OPCML Antibody, Biotin conjugated

What is OPCML and what is its significance in cancer research?

OPCML (also called OBCAM) belongs to the IgLON family of GPI-anchored cell adhesion molecules. It functions as a tumor suppressor that is frequently epigenetically inactivated in epithelial ovarian cancers, with demonstrated suppressor function both in vitro and in vivo . The significance of OPCML in cancer research stems from its mechanism of action: it negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) including EPHA2, FGFR1, FGFR3, HER2, and HER4 in ovarian cancer cell lines and normal ovarian surface epithelial cells . OPCML binds to the extracellular domains of these RTKs, alters their trafficking via non-clathrin-dependent endocytosis, and promotes their degradation via a polyubiquitination-associated proteasomal mechanism .

What is biotin conjugation and why is it useful for OPCML antibodies?

Biotin conjugation refers to the chemical process of attaching biotin molecules to antibodies. This conjugation is valuable for immunochemical procedures as it allows for the use of secondary reagents coupled to biotin-binding proteins such as avidin . For OPCML antibodies specifically, biotin conjugation creates a versatile tool that can be used in conjunction with streptavidin-based detection systems, enabling higher sensitivity in various applications such as immunohistochemistry, ELISA, and Western blotting. This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, making them ideal for antibody labeling, functional and cell-based assays, flow-based assays, and multiplex imaging applications .

What are the optimal methods for conjugating OPCML antibodies to biotin?

There are several approaches to conjugating OPCML antibodies to biotin, each with distinct advantages:

The choice of method depends on the specific application, desired stability, and downstream applications. For most research applications, the direct conjugation method using NHS-biotin is recommended for its balance of simplicity and effectiveness.

What are the recommended validation assays for biotin-conjugated OPCML antibodies?

A comprehensive validation approach should include:

• Western Blot Analysis: Verify specific binding to OPCML protein (expected MW ~45 kDa) .

  • Positive controls: mouse pancreas tissue, rat pancreas tissue

  • Recommended dilution: 1:500-1:1000

• Immunohistochemistry: Confirm tissue distribution pattern.

  • Recommended tissue samples: human cerebral cortex, ovarian tissue

  • Expected staining pattern: cytoplasmic and membrane staining

  • Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes is recommended

• Streptavidin-Binding Assay: Confirm successful biotin conjugation.

  • Incubate conjugated antibody with streptavidin-coated beads

  • Analyze bound fraction for OPCML antibody presence

• Functional Assays: Verify the antibody retains its binding properties after conjugation.

  • Compare binding affinity of conjugated vs. unconjugated antibody

  • Assess impact on epitope recognition

• Degree of Labeling (DOL) Determination: Calculate the biotin:antibody ratio.

  • Spectrophotometric methods using HABA (4'-hydroxyazobenzene-2-carboxylic acid) assay

  • Optimal DOL typically ranges from 3-8 biotin molecules per antibody

How can biotin-conjugated OPCML antibodies be used to study tumor suppressor mechanisms?

Biotin-conjugated OPCML antibodies provide powerful tools for investigating the tumor suppressor mechanisms of OPCML:

• RTK Regulation Studies:

  • Co-immunoprecipitation experiments to identify interactions between OPCML and RTKs

  • Pull-down assays similar to those performed with GST-OPCML fusion proteins

  • Quantification of surface RTK levels via biotinylation of surface proteins followed by streptavidin capture

• Proteasomal Degradation Pathway Analysis:

  • Biotin-conjugated OPCML antibodies can be used to track OPCML-mediated polyubiquitination

  • Combined with ubiquitin assays to monitor effects on RTK degradation

  • Studies show OPCML restoration is associated with increased ubiquitination of HER2

• Trafficking Visualization:

  • Coupled with streptavidin-fluorophore conjugates for live-cell imaging

  • Enables visualization of OPCML-mediated endocytosis of RTKs

  • Can track accelerated loss of surface-labeled RTKs (such as HER2) in OPCML-expressing cells

• In vivo Tumor Models:

  • Biotin-conjugated OPCML antibodies can be used with avidin-based in vivo imaging systems

  • Allows tracking of OPCML expression in xenograft models

  • Can monitor response to recombinant OPCML protein therapy in ovarian cancer models

How do biotin-conjugated OPCML antibodies compare with antibody-oligonucleotide conjugates (AOCs) for targeted delivery applications?

Biotin-conjugated OPCML antibodies and antibody-oligonucleotide conjugates (AOCs) represent two different approaches for targeted delivery applications with distinct characteristics:

For targeting OPCML-expressing cells in ovarian cancer research, biotin-conjugated OPCML antibodies provide an advantage for diagnostic applications and protein-based therapeutics delivery, while AOCs would be more suitable for delivering gene-silencing payloads to modulate OPCML-related pathways.

What are common technical challenges when working with biotin-conjugated OPCML antibodies?

Researchers commonly encounter these challenges:

• Over-biotinylation Effects:

  • Excessive biotin conjugation can interfere with antigen recognition

  • May cause aggregation or precipitation of the antibody

  • Solution: Optimize biotin:antibody ratio (3-8 biotin molecules per antibody typically optimal)

• Tissue-Specific Expression Variations:

  • OPCML expression varies across tissues (brain, ovary, heart, placenta, testis, kidney, liver, pancreas, and colon)

  • Solution: Include appropriate positive controls for each tissue type; normal rete ovarii and uterus show strong OPCML immunoreactivity

• Non-specific Binding:

  • Endogenous biotin can cause background in biotin-rich tissues

  • Solution: Block endogenous biotin using avidin-biotin blocking reagents prior to antibody application

• Antigen Retrieval Optimization:

  • OPCML detection may require specific antigen retrieval methods

  • Solution: Heat-mediated antigen retrieval with 0.01M citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0)

• Antibody Storage Stability:

  • Biotin conjugates may lose activity during storage

  • Solution: Store at -20°C with 50% glycerol; stable for one year after shipment

How can researchers optimize immunohistochemistry protocols for biotin-conjugated OPCML antibodies?

Optimized IHC protocol for biotin-conjugated OPCML antibodies:

• Section Preparation:

  • Cut sections at 4 μm thickness

  • Dewax and rehydrate using standard protocols

• Antigen Retrieval:

  • Heat-mediated retrieval in 0.01M citrate buffer (pH 6.0)

  • Boil in microwave for 10 minutes

  • Allow to stand for 15 minutes in hot buffer before washing in PBS

• Endogenous Peroxidase and Biotin Blocking:

  • Block endogenous peroxidase with 3% hydrogen peroxide in methanol (5 minutes)

  • Wash in PBS containing 1% Triton X-100

  • Apply avidin-biotin blocking reagents

  • Block with 20% normal goat serum (1:20 in PBS, 30 minutes)

• Antibody Incubation:

  • Primary antibody dilution: 1:30 overnight at 4°C

  • For detection, use a streptavidin-HRP system rather than biotinylated secondary antibody to avoid cross-reactivity

• Signal Development and Counterstaining:

  • Detect antibody complex with diaminobenzidine (DAB)

  • Counterstain with Gill's hematoxylin

• Assessment:

  • Grade intensity of immunoreactive staining on a scale of 0-3:

    • 0: negative

    • 1: mild, cytoplasmic staining

    • 3: strong, membrane-situated OPCML immunoreactivity

What are the key findings on OPCML expression patterns relevant to researchers using these antibodies?

Important expression patterns to consider when using OPCML antibodies:

• Tissue Distribution:

  • Strong OPCML immunoreactivity observed in oviduct, rete ovarii, and uterus

  • In ovary, more immunoreactivity is seen in granulosa cells than ovarian surface epithelium (OSE)

  • No staining observed in OSE around ovulation sites (where OSE cells divide)

  • Originally isolated from brain, but also expressed in stomach, ovary, heart, placenta, testis, kidney, liver, pancreas, and colon

• Cancer-Related Patterns:

  • Frequently epigenetically inactivated in over 80% of human epithelial ovarian cancers

  • Weaker immunoreactivity in more dysplastic cells lining large ovarian inclusion cysts compared with normal rete ovarii

  • Significant correlation between OPCML hypermethylation and loss of expression in cancer cell lines

• Subcellular Localization:

  • Membrane-situated in normal cells (GPI-anchored protein)

  • Cytoplasmic staining also observed in some tissues

  • Functions at the cell surface to interact with RTKs

This expression data is crucial for selecting appropriate positive controls and interpreting results when using biotin-conjugated OPCML antibodies.

How might biotin-conjugated OPCML antibodies contribute to developing new therapeutic approaches for ovarian cancer?

Biotin-conjugated OPCML antibodies could enable several innovative therapeutic strategies:

• Antibody-Based Therapeutic Development:

  • Biotin-conjugated OPCML antibodies can be used to identify cells with low OPCML expression for targeted therapy

  • The biotin-streptavidin system provides a versatile platform for attaching therapeutic payloads

  • Studies have shown that recombinant OPCML domain 1-3 protein inhibits ovarian cancer cell growth in vitro and in vivo

• RTK-Targeted Interventions:

  • Biotin-conjugated OPCML antibodies can help identify which RTKs are upregulated in OPCML-deficient tumors

  • This knowledge could guide selection of appropriate RTK inhibitors for combination therapy

  • OPCML negatively regulates EPHA2, FGFR1, FGFR3, HER2, and HER4, making these potential therapeutic targets

• Biomarker Development:

  • Biotin-conjugated OPCML antibodies can be used in multiplexed assays to correlate OPCML expression with other cancer markers

  • This could lead to development of diagnostic panels for early detection or prognosis

  • Research shows that OPCML expression inhibits ovarian cancer cell growth, enhances inter-cellular attachment, and abrogates tumorigenicity

• Theranostic Applications:

  • The biotin-streptavidin system allows for both imaging and therapeutic applications

  • Could enable simultaneous detection and treatment of OPCML-deficient tumors

  • Potential for development of nanoparticle-based delivery systems targeting OPCML-regulated pathways

These approaches align with emerging data showing that recombinant OPCML protein therapy shows promise in epithelial ovarian cancers, offering a novel "extracellular tumor-suppressor replacement strategy" .

How do different commercially available OPCML antibodies compare for research applications?

Comparison of commercially available OPCML antibodies:

When selecting an antibody for biotin conjugation, researchers should consider:

• Monoclonal antibodies may provide more consistent results after conjugation

• Higher concentration formulations simplify the conjugation process

• Carrier-free options eliminate interference from stabilizing proteins

What are the advantages and limitations of using biotin-conjugated OPCML antibodies compared to other detection methods?

For optimal detection of OPCML in cancer research applications, biotin-conjugated antibodies provide an excellent balance of sensitivity, versatility, and established protocols, particularly when endogenous biotin is properly blocked.