CDH15 Antibody, Biotin conjugated

What is CDH15 and what cellular functions does it mediate?

CDH15, also known as M-cadherin or Muscle cadherin, is a 124-127 kDa type I transmembrane glycoprotein belonging to the Cadherin superfamily of calcium-dependent homotypic adhesion molecules . As an atypical member of the classical cadherin family, CDH15 plays critical roles in cellular adhesion processes. Research has identified CDH15 expression in endothelial cells, where it serves as a receptor for PEPITEM, a peptide proteolytically derived from 14.3.3.ζδ protein . This interaction promotes the synthesis and release of sphingosine-1 phosphate, which regulates T cell trafficking across the endothelium without affecting other leukocytes . CDH15 expression is endogenously present in endothelial cells and can be upregulated when these cells are stimulated with inflammatory cytokines, suggesting its importance in inflammation and immune regulation .

How are biotin-conjugated CDH15 antibodies generated and what makes them useful for research?

Biotin-conjugated CDH15 antibodies are generated through chemical conjugation processes that attach biotin molecules to antibodies raised against CDH15. These antibodies can be either monoclonal (derived from a single B-cell clone) or polyclonal (derived from multiple B-cell clones) depending on the research requirements . The production typically involves immunizing host animals (such as mice, rabbits, or sheep) with an immunogen corresponding to CDH15 protein or specific peptide sequences derived from CDH15 . For instance, some commercial anti-CDH15 antibodies use recombinant Human Cadherin-15 protein (specifically amino acids 639-766) as the immunogen .

After harvesting and purifying the antibodies, biotin molecules are conjugated to them through various chemical processes. The resulting biotin-conjugated antibodies are then purified to >95% purity using techniques such as Protein G affinity chromatography . This biotin conjugation enables researchers to leverage the strong biotin-streptavidin interaction for detection systems, significantly enhancing signal amplification in assays like flow cytometry and immunohistochemistry .

What are the main differences between polyclonal and monoclonal CDH15 antibodies?

The choice between polyclonal and monoclonal CDH15 antibodies depends on research objectives and application requirements:

Both antibody types have been successfully used for CDH15 detection across various experimental platforms; polyclonal antibodies often provide stronger signals while monoclonal antibodies offer greater specificity and reproducibility .

How should biotin-conjugated CDH15 antibodies be optimally used in flow cytometry experiments?

For optimal use of biotin-conjugated CDH15 antibodies in flow cytometry, researchers should follow these methodological approaches:

What are the validated applications for CDH15/Cadherin-15 antibodies beyond flow cytometry?

Biotin-conjugated CDH15 antibodies have been validated for multiple applications beyond flow cytometry:

• Western blotting: CDH15 antibodies have successfully detected the protein in cell lysates from cell lines including HeLa (human cervical epithelial carcinoma), C2C12 (mouse myoblast), and L6 (rat myoblast) . Under reducing conditions, CDH15 appears as a specific band at approximately 125-130 kDa . Recommended protocols include using PVDF membranes and Immunoblot Buffer Group 1 or 8 .

• Immunofluorescence/Immunocytochemistry: CDH15 antibodies have been used to visualize protein expression and localization in fixed cells. For instance, in C2C12 mouse myoblast cells, CDH15 staining localizes to cell surfaces and cytoplasm . Protocols typically involve:

  • Fixation with 4% paraformaldehyde

  • Permeabilization with 0.1% Triton X-100

  • Blocking with appropriate serum

  • Primary antibody incubation (e.g., 10 μg/mL for 3 hours at room temperature)

  • Detection with fluorescently-labeled secondary antibodies

  • Counterstaining nuclei with DAPI

• ELISA: Biotin-conjugated polyclonal antibodies against CDH15 have been specifically tested and validated for ELISA applications, allowing quantitative measurement of CDH15 in biological samples .

• Immunohistochemistry on frozen sections (IHC-Fr): CDH15 antibodies have been used successfully for detection in frozen tissue sections, particularly in studies examining human tissues .

• Protein-protein interaction studies: CDH15 antibodies have been utilized in studies examining the interaction between CDH15 and PEPITEM, including Biacore assays to determine binding kinetics (KD=108.9 μM) .

What storage and handling conditions are critical for maintaining biotin-conjugated CDH15 antibody integrity?

Proper storage and handling of biotin-conjugated CDH15 antibodies is crucial for maintaining their integrity and functionality:

• Storage temperature: Store at -20°C for long-term preservation. Avoid storing at 4°C for extended periods .

• Aliquoting: Upon receipt, divide the antibody into small single-use aliquots to minimize freeze-thaw cycles, which can degrade both the antibody and the biotin conjugate .

• Light exposure: Minimize exposure to light, especially if the biotin is part of a detection system with fluorophores .

• Freeze-thaw cycles: Limit freeze-thaw cycles as repeated cycles can lead to protein denaturation and biotin degradation. Each cycle may reduce activity by 10-15% .

• Buffer composition: Commercial preparations typically contain stabilizing buffers such as 0.01 M PBS, pH 7.4, with preservatives like 0.03% Proclin-300 and 50% Glycerol . Avoid introducing contaminants by using sterile technique when handling antibodies.

• Working solution preparation: When preparing working solutions, use high-quality, sterile buffers. For flow cytometry applications, PBS containing 1-2% BSA is commonly used .

• Shelf-life considerations: Even with optimal storage, monitor for signs of degradation such as precipitation, cloudiness, or diminished performance in control assays if antibodies are used beyond the manufacturer's expiration date.

How can researchers confirm the specificity of CDH15 antibody binding in their experimental systems?

Confirming antibody specificity is crucial for reliable research findings. For CDH15 antibodies, implement these validation approaches:

• siRNA-mediated knockdown: As demonstrated in published studies, siRNA targeting CDH15 can efficiently knockdown expression, providing a negative control to confirm antibody specificity. Both mRNA levels and protein expression should decrease following successful knockdown . For instance, researchers have confirmed specificity by showing reduced CDH15 staining in endothelial cells (HDMEC) following siRNA-mediated silencing of CDH15 .

• Positive and negative controls: Use cell lines known to express CDH15 (positive controls) such as C2C12 mouse myoblast cells, skeletal muscle cells (SkMC), and HeLa cells . As negative controls, use cells with low or no CDH15 expression or isotype-matched control antibodies .

• Peptide competition assay: Pre-incubating the CDH15 antibody with the immunizing peptide or recombinant CDH15 protein should block specific binding. This approach can be particularly useful for polyclonal antibodies like those generated against recombinant Human Cadherin-15 protein (639-766AA) .

• Orthogonal detection methods: Compare results from multiple detection methods (e.g., western blot, immunofluorescence, and flow cytometry) and across antibodies raised against different epitopes of CDH15 .

• Cross-species validation: Examine antibody performance across species when appropriate. For instance, some CDH15 antibodies demonstrate cross-reactivity between human and mouse samples, which can be used to assess consistency of detection patterns .

What are common pitfalls in CDH15 detection experiments and how can they be addressed?

Researchers commonly encounter several challenges when working with CDH15 antibodies:

• High molecular weight of CDH15: The 124-130 kDa size of CDH15 can make it challenging to transfer efficiently in western blotting. To address this:

  • Use lower percentage gels (6-8%) for better separation

  • Extend transfer time or use specialized transfer systems optimized for high molecular weight proteins

  • Consider using Immunoblot Buffer Group 1 or 8 as recommended in published protocols

• Variable expression levels: CDH15 expression can be regulated by cytokines and inflammatory signals . To account for this:

  • Standardize cell culture conditions

  • Document treatment conditions precisely

  • Consider testing different time points after stimulation

  • Include positive controls with known CDH15 expression levels

• Non-specific binding: To reduce background:

  • Optimize blocking conditions (typically 5% BSA or 5% non-fat dry milk)

  • Titrate antibody concentration carefully

  • Increase washing steps between incubations

  • Use appropriate isotype controls

• Epitope masking: Certain fixation methods may mask the CDH15 epitope. Consider:

  • Testing multiple fixation protocols (PFA, methanol, acetone)

  • Implementing antigen retrieval methods when working with tissue sections

  • Using fresh samples when possible

• Biotin endogenous expression: Endogenous biotin can interfere with biotin-streptavidin detection systems. To mitigate this:

  • Use commercial biotin-blocking kits before applying biotin-conjugated antibodies

  • Consider alternative conjugation systems when working with biotin-rich tissues

How should researchers interpret CDH15 experimental data in the context of inflammatory and autoimmune conditions?

Interpreting CDH15 data in inflammation and autoimmune contexts requires careful consideration:

• Age-dependent expression effects: Research indicates that the functional pathway involving CDH15 undergoes immune senescence with age. Specifically, expression of adiponectin receptors on B cells and adiponectin-induced PEPITEM secretion diminishes with aging . When designing experiments:

  • Include age-matched controls

  • Consider stratifying analyses by age groups

  • Document age-related variables in experimental subjects

• Disease-specific alterations: CDH15 expression and function appear altered in autoimmune conditions like type-1 diabetes and rheumatoid arthritis . When analyzing experimental data:

  • Compare CDH15 expression patterns between healthy controls and disease samples

  • Consider measuring circulating PEPITEM levels alongside CDH15 expression

  • Analyze correlation between CDH15 expression and disease severity markers

• Inflammatory cytokine regulation: CDH15 expression in endothelial cells is regulated by inflammatory cytokines . For comprehensive data interpretation:

  • Document cytokine exposure in experimental systems

  • Consider measuring key cytokines alongside CDH15 expression

  • Examine temporal relationships between cytokine exposure and CDH15 expression changes

• Tissue-specific expression patterns: While traditionally associated with muscle tissue (hence the name M-cadherin), CDH15 expression has now been confirmed in endothelial cells and other tissues . For accurate data interpretation:

  • Validate CDH15 expression in the specific tissue or cell type under investigation

  • Consider tissue-specific functions when interpreting phenotypic effects

  • Use appropriate tissue-specific positive controls

What recent studies have implicated CDH15 in disease pathogenesis?

Recent research has revealed significant roles for CDH15 in various disease processes:

• Interstitial Lung Disease (ILD): A 2025 proteome-wide Mendelian randomization study identified CDH15 as one of three plasma proteins genetically associated with ILD and its subtypes. This study proposed for the first time that CDH15 holds promise as a novel potential circulating protein biomarker and therapeutic target for ILD diagnosis and treatment . The researchers applied rigorous sensitivity analyses, including Bayesian colocalization and bidirectional Mendelian randomization, to establish reliable causal associations .

• Autoimmune Conditions: Research published in Nature Medicine demonstrated that the CDH15 pathway is dysregulated in autoimmune diseases, particularly type-1 diabetes and rheumatoid arthritis. Patients with these conditions showed altered PEPITEM-CDH15 signaling and reduced circulating PEPITEM in serum compared to healthy age-matched donors . This suggests CDH15 involvement in the dysregulated lymphocyte trafficking characteristic of these diseases.

• Inflammatory Regulation: CDH15 has been implicated in the homeostatic regulation of T cell trafficking during inflammation. By binding PEPITEM (a B cell-derived peptide), CDH15 on endothelial cells promotes sphingosine-1-phosphate synthesis and release, which in turn inhibits T cell trafficking without affecting other leukocytes . This mechanism represents a potential therapeutic target for inflammatory conditions.

What are emerging applications of biotin-conjugated CDH15 antibodies in cutting-edge research?

Biotin-conjugated CDH15 antibodies are finding applications in several innovative research areas:

• Multiplexed Single-Cell Analysis: Biotin-conjugated antibodies enable detection in multi-parameter flow cytometry and mass cytometry (CyTOF) panels. These approaches allow researchers to simultaneously examine CDH15 expression alongside dozens of other markers at the single-cell level, providing insights into cell heterogeneity and complex phenotypes.

• Protein-Protein Interaction Studies: Biotin-conjugated CDH15 antibodies can be used in proximity ligation assays and pull-down experiments to investigate protein interactions. For example, researchers have used such approaches to study the interaction between CDH15 and PEPITEM, determining a binding kinetic with KD=108.9 μM using Biacore assays .

• Biomarker Development: Following the identification of CDH15 as a potential biomarker for ILD , biotin-conjugated antibodies could be developed into diagnostic tools for clinical applications. Biotin conjugation offers advantages for signal amplification and assay sensitivity in such applications.

• Therapeutic Target Validation: As CDH15 emerges as a potential therapeutic target , biotin-conjugated antibodies can facilitate target validation studies, including assessment of expression levels, accessibility, and functional consequences of target modulation.

How might functional studies of CDH15 contribute to development of novel therapeutics?

Understanding CDH15's functions could lead to innovative therapeutic approaches:

• Peptide Mimetics: Knowledge of the PEPITEM-CDH15 interaction could inform the development of peptide mimetics that modulate this pathway. Such therapeutics might restore normal T cell trafficking regulation in autoimmune and inflammatory conditions where this mechanism is impaired.

• Targeted Drug Delivery: Biotin-conjugated CDH15 antibodies could potentially be used to direct therapeutic payloads to cells expressing CDH15, such as endothelial cells at sites of inflammation or other CDH15-expressing diseased tissues.

• Biomarker-Guided Therapy: The identification of CDH15 as a potential biomarker for ILD suggests its utility in patient stratification for clinical trials and personalized medicine approaches. Monitoring changes in CDH15 expression or associated pathway components could help assess therapeutic responses.

• Combined Therapeutic Approaches: The mechanistic insights linking CDH15 to sphingosine-1-phosphate signaling suggest that combination therapies targeting multiple components of this pathway might yield synergistic benefits in treating inflammatory and autoimmune conditions.

• Gene Therapy Approaches: As gene editing technologies advance, precise modification of CDH15 expression or function in specific tissues could represent a future therapeutic strategy for conditions where CDH15 dysregulation contributes to pathology.