ADAM10 Recombinant Monoclonal Antibody

What is ADAM10 and what biological functions does it regulate?

ADAM10 is a transmembrane metalloprotease that functions as a major shedding enzyme, cleaving the ectodomains of various cell surface proteins. It plays critical roles in multiple biological processes including inflammation, apoptosis, cell adhesion, cell metabolism, and development. ADAM10 has over 40 identified substrates involved in diverse cellular functions . It is crucial for embryonic development, as evidenced by the fact that ADAM10-deficient mice die around day 9.5 during early embryonic stages .

Among its most well-studied functions, ADAM10 is known for its role in Notch signaling and for cleaving the amyloid precursor protein (APP) involved in Alzheimer's disease pathophysiology. Other important substrates include E-cadherin, L-selectin, EGF, FASL, CD40L, ICOS-L, MICA, MICB, and ULBP2 . In the nervous system, it plays a particularly important role in neuronal development and axon targeting .

How is ADAM10 expressed across different tissues and cell types?

The ubiquitous expression of ADAM10 presents both challenges and opportunities for targeted therapeutic approaches, as researchers must consider potential off-target effects when developing ADAM10-targeting strategies.

What distinguishes recombinant monoclonal antibodies against ADAM10 from traditional antibodies?

Recombinant monoclonal antibodies against ADAM10, such as the ZooMAb® rabbit recombinant monoclonal antibodies, represent a new generation of antibody technology with several distinct advantages. Unlike traditional antibodies produced through animal immunization, these antibodies are manufactured using proprietary recombinant expression systems .

Key differences include:

• Production without animal sacrifice or harm, aligning with ethical "Waste Prevention" principles

• Precise manufacturing resulting in exceptional lot-to-lot consistency

• Superior stability allowing for ambient shipping and storage in many cases

• Rigorous validation for high specificity and affinity across multiple applications

• Reliable availability due to recombinant production methods

• Selection of only top-performing clones for research use

These properties make recombinant monoclonal antibodies particularly valuable for longitudinal studies where consistent antibody performance is critical.

What are the optimal applications for ADAM10 recombinant monoclonal antibodies in research?

ADAM10 recombinant monoclonal antibodies have been validated for several key applications in research settings:

• Western Blotting: Typically used at 1:1,000 dilution to detect ADAM10 in various cell lysates including NIH3T3, HeLa, and Jurkat cells .

• Immunohistochemistry: Effective at 1:100 dilution on paraffin-embedded tissue sections, particularly for human cerebral cortex and kidney tissues .

• Immunocytochemistry: Successfully applied at 1:100 dilution for detecting ADAM10 in cultured cells like Jurkat cell lines .

• Affinity Binding Assays: High-quality antibodies demonstrate strong affinity binding, with representative lots binding ADAM10 with KD values in the nanomolar range (e.g., 2.3 x 10-9) .

• Substrate Identification Studies: Used in comparative proteomic analyses to identify ADAM10 substrates by comparing secretomes with and without ADAM10 activity .

It's important to note that optimal working dilutions must be determined by each researcher as specimens and experimental conditions can vary significantly between laboratories.

How should researchers validate the specificity of ADAM10 antibodies?

Validating specificity of ADAM10 antibodies requires multiple complementary approaches:

• Positive and Negative Controls: Use cell lines known to express ADAM10 (e.g., NIH3T3, HeLa, Jurkat) as positive controls, and implement ADAM10 knockout or knockdown models as negative controls.

• Multiple Detection Methods: Confirm specificity using at least two independent detection methods (e.g., western blotting plus immunocytochemistry).

• Epitope Mapping: Understand the specific epitope recognized by the antibody. For example, some recombinant monoclonal antibodies target epitopes within the C-terminal cytoplasmic domain of ADAM10 .

• Competitive Binding Assays: Perform peptide competition assays with the immunization peptide to confirm binding specificity.

• Cross-Reactivity Testing: Test the antibody against related ADAM family members, particularly ADAM17, which has the highest structural and functional similarity to ADAM10 .

• Knockout Validation: The most stringent specificity control involves using genetic models with ADAM10 deletion, such as conditional Adam10 knockout mouse models .

What protocols are recommended for sample preparation when using ADAM10 antibodies?

Effective sample preparation protocols vary by application but share critical considerations:

For Western Blotting:

• Efficient lysis buffers typically contain 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitor cocktails

• Include specific inhibitors for metalloproteases if attempting to preserve the intact form of ADAM10

• Avoid excessive heating of samples to prevent protein aggregation

• Consider non-reducing conditions when analyzing the active conformation of ADAM10

For Immunohistochemistry:

• Optimal fixation with 4% paraformaldehyde

• Antigen retrieval methods (typically heat-induced epitope retrieval in citrate buffer pH 6.0)

• Blocking with appropriate sera (5-10% normal serum from the same species as the secondary antibody)

• Antibody concentration optimization (starting with 1:100 dilution for paraffin sections)

For Immunocytochemistry:

• Mild fixation (4% paraformaldehyde for 10-15 minutes)

• Permeabilization with 0.1-0.3% Triton X-100

• Thorough blocking (1-2 hours with 5% normal serum)

• Overnight primary antibody incubation at 4°C

How can researchers distinguish between active and inactive forms of ADAM10 using antibodies?

Distinguishing between active and inactive ADAM10 conformations represents an advanced research challenge. Recent studies have developed antibodies that specifically recognize the active conformation of ADAM10, enabling more precise investigation of ADAM10 activity states in different tissues and disease conditions.

For example, the monoclonal antibody 8C7 preferentially recognizes an active form of ADAM10 in human and mouse tumors . This antibody has been used to demonstrate that an active form of ADAM10 is elevated in tumors compared to normal tissues. The specificity appears to be based on recognition of a conformation-specific epitope that is exposed in the active form but hidden in the inactive form of ADAM10 .

Researchers can implement the following approaches:

• Use conformation-specific antibodies like 8C7 that preferentially bind to active ADAM10

• Combine antibody-based detection with activity-based assays using fluorogenic peptide substrates

• Use comparative immunoprecipitation followed by activity assays to correlate antibody binding with functional activity

• Implement proximity ligation assays to detect ADAM10 interaction with known substrate partners as an indirect measure of activity

What methodologies are effective for identifying novel ADAM10 substrates?

The identification of novel ADAM10 substrates requires sophisticated methodological approaches. Based on successful strategies from published research, the following approaches are recommended:

• Comparative Secretome Analysis: Compare the secretome (collection of secreted proteins) between wild-type cells and those with ADAM10 knockout or inhibition. This can reveal proteins whose shedding depends on ADAM10 activity .

• SPECS (Secretome Protein Enrichment with Click Sugars) Method:

  • Culture primary neurons from conditional Adam10 knockout mouse models

  • Transduce with control or iCre-encoding lentivirus

  • Incubate with ManNAz (a metabolically incorporated sugar)

  • Enrich secreted glycoproteins and analyze by mass spectrometry

  • Compare abundance profiles between ADAM10-positive and ADAM10-negative conditions

• Quantitative Proteomic Analysis: Using techniques such as SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling can enhance quantitative accuracy.

• Validation Studies: Confirm candidate substrates using:

  • Western blot analysis of conditioned media

  • ELISA-based quantification of shed ectodomains

  • Inhibitor studies with selective ADAM10 inhibitors like GI254023X

  • siRNA-mediated knockdown or CRISPR-Cas9 genome editing of ADAM10

This methodology has proven successful in identifying almost 100 new substrates of ADAM10, with 90% possessing a type-I membrane orientation, consistent with known ADAM10 substrate preferences .

How can researchers address the challenges of functional redundancy between ADAM10 and other proteases?

Functional redundancy, particularly between ADAM10 and ADAM17, presents significant challenges for researchers studying specific ADAM10 functions. Several methodological approaches can help address this challenge:

• Use of Selective Inhibitors:

  • GI254023X shows approximately 100-fold selectivity for ADAM10 over ADAM17

  • Compare with dual inhibitors like INCB3619 and INCB7839 that target both enzymes

  • Implement dose-response studies to identify concentration ranges with optimal selectivity

• Genetic Models with Conditional Knockout:

  • Utilize conditional Adam10 knockout mouse models (e.g., Adam10fl/fl crossed with tissue-specific Cre lines)

  • Compare phenotypes with ADAM17 knockout models to distinguish specific functions

  • Implement double knockout approaches to identify compensatory mechanisms

• Substrate-Specific Assay Systems:

  • Focus on "exclusive" ADAM10 substrates like Neuroligin-1 that show nearly complete abolishment of shedding upon ADAM10 deletion

  • Contrast with "shared" substrates like APP, where shedding is only partially reduced in ADAM10-/- models

• Time-Resolved Analysis:

  • Implement acute inhibition studies to identify immediate versus compensatory effects

  • Use inducible knockout systems to distinguish between developmental versus adult functions

A practical experimental design might involve parallel treatment groups with selective ADAM10 inhibitors, selective ADAM17 inhibitors, dual inhibitors, and vehicle controls, followed by quantitative analysis of multiple substrate cleavage events.

What considerations are important when designing therapeutic targeting strategies for ADAM10?

Designing therapeutic strategies targeting ADAM10 requires careful consideration of several factors due to its ubiquitous expression and diverse substrate profile:

• Target Specificity:

  • Distinguishing between ADAM10 and its close homolog ADAM17 is critical

  • Consider developing antibodies that recognize specific conformational states of ADAM10, such as the 8C7 antibody that preferentially binds active ADAM10 in tumors

• Tissue and Context Specificity:

  • ADAM10 shows differential activity patterns across tissues despite ubiquitous expression

  • Evidence suggests minimal ADAM10 activity in some normal tissues compared to disease states (e.g., normal brain versus glioblastoma)

  • Design strategies that exploit disease-specific ADAM10 activation patterns

• Delivery Methods:

  • For antibody-based approaches, consider antibody-drug conjugates (ADCs) that can deliver cytotoxic payloads specifically to cells with high ADAM10 activity

  • The 8C7 antibody-drug conjugate has demonstrated ability to preferentially kill cells displaying the 8C7 epitope and inhibit tumor growth in mice

• Considerations of Potential Side Effects:

  • ADAM10 is crucial for development, so targeting strategies must carefully consider developmental processes

  • Potential impacts on Notch signaling pathways must be monitored

  • Effects on neuronal connectivity and axon targeting should be evaluated

• Biomarker Development:

  • Implement methods to monitor ADAM10 activity as a biomarker

  • Serum ADAM10 levels have shown promise as predictive biomarkers for treatment response in rheumatoid arthritis

  • Consider developing companion diagnostics that can identify patients most likely to benefit from ADAM10-targeting approaches

What are common pitfalls when working with ADAM10 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with ADAM10 antibodies:

• Non-specific Binding:

  • Problem: Cross-reactivity with other ADAM family members, particularly ADAM17

  • Solution: Use recombinant monoclonal antibodies validated for specificity against ADAM10 ; implement appropriate negative controls including ADAM10 knockout samples

• Variable Detection of Processed Forms:

  • Problem: ADAM10 undergoes complex processing including prodomain removal and potential C-terminal fragmentation

  • Solution: Use antibodies targeting specific domains; understand the epitope location relative to processing sites; consider using multiple antibodies targeting different domains

• Conformational Sensitivity:

  • Problem: Some antibodies may recognize only specific conformations of ADAM10

  • Solution: Be aware of the conformational specificity of your antibody; use native conditions for applications where conformation is important

• Fixation Sensitivity in Immunohistochemistry:

  • Problem: Overfixation can mask epitopes or alter protein conformation

  • Solution: Optimize fixation protocols; implement appropriate antigen retrieval methods; test a range of antibody dilutions (e.g., 1:50-1:200)

• Inconsistent Results Across Experiments:

  • Problem: Lot-to-lot variation in traditional antibodies

  • Solution: Use recombinant monoclonal antibodies which offer superior lot-to-lot consistency ; maintain consistent experimental conditions; implement positive controls in each experiment

How can researchers optimize detection of ADAM10 in different experimental systems?

Optimizing ADAM10 detection across experimental systems requires tailored approaches:

For Western Blotting:

• Optimize lysis conditions to effectively solubilize membrane proteins

• Consider non-reducing conditions if detecting the active conformation

• Use 7-10% gels for optimal resolution of ADAM10 (~90 kDa)

• Test a range of antibody dilutions, typically starting with 1:1,000

• Include positive control lysates (e.g., NIH3T3, HeLa, or Jurkat cells)

For Immunohistochemistry:

• Test multiple fixation protocols (formalin, paraformaldehyde)

• Optimize antigen retrieval methods (heat-induced vs. enzymatic)

• Start with 1:100 antibody dilution and adjust as needed

• Use positive control tissues with known ADAM10 expression

• Implement blocking steps to reduce background staining

For Flow Cytometry:

• Use mild fixation to preserve epitope accessibility

• Test multiple cell dissociation methods (enzymatic vs. non-enzymatic)

• Optimize staining buffer composition

• Consider native (non-permeabilized) vs. permeabilized detection depending on epitope location

Universal Considerations:

• Temperature and incubation time optimization

• Buffer composition adjustment

• Signal amplification techniques for low expression systems

• Consider using secondary detection systems optimized for rabbit monoclonal antibodies

What experimental controls are essential when studying ADAM10 function?

Rigorous experimental controls are essential for reliable ADAM10 functional studies:

• Genetic Controls:

  • ADAM10 knockout or knockdown models as negative controls

  • Rescue experiments with ADAM10 re-expression to confirm specificity

  • Use of conditional knockout systems (e.g., Adam10fl/fl with Cre expression)

• Pharmacological Controls:

  • Selective ADAM10 inhibitors (e.g., GI254023X)

  • Broad-spectrum metalloprotease inhibitors (e.g., GM6001)

  • Inactive structural analogs of inhibitors as negative controls

• Antibody Controls:

  • Isotype control antibodies to assess non-specific binding

  • Multiple validated antibodies targeting different ADAM10 epitopes

  • Peptide competition controls to verify epitope specificity

• Substrate Controls:

  • Known ADAM10 substrates (e.g., APP, Notch, E-cadherin) as positive controls

  • Non-ADAM10 substrates as negative controls

  • Artificial substrates with known cleavage sites

• Cell Type Controls:

  • Cell lines with high endogenous ADAM10 expression (e.g., NIH3T3, HeLa, Jurkat)

  • Comparison across multiple cell types to account for cell-specific differences

  • Primary cells vs. immortalized cell lines to verify physiological relevance

How are ADAM10 antibodies being developed for targeted cancer therapies?

Recent research has made significant progress in developing ADAM10-targeted antibody therapies for cancer:

• Conformational-Specific Antibodies:
  Research has identified antibodies like 8C7 that preferentially recognize an active form of ADAM10 that is elevated in tumors compared to normal tissues. This conformational specificity provides a crucial targeting advantage .

• Antibody-Drug Conjugates (ADCs):
  The development of 8C7 antibody-drug conjugates has shown promise in preferentially binding and killing tumor cells with active ADAM10. These conjugates have demonstrated ability to inhibit tumor growth in mouse models without significant side effects .

• Tumor-Specific Targeting:
  Biodistribution studies in mice bearing human tumor xenografts have shown preferential targeting of ADAM10 antibodies to tumors compared to normal tissues, further supporting the potential of these approaches for targeted therapy .

• Correlation with Patient Outcomes:
  Studies have shown that ADAM10 expression levels in tumors can correlate with patient outcomes. For example, in glioblastoma patients, low ADAM10 expression levels positively correlated with increased survival, particularly when combined with tumor resection .

These advances suggest significant potential for ADAM10-targeted antibody therapies in cancer, particularly using approaches that can distinguish the active, disease-associated form of ADAM10 from the inactive form found in normal tissues.

What are the latest advancements in understanding ADAM10's role in neurological disorders?

Research into ADAM10's role in neurological disorders has yielded several important insights:

• Alzheimer's Disease:
  ADAM10 is recognized as an α-secretase that cleaves the amyloid precursor protein (APP) within the Aβ domain, thereby preventing the formation of amyloidogenic peptides. This has led to interest in developing drugs that activate ADAM10 as potential Alzheimer's disease therapies .

• Brain Development and Connectivity:
  Studies in conditional Adam10 knockout mice have revealed that ADAM10 plays critical roles in axon targeting and brain connectivity. Analysis of these models showed:

  • 40% diffuse olfactory glomeruli in Adam10-/- mice compared to 10% in controls

  • Mistargeted axon projections connecting to multiple glomeruli

  • These defects resemble those seen in mice lacking ADAM10 substrates like NRCAM

• Substrate Identification in Neuronal Systems:
  Proteomic studies comparing the secretome of neurons with and without ADAM10 activity have identified numerous neuronal substrates, many of which are involved in:

  • Axon guidance

  • Neuronal positioning

  • Cell-cell communication

  • Synapse formation and maintenance

• Therapeutic Considerations:
  The diverse roles of ADAM10 in neuronal development and function highlight both opportunities and challenges for therapeutic targeting. While activating ADAM10 might benefit Alzheimer's patients, potential impacts on neuronal connectivity must be carefully considered .

How are researchers addressing the challenge of selective ADAM10 inhibition versus ADAM17?

The challenge of selectively targeting ADAM10 over its close homolog ADAM17 is being addressed through several innovative approaches:

• Structure-Based Drug Design:
  Leveraging structural differences between ADAM10 and ADAM17 to design inhibitors with improved selectivity. This approach has led to compounds like GI254023X that show approximately 100-fold selectivity for ADAM10 over ADAM17 .

• Conformation-Specific Antibodies:
  Development of antibodies that recognize specific conformational states of ADAM10 that may differ from ADAM17. The 8C7 antibody represents an example of this approach, binding preferentially to an active conformation of ADAM10 .

• Substrate-Guided Approaches:
  Designing inhibitors based on the unique substrate preferences of ADAM10 compared to ADAM17. The observation that 90% of ADAM10 substrates possess a type-I orientation provides valuable information for substrate-guided inhibitor design .

• Tissue-Specific Delivery Systems:
  Developing delivery strategies that target tissues where ADAM10, but not ADAM17, plays a dominant role in disease pathology.

• Pharmacokinetic Optimization:
  Engineering molecules with distribution profiles that favor tissues where ADAM10 is the primary therapeutic target relative to ADAM17.

• Combined Inhibition Strategies:
  In some disease contexts, dual inhibition of both ADAM10 and ADAM17 may be advantageous. Compounds like INCB3619 and INCB7839 that target both proteases have shown promise in clinical settings where both enzymes contribute to pathology .

Comparison of ADAM10 with other related proteases