactivity with other phosphorylated proteins

  • Challenge: Phospho-Met (Tyr1234/1235) biotinylated antibodies may cross-react with overexpressed tyrosine phosphorylated Src proteins in western blot analysis .

  • Solution: Include appropriate negative controls and validate findings with complementary approaches such as immunoprecipitation followed by western blotting with alternative antibodies.

By addressing these challenges with appropriate methodological solutions, researchers can enhance the reproducibility and reliability of their studies using biotin-conjugated Met antibodies.

What future directions exist for biotin-conjugated Met antibody applications in personalized medicine?

Biotin-conjugated Met antibodies show significant promise for advancing personalized medicine approaches in several key directions:

• Enhanced diagnostic capabilities: The ability of these antibodies to specifically detect Met expression and activation status could lead to more precise patient stratification for Met-targeted therapies. By developing standardized immunohistochemical or liquid biopsy assays using biotinylated antibodies, researchers can better identify patients likely to respond to Met inhibitors or Met-targeting antibody-drug conjugates .

• Improved therapeutic monitoring: Biotin-conjugated phospho-specific Met antibodies could facilitate real-time monitoring of treatment response through minimally invasive sampling. This would allow clinicians to rapidly detect emerging resistance mechanisms and adjust treatment strategies accordingly .

• Development of novel theranostic approaches: The combination of diagnostic and therapeutic capabilities in a single molecule is increasingly valuable. Biotin-conjugated Met antibodies could be developed as theranostic agents, where the biotin moiety enables detection while the antibody component delivers therapeutic payloads or facilitates receptor downregulation .

• Mechanism-based combination therapies: Research showing that biotin-conjugated anti-Met antibodies induce receptor clustering and internalization provides insights for developing more effective combination therapies. Triggering Met internalization could potentiate the effects of kinase inhibitors or enhance the delivery of antibody-drug conjugates .

• Broader application across cancer types: Met-targeting ADCs like BYON3521 have shown good efficacy in cell lines and in vivo tumor models with varying levels of Met expression. This suggests potential applicability across a wider range of cancer types than therapies dependent on Met amplification or mutation, expanding personalized medicine options for patients whose tumors express Met without genetic alterations .

As research continues to advance our understanding of Met biology and improve biotinylation technologies, these antibodies will likely play an increasingly important role in delivering personalized cancer treatments based on individual tumor characteristics rather than traditional histological classifications.

How do the methodological insights from Met antibody biotinylation translate to other receptor tyrosine kinase systems?

The methodological insights gained from Met antibody biotinylation research provide valuable translatable principles for studying other receptor tyrosine kinase (RTK) systems:

• Receptor clustering as an activation mechanism: The discovery that biotin-conjugated bivalent antibodies can induce Met clustering and activation mimicking ligand effects suggests a common mechanism potentially applicable to other RTKs. This approach could be adapted to study receptor dynamics of EGFR, HER2, VEGFR, and other clinically relevant RTKs, potentially revealing new activation mechanisms independent of ligand binding .

• Temporal resolution of receptor trafficking: The detailed characterization of Met internalization kinetics following antibody binding (clustering at 2-5 minutes, strong patching at 7-10 minutes, internalization at 15-30 minutes) provides a methodological framework for studying the trafficking of other RTKs with similar temporal resolution .

• Multi-channel imaging approaches: The three-channel detection strategy used to simultaneously visualize biotinylated antibodies, activated receptors, and total receptor pools offers a powerful approach for real-time monitoring of RTK activation and trafficking that could be directly applied to other receptor systems .

• Biotinylation optimization protocols: The characterization methods developed for determining biotin-to-protein ratios, including modified Quant*Tag assays and capillary isoelectric focusing, provide standardized approaches for optimizing biotinylation of antibodies targeting any RTK .

• Antibody-drug conjugate development strategy: The successful approach demonstrated with BYON3521, a site-specifically conjugated duocarmycin-based ADC targeting Met, offers a blueprint for developing ADCs against other RTKs with improved efficacy and safety profiles .

• Biotin interference mitigation strategies: The methods developed to overcome biotin interference in assays, such as increasing streptavidin concentration, are broadly applicable to any biotin-streptavidin detection system used in RTK research .