CHR24 Antibody

What is CD24 and why is it significant in cancer research?

CD24, also known as heat stable antigen (HSA), is a heavily and variably glycosylated 35-60 kDa GPI-linked sialoprotein expressed on B cells, T cells, keratinocytes, and myofiber synaptic nuclei. Its significance in cancer research stems from its upregulation in a wide variety of cancers and its role as a functional liver tumor-initiating cell (T-IC) marker . CD24 binds to P-Selectin on activated platelets and vascular endothelial cells, and CD24 ligation induces tumor cell apoptosis. It's particularly relevant for hepatocellular carcinoma (HCC), where it's involved in tumorigenesis and progression, and is upregulated in chemoresistant residual liver tumors .

What are the main types of CD24 antibodies available for research?

CD24 antibodies for research fall into two main categories: murine (mouse-derived) antibodies and humanized antibodies. Murine antibodies, while effective for many research applications, have inherent immunogenicity concerns for therapeutic applications. Humanized antibodies, such as hG7-BM3, are engineered through complementarity determining region (CDR) grafting to reduce immunogenicity while maintaining binding specificity and affinity . Both types are available in various formats including monoclonal antibodies for applications such as immunohistochemistry, immunofluorescence, ELISA, and flow cytometry .

How does CD24 expression vary across different tissue types?

CD24 is expressed across multiple cell types including B cells, T cells, and keratinocytes in normal tissues . In pathological contexts, CD24 is upregulated in numerous cancer types, particularly in hepatocellular carcinoma (HCC) . In research applications, CD24 has been detected in human colon tissue and ovarian cancer tissue using immunohistochemistry techniques . The expression patterns can be heterogeneous, which makes CD24 a useful marker for distinguishing certain cancer subtypes and for targeting therapeutic strategies.

What are the optimal protocols for CD24 antibody use in immunohistochemistry?

For optimal immunohistochemical detection of CD24, the following protocol has shown efficacy: Use 15 μg/mL of CD24 antibody on immersion-fixed paraffin-embedded tissue sections, with overnight incubation at 4°C. Follow with an appropriate detection system such as HRP-DAB staining and counterstain with hematoxylin . For successful staining:

• Perform proper antigen retrieval, typically heat-mediated in citrate buffer

• Include positive controls (such as colon or ovarian cancer tissue known to express CD24)

• Optimize antibody concentration for each tissue type

• Ensure sufficient incubation time to allow complete antibody binding

• Use validated detection systems compatible with your primary antibody species

Application-specific optimizations may be required based on tissue type and fixation methods.

How can I validate the specificity of CD24 antibody binding in my samples?

Validating CD24 antibody specificity requires multiple complementary approaches:

• Knockdown validation: Use RNA interference to reduce CD24 expression in your cell line, then perform flow cytometry or Western blotting. A reduction in signal proportional to the knockdown efficiency (e.g., 55.3% and 55.7% knockdown resulting in corresponding signal reduction) confirms specificity

• Blocking experiments: Pre-incubate the antibody with recombinant CD24 protein before application to samples; a significant reduction in signal indicates specific binding

• Negative controls: Test the antibody on cell lines or tissues known not to express CD24, such as HL-7702 normal human hepatic cells compared to CD24-positive Huh-7 and BEL-7402 hepatoma cells

• Multiple antibody validation: Use different antibody clones targeting different CD24 epitopes to confirm consistent staining patterns

This multi-pronged approach ensures that your observed signals are genuinely attributable to CD24 expression.

What concentration of CD24 antibody is optimal for flow cytometry applications?

For flow cytometry applications, the optimal CD24 antibody concentration typically ranges from 5-15 μg/mL, though this must be determined empirically for each specific antibody and cell type. When working with hepatoma cell lines such as Huh-7 and BEL-7402, antibody concentrations of approximately 10 μg/mL have demonstrated effective binding and discrimination between CD24-positive cells and negative controls . A titration experiment is recommended for each new cell line, where serial dilutions (e.g., 1, 2.5, 5, 10, and 20 μg/mL) of antibody are tested to determine the concentration that provides the optimal signal-to-noise ratio and separation between positive and negative populations.

What is the rationale behind humanizing CD24 antibodies and how does it impact research applications?

The humanization of CD24 antibodies addresses a critical challenge in translational research - reducing immunogenicity while maintaining binding specificity and affinity. Murine antibodies can induce a human anti-mouse antibody (HAMA) response in therapeutic applications, limiting their clinical utility . The humanization process involves grafting the complementarity determining regions (CDRs) from murine antibodies onto human antibody frameworks, significantly reducing immunogenic potential.

For research applications, humanized antibodies offer several advantages:

• Better modeling of potential therapeutic antibodies in preclinical studies

• Reduced background in assays involving human samples or human immune components

• Maintenance of critical binding characteristics while improving pharmacokinetic properties

• Preservation of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) that might be relevant to mechanism studies

The impact of humanization on research is particularly significant when studying CD24 as a therapeutic target in cancer models, as seen with the successful development of humanized antibodies like hG7-BM3 that retain binding capacity to CD24 while demonstrating reduced immunogenicity .

What are the key steps in the CDR grafting process for CD24 antibodies?

The complementarity determining region (CDR) grafting process for CD24 antibodies involves several sophisticated steps:

• Selection of appropriate human framework templates with high sequence homology to the murine variable domains

• Identification and precise transfer of the six CDRs (three from VH and three from VL) from the murine antibody to the human framework

• Critical back-mutation of specific canonical residues that support CDR loop conformation:

  • Residues in the VL/VH interface core

  • Residues in the loop foundation

  • Residues that interact with loop residues

• Identification and mutation of framework residues involved in the antigen-binding interface based on molecular docking analysis

• Assessment of stability changes using computational methods like dStability values to identify potentially destabilizing mutations

• Construction of multiple variant antibodies with different combinations of back-mutations

• Expression and purification of candidate humanized antibodies

• Comparative analysis of binding affinity and 3D structure against the parent antibody

This structured approach has successfully generated humanized antibodies like hG7-BM3 that maintain high binding affinity (KD of 5.70×10^-10 M) comparable to their parental chimeric antibodies .

How can computational modeling improve CD24 antibody humanization outcomes?

Computational modeling significantly enhances CD24 antibody humanization through several advanced approaches:

• Structural analysis and precise modeling of antibody variable domains using platforms like Molecular Operating Environment (MOE) to ensure accurate identification of critical residues

• Quantitative assessment of structural stability changes (dStability values) to identify potentially destabilizing mutations that require back-mutation

• Molecular docking simulations between the antibody and CD24 antigen to identify key interface residues beyond the CDRs that contribute to binding

• Root Mean Square Deviation (RMSD) calculation between parental and humanized antibody structures to quantify conformational preservation

• In silico prediction of immunogenic epitopes in the framework regions to prioritize modifications that reduce potential immunogenicity

• Computational assessment of developability parameters, including aggregation propensity, based on sequence characteristics

This computational approach streamlines the humanization process by providing rational guidance for engineering decisions, reducing the number of variants requiring experimental testing, and increasing the probability of generating high-affinity humanized antibodies with favorable developability profiles .

How effective are CD24 antibodies in distinguishing cancerous from normal tissue?

CD24 antibodies demonstrate significant efficacy in distinguishing cancerous from normal tissue due to the differential expression patterns of CD24. In hepatocellular carcinoma research, humanized CD24 antibodies like hG7-BM3 show markedly higher binding to CD24-positive hepatoma cell lines (Huh-7 and BEL-7402) compared to normal human hepatic cell lines (HL-7702) . This specificity extends to in vivo studies where fluorescently-labeled antibodies showed preferential accumulation in tumor xenografts with a tumor/normal tissue ratio of 2.50 at 4 hours post-injection, compared to 1.14 for blocked controls .

The discriminatory capability of CD24 antibodies can be observed in multiple applications:

• Immunohistochemistry studies clearly distinguish CD24-positive cancer tissue from surrounding normal tissue

• Flow cytometry applications show distinct population separation between CD24-positive and negative cells

• In vivo imaging demonstrates specific tumor targeting with minimal background in normal tissues

This targeted specificity makes CD24 antibodies valuable tools for both diagnostic applications and potential therapeutic development for CD24-expressing tumors.

What is the mechanism of CD24 antibody-mediated tumor cell killing?

CD24 antibody-mediated tumor cell killing occurs through multiple mechanisms:

• Antibody-Dependent Cellular Cytotoxicity (ADCC): Humanized CD24 antibodies like hG7-BM3 retain Fc-mediated ADCC functionality. Studies with PBMCs at an effector/target ratio of 100:1 and NK92-FcR cells at a 10:1 ratio demonstrated significant cytotoxicity (approximately 40-42% lysis) against CD24-positive hepatoma cell lines . This effect relies on the antibody's Fc region engaging with Fc receptors on immune effector cells.

• Receptor-Mediated Internalization: CD24 antibodies undergo rapid internalization into CD24-positive cells (approximately 58.3% internalization rate for Huh-7 cells and 47.5% for BEL-7402 cells within 90 minutes) . This internalization property is crucial for antibody-drug conjugate (ADC) approaches, where the antibody delivers cytotoxic payloads intracellularly.

• Direct Apoptosis Induction: CD24 ligation can induce tumor cell apoptosis directly, contributing to the cytotoxic effect of anti-CD24 antibodies .

• ADC-Mediated Killing: Conjugates like hG7-BM3-VcMMAE combine the targeting specificity of CD24 antibodies with potent cytotoxic agents that work upon internalization to effectively suppress tumor growth in xenograft models .

These diverse mechanisms make CD24 antibodies versatile tools in cancer research and potential therapeutic development.

How do antibody-drug conjugates utilizing CD24 antibodies compare to unconjugated antibodies?

Antibody-drug conjugates (ADCs) utilizing CD24 antibodies offer several distinct advantages over unconjugated antibodies in research and potential therapeutic applications:

• Enhanced Cytotoxicity: ADCs like hG7-BM3-VcMMAE combine the specific targeting of CD24 antibodies with potent cytotoxic payloads, resulting in significantly increased tumor cell killing compared to unconjugated antibodies. This is especially valuable for tumors with moderate CD24 expression levels .

• Leveraging Internalization: CD24 antibodies demonstrate substantial internalization rates (47.5-58.3% within 90 minutes depending on cell line) . ADCs capitalize on this property by delivering cytotoxic payloads intracellularly, where they can exert maximum effect.

• Reduced Systemic Toxicity: The targeted nature of ADCs potentially allows for lower systemic exposure to cytotoxic agents compared to conventional chemotherapy, making them valuable research tools for studying precision targeting in tumor models.

• Complementary Mechanisms: While unconjugated antibodies rely primarily on immune-mediated mechanisms like ADCC, ADCs add direct cytotoxic action through their payloads, providing multiple mechanisms of action in research models.

• In vivo Efficacy: Studies demonstrate that CD24-targeted ADCs effectively suppress nude mice bearing HCC xenografts , offering robust models for studying targeted therapy approaches.

For research applications, this comparison provides important insights into different therapeutic modalities and their potential efficacy in CD24-positive cancer models.

What are common causes of non-specific binding with CD24 antibodies and how can they be mitigated?

Non-specific binding with CD24 antibodies can compromise experimental results. Common causes and their mitigations include:

• Fc Receptor Binding:

  • Cause: Fc receptors on certain cell types bind to the Fc portion of antibodies

  • Mitigation: Use Fc receptor blocking reagents (Human BD Fc Block™) prior to antibody incubation, or use F(ab')2 fragments that lack the Fc region

• High Antibody Concentration:

  • Cause: Excessive antibody concentration increases background binding

  • Mitigation: Perform careful titration experiments to determine the minimum concentration required for specific signal detection

• Insufficient Blocking:

  • Cause: Inadequate blocking of non-specific binding sites

  • Mitigation: Optimize blocking protocols using appropriate blocking buffers (BSA, serum, or commercial blocking solutions) and sufficient incubation times

• Cross-Reactivity with Similar Epitopes:

  • Cause: Antibody recognizes similar epitopes on other proteins

  • Mitigation: Validate antibody specificity through knockdown experiments (as demonstrated with CD24 knockdown resulting in 55.3-55.7% reduction in binding) and use blocking peptides specific to CD24

• Sample Processing Artifacts:

  • Cause: Fixation or permeabilization can expose epitopes that cause non-specific binding

  • Mitigation: Optimize fixation and permeabilization protocols for each sample type and include appropriate isotype controls

Implementing these strategies will enhance signal specificity and experimental reliability when working with CD24 antibodies.

How can I optimize CD24 antibody storage and handling to maintain long-term activity?

Optimizing CD24 antibody storage and handling is critical for maintaining activity and ensuring experimental reproducibility. Based on established protocols for antibodies like Human CD24 Antibody (MAB5248), follow these guidelines:

• Storage Temperature Management:

  • Store unopened antibody at -20°C to -70°C for up to 12 months from date of receipt

  • After reconstitution, store at 2-8°C for short-term use (up to 1 month) under sterile conditions

  • For long-term storage (up to 6 months), aliquot and store at -20°C to -70°C under sterile conditions

• Freeze-Thaw Management:

  • Use a manual defrost freezer rather than auto-defrost

  • Avoid repeated freeze-thaw cycles by preparing appropriate aliquot sizes for single use

  • Record the number of freeze-thaw cycles for each aliquot

• Reconstitution Best Practices:

  • Use sterile techniques when reconstituting lyophilized antibodies

  • Allow antibody vials to equilibrate to room temperature before opening

  • Reconstitute with the recommended buffer at the specified concentration

• Working Solution Preparation:

  • Prepare working dilutions on the day of experiment whenever possible

  • Use high-quality, sterile-filtered buffers appropriate for the application

  • Maintain cold chain during handling to prevent degradation

• Quality Control:

  • Periodically test antibody activity using positive control samples

  • Monitor changes in required concentration as an early indicator of degradation

Following these guidelines will help maintain antibody functionality and extend its useful lifespan for research applications.

What strategies can improve reproducibility in quantitative CD24 expression analyses?

Improving reproducibility in quantitative CD24 expression analyses requires systematic attention to multiple experimental variables:

• Standardized Sample Processing:

  • Implement consistent protocols for sample collection, storage, and preparation

  • For cell lines, standardize culture conditions, passage number, and harvesting procedures

  • For tissue samples, standardize fixation times, processing protocols, and sectioning thickness

• Antibody Validation and Calibration:

  • Use the same antibody clone, lot, and concentration across experiments

  • Include calibration standards in each experiment (quantitative beads for flow cytometry or standardized positive controls for IHC)

  • Validate antibody performance regularly using positive and negative controls

• Instrumentation Calibration:

  • For flow cytometry, use calibration beads to standardize voltage settings and fluorescence intensity

  • For imaging-based methods, implement standard exposure settings and calibration slides

  • Perform regular quality control checks on all instruments

• Data Normalization and Analysis:

  • Use consistent gating strategies in flow cytometry

  • Implement automated image analysis algorithms to reduce subjective interpretation

  • Include housekeeping markers or internal standards for normalization

• Biological Controls:

  • Include cell lines with known CD24 expression levels (e.g., Huh-7 and BEL-7402 as positive controls, HL-7702 as negative control)

  • Use isotype controls to establish background thresholds

  • Consider spike-in controls for complex samples

• Quantitative Reporting:

  • Report absolute values rather than relative measures where possible

  • Include measures of variability (standard deviation, coefficient of variation)

  • Document all experimental parameters comprehensively

Implementing these strategies creates a robust framework for generating reproducible quantitative data on CD24 expression across different experimental conditions and research laboratories.

How is antibody engineering advancing CD24-targeted therapeutic approaches?

Antibody engineering is significantly advancing CD24-targeted therapeutic approaches through multiple sophisticated strategies:

• Humanization Optimization: Advanced CDR grafting techniques combined with computational modeling are producing humanized antibodies like hG7-BM3 with reduced immunogenicity while maintaining high binding affinity (KD of 5.70×10^-10 M) . These engineered antibodies demonstrate tumor-targeting capabilities in vivo with tumor/normal tissue ratios of 2.50 .

• Antibody-Drug Conjugates: Engineering of CD24 antibodies into ADCs (such as hG7-BM3-VcMMAE) leverages the rapid internalization properties of CD24 (47.5-58.3% internalization within 90 minutes) to deliver cytotoxic payloads specifically to tumor cells, inducing apoptosis and suppressing tumor growth in xenograft models.

• Developability Optimization: Implementation of early-stage screening for parameters like self-interaction, aggregation propensity, thermal stability, and colloidal stability helps identify and engineer antibody candidates with superior pharmaceutical properties .

• Fc Engineering: Modification of the Fc region enhances effector functions like ADCC or extends half-life, improving the therapeutic potential of CD24-targeted antibodies against cancer cells.

• Bispecific Antibody Development: Creation of bispecific antibodies that simultaneously target CD24 and immune effector cells or other tumor antigens to enhance therapeutic efficacy through multiple mechanisms.

These engineering advances are expanding the potential of CD24 antibodies beyond research tools to promising therapeutic candidates for cancers with elevated CD24 expression.

What emerging technologies are improving the specificity and sensitivity of CD24 detection?

Emerging technologies are revolutionizing the specificity and sensitivity of CD24 detection across multiple research platforms:

• Single-Cell Analysis Technologies:

  • High-dimensional cytometry (mass cytometry/CyTOF) allows simultaneous detection of CD24 alongside dozens of other markers with minimal spectral overlap

  • Single-cell RNA sequencing provides correlation between CD24 protein expression and transcriptional profiles at individual cell resolution

• Advanced Imaging Modalities:

  • Super-resolution microscopy techniques overcome diffraction limits to visualize CD24 distribution at nanometer scale

  • Multiplexed ion beam imaging (MIBI) and imaging mass cytometry enable visualization of CD24 alongside numerous other markers in tissue sections

• Proximity-Based Detection Methods:

  • Proximity ligation assays (PLA) detect interactions between CD24 and binding partners with superior sensitivity

  • FRET (Förster Resonance Energy Transfer)-based approaches allow real-time monitoring of CD24 interactions

• Engineered Detection Reagents:

  • Nanobodies and single-domain antibodies provide improved tissue penetration and reduced background

  • Aptamer-based detection offers alternatives to traditional antibodies with potentially superior specificity

• Computational Analysis Enhancements:

  • Machine learning algorithms improve signal extraction from complex datasets

  • Automated image analysis reduces operator variability and enhances quantitative precision

These technologies are enabling researchers to study CD24 biology with unprecedented resolution and sensitivity, advancing our understanding of its role in normal physiology and disease processes.

What are the most promising applications of CD24 antibodies in cancer research beyond hepatocellular carcinoma?

CD24 antibodies show promising applications across multiple cancer types beyond hepatocellular carcinoma, expanding their utility in oncological research:

• Ovarian Cancer Research:

  • CD24 has been detected in ovarian cancer tissue using immunohistochemistry

  • CD24 antibodies can help identify and isolate cancer stem cell populations that contribute to chemoresistance and tumor recurrence

• Breast Cancer Investigation:

  • CD24 expression patterns help distinguish breast cancer subtypes

  • Anti-CD24 antibodies are valuable in studying mammospheres of letrozole-resistant breast cancer, which enhance breast cancer aggressiveness

• Colorectal Cancer Studies:

  • CD24 antibodies facilitate identification of CD24-positive tumor cells in colon tissues

  • They enable investigation of CD24's role in metastasis and tumor progression

• Pancreatic Cancer Research:

  • CD24 is a marker for pancreatic cancer stem cells

  • Antibodies help isolate and characterize these populations for therapeutic targeting studies

• Tumor Microenvironment Interactions:

  • CD24 antibodies are crucial for studying how CD24 on tumor cells interacts with Siglec-10 on immune cells, revealing immune evasion mechanisms

  • Research shows malignant ascite-derived extracellular vesicles inhibit T cell activity by upregulating Siglec-10 expression

• Liquid Biopsy Development:

  • CD24 antibodies can detect circulating tumor cells and tumor-derived exosomes in blood samples

  • They enable monitoring of treatment response and disease progression through minimally invasive means

These diverse applications highlight the versatility of CD24 antibodies as valuable tools across the cancer research spectrum, potentially leading to novel diagnostic and therapeutic approaches.