ML5 Antibody

What is the ML5 antibody and what specific target does it recognize?

ML5 is a monoclonal mouse IgG2a kappa antibody that specifically binds to human CD24, a 35-70 kDa glycophosphatidylinositol (GPI)-linked glycoprotein . CD24 is also known as CD24A, signal transducer CD24, or small cell lung carcinoma cluster 4 antigen . The antibody recognizes the full-length version of the CD24 protein and has been validated extensively for reactivity with human samples . Based on immunogen sequence analysis, ML5 is expected to show cross-reactivity with CD24 from chimpanzees and baboons, making it valuable for comparative studies across primate species .

What validated applications can ML5 antibody be used for in research?

ML5 antibody has been validated for multiple research applications including:

• Flow Cytometry (recommended dilution: 1:10-1:1000)

• Immunocytochemistry/Immunofluorescence (recommended usage: <0.125 μg/10^6 cells)

• Immunohistochemistry (recommended dilution: 1:10-1:500)

• Immunohistochemistry-Frozen (recommended concentration: 5-25 μg/ml)

What cell types express CD24 that can be detected using ML5 antibody?

CD24 shows expression on multiple cell types that can be detected using ML5 antibody:

• B lymphocyte lineage cells (except plasma cells)

• Neutrophils

• Eosinophils

• Dendritic cells

• Neural cells

• Epithelial cells

• Muscle cells

• Various cancer cells including hepatocellular carcinoma

CD24 is variably expressed on B lineage cells during development and differentiation, making ML5 a valuable marker for distinguishing between lymphocyte developmental stages .

How should I optimize ML5 antibody for flow cytometry applications?

For optimal results in flow cytometry applications with ML5 antibody:

• Titration: While the recommended range is 1:10-1:1000, perform a titration experiment with different antibody concentrations (e.g., 0.5 μg/10^6 cells, 0.25 μg/10^6 cells, 0.125 μg/10^6 cells) to determine optimal signal-to-noise ratio .

• Controls: Include appropriate isotype controls (Mouse IgG2a kappa) to assess non-specific binding, and include both CD24 positive (e.g., MCF-7 cells) and negative cell populations .

• Multiparameter analysis: ML5 has been successfully used in multiparameter flow cytometry panels. When designing panels, consider fluorophore brightness and spectral overlap with other markers .

• Sample preparation: Fresh samples yield optimal results. For peripheral blood lymphocytes, use either purified ML5 detected with anti-mouse IgGs FITC or directly conjugated ML5-FITC, depending on your experimental design .

• Data analysis: When analyzing CD24 expression, consider using biexponential display for proper visualization of the full range of expression levels.

What are the recommended protocols for immunocytochemistry/immunofluorescence with ML5 antibody?

For optimal immunocytochemistry/immunofluorescence results:

• Fixation: Use 4% paraformaldehyde fixation for 10-15 minutes at room temperature .

• Permeabilization: If intracellular staining is required, permeabilize with 0.1% Triton X-100 for 5-10 minutes .

• Blocking: Block non-specific binding with appropriate blocking buffer (typically 5-10% normal serum from the same species as the secondary antibody).

• Primary antibody: Apply ML5 at concentrations <0.125 μg/10^6 cells. For adherent cells, this typically translates to 1-5 μg/ml .

• Secondary detection: Use appropriate fluorophore-conjugated anti-mouse IgG secondary antibodies.

• Counterstaining: Include nuclear counterstain (e.g., DAPI) and any additional markers of interest.

• Validation: MCF-7 breast cancer cells have been validated as a positive control for CD24 detection using ML5 in immunofluorescence applications .

How stable is the ML5 antibody and what are the optimal storage conditions?

The ML5 antibody should be stored at 4°C and should not be frozen to maintain its stability and functionality . The typical formulation includes:

• PBS buffer

• 0.02% Sodium Azide as a preservative (note that sodium azide yields highly toxic hydrazoic acid under acidic conditions; dilute azide compounds in running water before discarding)

How can ML5 antibody be used to identify and isolate cancer stem cells?

CD24 expression is a key marker for cancer stem cells in several tumor types. For identification and isolation of CD24-expressing cancer stem cells:

• Flow cytometry/FACS isolation: Use ML5 in combination with other stem cell markers. For example, CD44+/CD24- phenotype in breast cancer or CD24+/CD44+ in other cancer types can identify stem-like populations .

• Imaging applications: ML5 has been conjugated to near-infrared fluorescent dye multiplex probe amplification (MPA) to form G7mAb-MPA for in vivo imaging of CD24+ tumors, such as Huh7 hepatocellular carcinoma xenografts .

• Functional validation: After isolation of CD24+ cells using ML5 antibody, confirm stem cell properties through:

  • Sphere formation assays

  • In vivo tumorigenicity assays

  • Differentiation capacity assays

  • Drug resistance assessments

• Comparative analysis: Compare ML5 with other anti-CD24 antibodies (such as G7mAb) for specific cancer applications. Research has shown that custom-developed antibodies like G7mAb have similar binding capacity to ML5 in immunohistochemical assays .

How can I use ML5 antibody to study CD24's role in immune cell differentiation?

CD24 plays a significant role in immune cell differentiation and function:

• B cell development tracking: Use ML5 in flow cytometry panels with additional B cell markers (CD19, CD21, CD27, IgD) to track B cell maturation stages, as CD24 expression varies during B cell development .

• Functional assays: Combine ML5 staining with functional readouts (proliferation, cytokine production) to correlate CD24 expression with specific immune cell functions.

• Multiparameter analysis: Create comprehensive immunophenotyping panels including:

  • B cell markers: CD19, CD21, CD27, IgD

  • T cell markers: CD3, CD4, CD8

  • Activation markers: CD38, HLA-DR

  • Other markers: CD11c, CXCR3, CXCR5

• Time-course experiments: Monitor CD24 expression changes during immune cell activation or differentiation over time using ML5 antibody.

How can ML5 antibody be used in imaging applications for CD24+ tumors?

ML5 antibody can be effectively utilized for various imaging applications targeting CD24+ tumors:

• Near-infrared fluorescence imaging: Similar to the approach with G7mAb and G7S, ML5 can be conjugated to near-infrared fluorescent dyes for in vivo imaging of CD24+ tumor xenografts .

• Multiplex immunohistochemistry: ML5 can be incorporated into multiplex IHC panels to study CD24 expression in relation to other tumor markers and the tumor microenvironment.

• Protocol optimization:

  • For formalin-fixed paraffin-embedded tissues: Use heat-induced epitope retrieval

  • For frozen sections: Apply ML5 at 5-25 μg/ml concentration

  • For optimal signal-to-noise ratio: Titrate antibody concentrations and optimize incubation conditions

• Validation: Confirm specificity using positive controls (e.g., MCF-7 cells) and negative controls (CD24-negative cell lines or tissues) .

What are common issues when using ML5 antibody and how can they be resolved?

How can I validate the specificity of ML5 antibody in my experimental system?

To validate ML5 antibody specificity:

• Positive controls: Use cell lines known to express CD24, such as MCF-7 breast cancer cells which have been validated for ML5 antibody .

• Negative controls:

  • Isotype control (Mouse IgG2a kappa) to assess non-specific binding

  • CD24-negative cell lines or tissues

  • CD24 knockdown/knockout models if available

• Blocking experiments: Pre-incubate ML5 antibody with recombinant CD24 protein before staining to confirm binding specificity.

• Multiple detection methods: Confirm CD24 expression using alternative methods (e.g., RT-PCR, Western blot) and compare with ML5 antibody staining patterns.

• Comparison with other anti-CD24 clones: Compare staining patterns with other validated anti-CD24 antibodies to confirm target specificity .

What considerations should be made when using ML5 in multicolor flow cytometry panels?

When incorporating ML5 into multicolor flow cytometry panels:

• Panel design considerations:

  • Select fluorophores based on expression level of CD24 in your samples (brighter fluorophores for lower expressed antigens)

  • Consider spectral overlap and compensation requirements

  • Match fluorophore brightness with antigen density

• Titration: Titrate ML5 antibody for each specific application and fluorophore conjugate to determine optimal signal-to-noise ratio .

• Controls:

  • Include FMO (Fluorescence Minus One) controls

  • Use isotype controls with matching fluorophores

  • Include compensation controls for each fluorophore

• Sample preparation: Optimize fixation and permeabilization based on all markers in your panel, not just CD24.

• Example validated panel: ML5 has been successfully used in panels including markers such as CD3, CXCR5, HLA-DR, CD4, CD69, CCR7, CD45RA, IgD, CD11c, CD38, CD19, CD21, CXCR3, CD27, and others for comprehensive immunophenotyping .

How can ML5 antibody be used in cancer research beyond cell identification?

ML5 antibody applications in cancer research extend beyond basic cell identification:

• Prognostic biomarker evaluation: CD24 expression assessed by ML5 can be correlated with clinical outcomes in various cancer types.

• Treatment response monitoring: Track changes in CD24+ cell populations following therapy using flow cytometry with ML5.

• Therapeutic targeting validation: ML5 can be used to validate CD24 as a therapeutic target, similar to the approach with G7mAb and G7S antibodies in hepatocellular carcinoma models .

• Cancer stem cell research: Identify and isolate CD24+ cancer stem cell populations for:

  • Drug screening assays

  • Resistance mechanism studies

  • Tumor initiation capacity assessment

• In vivo imaging: When conjugated to appropriate imaging agents, ML5 can be used to track CD24+ tumors in vivo, similar to the G7mAb-MPA approach demonstrated in hepatocellular carcinoma xenografts .

What is the role of ML5 antibody in studying immune responses after vaccination?

ML5 antibody has applications in vaccination and immune response studies:

• B cell response tracking: Monitor changes in CD24 expression on B cells following vaccination to track B cell activation and differentiation.

• Post-vaccination analysis: ML5 has been used in studies examining antibody responses after SARS-CoV-2 mRNA vaccination, particularly in patients previously treated with anti-CD20 antibodies .

• Methodology:

  • Use ML5 in flow cytometry panels alongside markers of B cell activation and maturation

  • Track temporal changes in CD24 expression patterns following vaccination

  • Correlate CD24 expression with antibody production capacity

• Sample processing protocol:

  • Isolate PBMCs from peripheral blood

  • Stain with viability dye

  • Block with human FcX

  • Perform multiparameter staining including ML5 (anti-CD24)

  • Analyze using multiparameter flow cytometry

How does CD24 detection with ML5 contribute to understanding immune cell interactions?

CD24 plays important roles in immune cell interactions that can be studied using ML5 antibody:

• Adhesion and migration: CD24 functions as an adhesion receptor, with ML5 allowing researchers to track its role in cell-cell interactions.

• Ligand interactions: CD24 interacts with P-selectin (CD62P) on activated platelets and endothelium. ML5 can be used in binding inhibition studies to understand these interactions .

• Signaling studies: CD24 signaling may be triggered by lectin-like ligand binding to CD24 carbohydrates. ML5 can help track these interactions and subsequent signaling cascades.

• B cell regulation: CD24 promotes antigen-dependent B cell proliferation while preventing terminal differentiation into antibody-forming cells. ML5 allows researchers to track this role in B cell function .

• Autoimmunity research: CD24, in association with SIGLEC10, may be involved in suppressing immune responses to danger-associated molecular patterns (DAMPs). ML5 can be used to study this regulatory role in autoimmunity .