CD24 Antibody, Biotin conjugated

What is CD24 and what cell types express this marker?

CD24, also known as Heat Stable Antigen (HSA), is a 35-50 kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein that is anchored in the plasma membrane via phosphatidylinositol. It is expressed by multiple cell types including erythrocytes, thymocytes, peripheral lymphocytes, and cells of myeloid lineage . CD24 is particularly important in B-cell development, with expression beginning in the bone marrow pro-B-cell compartment and continuing through mature, surface Ig positive B-cells, though plasma cell expression is typically very low or negative . CD24 is also expressed on the majority of B-lineage acute lymphoblastic leukemias, B-cell CCLs, and B-cell non-Hodgkin's lymphomas . The variable glycosylation of CD24 results in heterogeneity of molecular mass on cells of different lineages, causing antibodies to CD24 to exhibit subtle differences in staining levels on different lymphocyte populations .

What are the key differences between mouse and human CD24 antibodies?

Mouse CD24 antibodies such as the M1/69 clone react specifically with mouse CD24 molecules, while human CD24 antibodies like the eBioSN3 (SN3 A5-2H10) clone are designed to recognize human CD24. The M1/69 antibody has been tested by flow cytometric analysis of mouse splenocytes , whereas the SN3 antibody has been tested on normal human peripheral blood cells . Both antibodies are available as biotin conjugates and are primarily used for flow cytometric analysis. When selecting an antibody, researchers must ensure they choose the appropriate species-specific variant based on their experimental model. While both antibodies target the same molecule conceptually, their epitope recognition, optimal concentrations, and performance characteristics may differ due to species-specific variations in CD24 structure and expression patterns.

How does CD24 function in cellular signaling pathways?

CD24 plays important roles in cellular signaling by interacting with multiple signaling molecules. One key interaction is between CD24 and P-selectin (CD62P) , which facilitates cell adhesion and migration processes. In the context of cancer immunotherapy, CD24 functions as a "do not eat me" signal on cancer cells by interacting with the inhibitory receptor Siglec-10 on tumor-associated macrophages (TAMs), effectively preventing macrophage-mediated phagocytosis of cancer cells . When this interaction is disrupted through anti-CD24 antibodies, it enhances macrophage-mediated phagocytosis and promotes cytotoxic T cell function in the tumor microenvironment . Additionally, CD24 appears to play a role in B-cell proliferation and maturation, as well as in the control of autoimmunity . CD24 is also necessary for steady-state T lymphocyte proliferation in lymphocytopenic environments .

What are the optimal conditions for using biotin-conjugated CD24 antibodies in flow cytometry?

For optimal flow cytometry results with biotin-conjugated CD24 antibodies, several key parameters should be considered. For the mouse M1/69 antibody, the recommended concentration is ≤0.125 μg per test, where a test is defined as the amount of antibody that will stain a cell sample in a final volume of 100 μL . For the human eBioSN3 antibody, the recommended concentration is ≤0.25 μg per test in the same final volume . Cell numbers should be determined empirically but can range from 10^5 to 10^8 cells/test for both antibodies .

Careful titration of the antibody is essential for optimal performance. When setting up the experiment, prepare a single-cell suspension in an appropriate buffer (PBS with 1-2% FBS is often sufficient), and include proper controls (isotype control, unstained sample, and positive control). Since these antibodies are biotin-conjugated, a secondary step with a streptavidin-fluorophore conjugate is necessary. Consider using compensation controls if performing multicolor flow cytometry. For optimal results, perform staining at 4°C in the dark for 20-30 minutes, followed by washing steps to remove unbound antibody.

How can I optimize detection of biotin-conjugated CD24 antibodies in various experimental systems?

To optimize detection of biotin-conjugated CD24 antibodies:

• Secondary reagent selection: Choose an appropriate streptavidin-conjugated fluorophore based on your flow cytometer configuration and other fluorophores in your panel. Common options include streptavidin-PE, streptavidin-APC, or streptavidin-FITC.

• Signal amplification: If needed, consider using a biotin-streptavidin amplification system where multiple fluorophore-conjugated streptavidin molecules can bind to each biotin molecule.

• Blocking approach: To reduce non-specific binding, include a blocking step with normal serum from the same species as the cells being analyzed, or commercially available Fc receptor blocking reagents.

• Titration optimization: Perform a titration experiment with both the primary antibody and the streptavidin conjugate to identify the optimal concentrations that provide the best signal-to-noise ratio.

• Sample processing: Ensure cells remain viable throughout the staining protocol, as dead cells can increase background fluorescence. Consider including a viability dye to exclude dead cells during analysis.

• Fixation considerations: If cells need to be fixed, do so after the staining is complete, as fixation before staining may alter epitope recognition. Ensure the fixative is compatible with the fluorophores used.

What control samples should be included when using CD24 antibodies in flow cytometry?

For rigorous flow cytometry experiments with CD24 antibodies, the following controls should be included:

• Unstained cells: To establish autofluorescence levels and set baseline parameters.

• Isotype control: Use a biotin-conjugated antibody of the same isotype, host species, and at the same concentration as the CD24 antibody to identify non-specific binding.

• Fluorescence-minus-one (FMO) control: Include all fluorophores in your panel except the streptavidin conjugate used to detect the CD24 antibody to establish proper gating boundaries.

• Positive control: Include a sample known to express CD24 (e.g., B cells for human samples or thymocytes for mouse samples).

• Negative control: Include a cell population known not to express CD24 (e.g., certain T cell subsets).

• Secondary-only control: Samples stained only with the streptavidin conjugate (no primary antibody) to assess background binding of the secondary reagent.

• Compensation controls: If performing multicolor flow cytometry, include single-color controls for each fluorophore to correct for spectral overlap.

These controls enable proper interpretation of results and help troubleshoot any unexpected findings in experimental samples.

How is CD24 being targeted in cancer immunotherapy research?

CD24 has emerged as a promising target for cancer immunotherapy because it functions as a "do not eat me" signal on cancer cells, similar to CD47 and PD-L1 checkpoints . Current advanced research approaches include:

• Monoclonal antibody development: Humanized anti-CD24 antibodies like ATG-031 are being investigated in clinical trials for their ability to block the CD24-Siglec-10 interaction . This blocking enhances macrophage-mediated phagocytosis of cancer cells and promotes cytotoxic T cell function in the tumor microenvironment.

• Fc-mediated mechanisms: Anti-CD24 antibodies like ATG-031 also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) towards target tumor cells through IgG1 Fc-dependent effects .

• Macrophage reprogramming: Anti-CD24 antibody treatment has been shown to transform macrophages from a tumor-tolerant M2 phenotype to an antitumor M1 phenotype , significantly inhibiting tumor growth both as monotherapy and synergistically with immune checkpoint inhibitors and/or chemotherapies in preclinical models.

• NK cell activation: Anti-CD24 antibodies can improve NK cell function. For example, the cG7 antibody has been shown to increase cytotoxicity and enhance secretion of IFN-γ and TNF-α by NK cells in a dose-dependent manner, resulting in reduced tumor growth and improved survival rates in mouse xenograft models .

• Targeting cancer stem cells: CD24 is increasingly correlated with cancer stem cells, making it a potential target for eliminating these therapy-resistant tumor subpopulations .

What are the challenges in targeting CD24 with antibody therapeutics?

Despite its promising potential, targeting CD24 with antibody therapeutics presents several challenges:

• Expression in normal tissues: CD24 is expressed not only on various cancer cells but also on normal tissues such as the esophagus and thyroid , raising concerns about potential on-target, off-tumor toxicity.

• Glycosylation heterogeneity: CD24 is a variably glycosylated molecule, and this glycosylation pattern differs between cancer cells and normal cells . The complex and elusive nature of these glycosylation patterns presents a significant challenge for developing antibodies that specifically target cancer-associated forms of CD24.

• Risk of inflammation: Therapeutic targeting of CD24 may potentially trigger inflammatory responses as it interferes with immunoregulatory pathways .

• Tumor resistance mechanisms: As with other immunotherapeutic approaches, tumors may develop resistance mechanisms to CD24-targeted therapies over time .

• Optimization of combination therapies: Determining the optimal combinations with other cancer therapies, such as immune checkpoint inhibitors or chemotherapies, requires extensive preclinical and clinical investigation .

• Dosing and safety considerations: Identifying the appropriate dosing strategies to minimize toxicity while maintaining efficacy is crucial, as demonstrated in the PERFORM clinical trial's implementation of a priming dose approach to mitigate cytokine release syndrome .

How does CD24 expression correlate with B cell development and cancer progression?

CD24 expression plays a dynamic role throughout B cell development and has significant implications in cancer progression:

• B cell development markers: CD24 is expressed at multiple stages of B-cell development, beginning with the bone marrow pro-B-cell compartment and continuing through mature, surface Ig positive B-cells . This expression pattern has made CD24 valuable for resolving stages of B lymphopoiesis in mouse bone marrow .

• Pro- and pre-B lymphocyte regulation: CD24 can encourage apoptosis in early differentiating B cells, and transgenic mice that overexpress CD24 show significantly reduced numbers of pro-B and pre-B lymphocytes , suggesting a regulatory role in early B cell development.

• Cancer stem cell marker: In various cancer types, CD24 serves as a marker for cancer stem cells, which are associated with tumor initiation, metastasis, and therapy resistance .

• Correlation with cancer progression: High CD24 expression has been observed in B-lineage acute lymphoblastic leukemias, B-cell CCLs, and B-cell non-Hodgkin's lymphomas , suggesting a potential role in malignant transformation or disease progression.

• Immunosuppressive tumor microenvironment: CD24 expression on tumor cells contributes to an immunosuppressive tumor microenvironment by inhibiting macrophage phagocytosis through the CD24-Siglec-10 interaction . Anti-CD24 monoclonal antibody treatment has been shown to reverse this immunosuppression in models of oral squamous cell carcinoma, leading to increased infiltration of CD4+ and CD8+ T cells .

• Potential therapeutic target: The differential expression and glycosylation patterns of CD24 between normal and malignant B cells make it a potential therapeutic target, particularly if antibodies can be developed that specifically recognize cancer-associated forms of CD24 .

What factors can affect CD24 antibody staining in flow cytometry?

Several factors can influence CD24 antibody staining in flow cytometry experiments:

• Antibody concentration: Suboptimal antibody concentrations can lead to either weak staining (too little antibody) or high background (too much antibody). Careful titration is recommended, starting with the suggested concentration (≤0.125 μg/test for mouse M1/69 or ≤0.25 μg/test for human eBioSN3) .

• Cell preparation: Harsh cell isolation procedures may damage surface proteins including CD24. Use gentle cell isolation techniques and maintain cells at 4°C during processing to preserve epitope integrity.

• Buffer composition: The staining buffer can affect antibody binding. PBS with 1-2% FBS or BSA works well for most applications. Ensure the pH is appropriate (typically 7.2-7.4).

• Viability issues: Dead or dying cells often show non-specific antibody binding. Include a viability dye to exclude dead cells from analysis.

• Expression heterogeneity: CD24 is a variably glycosylated molecule, resulting in heterogeneity of molecular mass on different cell lineages . This can cause variation in staining intensity across different cell populations.

• Fixation effects: If cells need to be fixed, certain fixatives may alter the CD24 epitope. If possible, perform a comparison of staining before and after fixation to assess any impact.

• Incubation conditions: Incubation time and temperature affect antibody binding kinetics. Typically, 20-30 minutes at 4°C is recommended, but optimization may be necessary.

• Washing efficiency: Insufficient washing can leave residual unbound antibody, increasing background. Include at least 2-3 washing steps with adequate buffer volumes.

How can I distinguish between specific and non-specific binding when using biotin-conjugated CD24 antibodies?

To distinguish between specific and non-specific binding:

• Proper controls: Always include an isotype control antibody that matches the species, isotype, and conjugation (biotin) of your CD24 antibody. This helps identify non-specific binding due to Fc receptor interactions or other non-specific mechanisms.

• Blocking strategy: Incorporate a blocking step using normal serum (2-5%) from the same species as your cells or commercially available Fc receptor blocking reagents before adding the primary antibody.

• Titration experiments: Perform antibody titration to identify the optimal concentration that provides the best signal-to-noise ratio. Plot the staining index (mean fluorescence intensity of positive population divided by the standard deviation of the negative population) against antibody concentration to identify the optimal concentration.

• Secondary reagent optimization: When using biotin-conjugated antibodies, the streptavidin-fluorophore conjugate concentration also needs optimization to minimize background.

• Comparison with known positive and negative populations: Include cell populations with known CD24 expression patterns to validate your staining protocol.

• Fluorescence-minus-one (FMO) controls: These help establish proper gating boundaries by including all fluorophores except the one conjugated to streptavidin for detecting CD24.

• Data analysis strategies: During analysis, use bivariate plots comparing CD24 expression with lineage markers to identify specific populations and facilitate gating strategies that distinguish specific from non-specific binding.

What technical considerations should be addressed when using CD24 antibodies in combination with other markers?

When using CD24 antibodies in multiparameter flow cytometry or other applications:

• Panel design considerations:

  • Ensure there is minimal spectral overlap between your selected fluorophores

  • Place the brightest fluorophores on markers with the lowest expression and vice versa

  • Consider co-expression patterns to facilitate proper gating strategies

• Compensation requirements:

  • Prepare single-color controls for each fluorophore in your panel

  • Use compensation beads or cells expressing high levels of each marker

  • Perform compensation either during acquisition or during analysis

• Titration of all antibodies individually:

  • Optimize each antibody separately before combining them

  • Re-validate the panel after combination, as some antibodies may compete for binding sites

• Order of antibody addition:

  • For some marker combinations, the order of antibody addition may affect binding

  • Consider testing simultaneous versus sequential staining approaches

• Effect of biotin conjugation:

  • When using other biotin-conjugated antibodies, consider potential competition for streptavidin binding

  • If using multiple biotin-conjugated antibodies, ensure sufficient streptavidin-fluorophore is available

  • Consider using different detection systems (direct fluorophore conjugates) for some markers to avoid competition

• Buffer compatibility:

  • Ensure all antibodies in your panel perform optimally in the selected buffer

  • Some specialized buffers may enhance staining of certain markers but interfere with others

• Epitope blocking or modulation:

  • Some antibody combinations may result in steric hindrance

  • Test different clones if you suspect epitope blocking is occurring

• Data analysis complexity:

  • With increasing parameters, data analysis becomes more complex

  • Consider using dimensionality reduction techniques like tSNE or UMAP for high-parameter datasets

  • Establish clear gating hierarchies based on lineage markers before examining CD24 expression

What emerging applications are being developed for CD24 antibodies beyond flow cytometry?

While flow cytometry remains the primary application for CD24 antibodies, several emerging applications show promise:

• Therapeutic antibody development: Anti-CD24 antibodies are being developed as cancer therapeutics, as exemplified by ATG-031, which is currently in clinical trials for advanced solid tumors and B-cell non-Hodgkin's lymphoma .

• Antibody-drug conjugates (ADCs): CD24's expression on various cancer cells makes it a potential target for ADCs, which combine the targeting precision of antibodies with the cytotoxic effects of chemotherapeutic agents .

• CAR-T and CAR-NK cell therapies: Chimeric antigen receptor T cells or NK cells directed against CD24 represent another potential therapeutic approach being explored in preclinical research .

• Imaging applications: CD24 antibodies conjugated to imaging agents could potentially be used for cancer detection and monitoring.

• Isolation of cancer stem cells: CD24 antibodies can be used to isolate and characterize cancer stem cell populations, which may lead to improved understanding of tumor initiation and progression.

• Combination immunotherapy approaches: Research is exploring the potential synergistic effects of combining anti-CD24 antibodies with established immunotherapies such as immune checkpoint inhibitors and/or conventional treatments like chemotherapy .

• Biomarker development: CD24 expression patterns may serve as prognostic or predictive biomarkers in various cancer types, informing treatment decisions and patient stratification.

How might advances in understanding CD24 glycosylation patterns impact antibody development?

The variable glycosylation of CD24 presents both challenges and opportunities for antibody development:

• Cancer-specific targeting: Identifying cancer-specific glycosylation patterns of CD24 could lead to the development of antibodies that selectively target malignant cells while sparing normal tissue, reducing potential toxicity .

• Enhanced binding specificity: Antibodies directed against specific glycoforms of CD24 could show improved binding specificity, potentially enhancing therapeutic efficacy.

• Novel epitope discovery: Mapping the glycosylation sites and patterns of CD24 may reveal novel epitopes that could be targeted by next-generation antibodies.

• Reduced immunogenicity: Understanding how glycosylation affects immunogenicity could help design antibodies with reduced potential for adverse immune reactions.

• Improved therapeutic index: Antibodies that recognize cancer-specific glycoforms of CD24 might have an improved therapeutic index, allowing for higher dosing with lower toxicity.

• Molecular imaging advances: Glycoform-specific antibodies could enable more precise molecular imaging of CD24-expressing tumors.

• Combination therapy optimization: Knowledge of how glycosylation affects CD24 function could inform optimal combinations with other therapeutic modalities.

The current research indicates there is "an urgent need to find the unique glycosylation pattern in cancer cells in order to design antibody drugs targeting this specific site of CD24" , highlighting the importance of this area for future development.