CD28 Antibody, Biotin

What is CD28 and what role does it play in immune cell function?

CD28 is a 44 kDa homodimeric surface glycoprotein expressed by thymocytes, mature T cells, and plasma cells. As a critical T cell costimulatory receptor, CD28 provides the essential second activation signal by binding to CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells. CD28 signaling significantly augments IL-2 and IL-2 receptor expression and enhances the cytotoxicity of CD3-activated T cells . Beyond co-stimulation, CD28 prevents T cells from entering an anergic state or undergoing premature apoptosis. In plasma cells, CD28 signaling plays a distinct role in supporting long-term survival and antibody production, particularly in bone marrow long-lived plasma cells (LLPCs) .

What are the typical applications for biotinylated CD28 antibodies in flow cytometry?

Biotinylated CD28 antibodies are valuable tools for flow cytometric analysis of human peripheral blood cells and other T cell populations. The CD28.2 monoclonal antibody specifically reacts with human CD28 and can be used at concentrations of ≤1 μg per test, where a test is defined as the amount needed to stain a cell sample in 100 μL final volume (with cell numbers ranging from 10^5 to 10^8 cells/test) . Applications include:

• Phenotyping of T cell subsets

• Assessing activation status of T cells

• Monitoring changes in CD28 expression during immune responses

• Detecting CD28+ plasma cells in bone marrow samples

The biotinylation allows for flexible secondary detection using various streptavidin-conjugated fluorophores, enabling integration into complex multi-color panels.

How does CD28 signaling differ between T cells and plasma cells?

CD28 signaling exhibits significant functional differences between T cells and plasma cells:

While both cell types express CD28, loss of CD28 or its ligands reduces LLPC half-life from 426 to 63 days and impairs the maintenance of long-term antibody titers, without affecting SLPC populations . This demonstrates the distinct role of CD28 in humoral immunity beyond its well-established function in T cell activation.

How should biotinylated CD28 antibodies be optimized for flow cytometry experiments?

Optimizing biotinylated CD28 antibodies for flow cytometry requires careful consideration of several technical parameters:

• Titration: Determine optimal concentration through serial dilutions (typically starting at 1 μg per test for CD28.2 clone), evaluating signal-to-noise ratio at each concentration .

• Streptavidin-fluorophore selection: Choose appropriate fluorophores based on:

  • Expected expression level of CD28 on target cells

  • Other markers in your panel to minimize spectral overlap

  • Instrument configuration

• Staining protocol optimization:

  • Sequential staining (biotinylated antibody followed by washing and then streptavidin addition) typically yields better results than premixed reagents

  • Include blocking steps for endogenous biotin when necessary

  • Maintain consistent staining temperature (typically 4°C) and time (30 minutes)

• Controls:

  • Include biotinylated isotype control antibodies

  • Use FMO (Fluorescence Minus One) controls for multi-color panels

  • Include known CD28+ and CD28- populations to verify staining efficacy

These optimization steps are critical for generating reliable and reproducible data in CD28 expression studies.

What experimental controls are essential when using biotinylated CD28 antibodies in functional assays?

When designing functional assays with biotinylated CD28 antibodies, include these essential controls:

• Non-biotinylated CD28 antibody (same clone): Distinguishes between CD28-specific effects and biotin-related effects.

• Non-specific biotinylated antibody (same isotype): Reveals any artifactual effects of biotin alone.

• CD3-only stimulation: Establishes baseline T cell response without CD28 co-stimulation.

• Combined CD3+CD28 stimulation: Demonstrates the full co-stimulatory effect.

• Unstimulated condition: Establishes baseline activation.

For CAR-T cell studies with CD28 domains, additional controls are critical as research has shown that UniCARCD28-T cells can be activated by biotin alone, independent of target antigen engagement . When testing third-generation constructs combining CD28 and 4-1BB domains, include conditions with non-specific biotinylated IgG to assess potential non-specific activation .

What is the optimal protocol for using biotinylated CD28 antibodies in T cell activation assays?

For T cell activation assays using biotinylated CD28 antibodies, the following protocol is recommended:

• Plate preparation:

  • Coat 96-well plates with sub-optimal concentrations of anti-CD3 (0.1-0.5 μg/mL) overnight at 4°C

  • Wash plates 3× with PBS before cell addition

• Cell preparation:

  • Isolate T cells or PBMCs using density gradient centrifugation or magnetic separation

  • Adjust to 1-2×10^6 cells/mL in complete medium

• Stimulation setup:

  • Add 100 μL cell suspension to wells (1-2×10^5 cells/well)

  • Add biotinylated CD28 antibody at predetermined optimal concentration

  • Include all necessary controls as outlined in section 2.2

• Readout options (time points typically 24-72 hours):

  • Flow cytometry: CD25, CD69 expression for activation markers

  • ELISA: IL-2, IFN-γ production

  • Proliferation: CFSE dilution or 3H-thymidine incorporation

• Analysis considerations:

  • Compare responses to non-biotinylated CD28 antibody stimulation

  • Assess dose-dependency of both antibody binding and functional outcomes

This protocol can be modified for different experimental objectives, such as long-term culture studies or combination with other costimulatory signals.

How do conventional and superagonistic CD28 antibodies differ, and what safety considerations apply?

The distinction between conventional and superagonistic CD28 antibodies is critical for research safety:

The TeGenero TGN1412 trial in 2006 resulted in life-threatening cytokine release syndrome in all volunteers, highlighting the dangers of superagonistic properties . Recent development of antibodies like E1P2 has focused on binding epitopes close to the apex of CD28, similar to its natural ligand, to avoid superagonistic properties while maintaining co-stimulatory function .

When working with any CD28 antibody, researchers should:

• Test for superagonistic properties using in vitro cytokine release assays

• Begin with extremely low concentrations for initial testing

• Include appropriate controls to distinguish co-stimulation from direct activation

• Implement rigorous safety monitoring for in vivo applications

How can biotinylated CD28 antibodies be utilized in CAR-T cell research?

Biotinylated CD28 antibodies serve several important functions in CAR-T cell research:

• Universal CAR Systems: Biotin-binding protein-based universal CAR (UniCAR) systems utilize the strong biotin-streptavidin interaction. Third-generation UniCAR-T cells combining CD28 and 4-1BB co-stimulatory domains show improved specificity compared to second-generation designs with either domain alone .

• Co-stimulatory Domain Comparisons: Studies show that UniCAR constructs with CD28 domains (UniCARCD28-T cells) exhibit different specificity profiles compared to 4-1BB domains. The CD28 domain sometimes leads to non-specific activation with biotinylated antibodies regardless of target specificity .

• Enhanced Tumor Cell Killing: The combination of biotinylated targeting antibodies with CAR-T cells containing CD28 domains can enhance tumor cell killing and T-cell proliferation. In experimental settings, this typically uses a co-culture with target cells at effector-to-target ratios of 5:1, measuring outcomes after 3-4 days .

• Domain Architecture Optimization: Biotinylated CD28 antibodies help evaluate how different CAR configurations impact T cell function, persistence, and exhaustion profiles.

When designing these experiments, researchers must include controls with non-specific biotinylated antibodies to distinguish between specific targeting and non-specific activation through the CD28 domain.

What mechanisms explain CD28's role in plasma cell longevity and antibody production?

CD28's crucial role in plasma cell longevity and sustained antibody production operates through several mechanisms:

• Selective Support of LLPC: While both long-lived plasma cells (LLPC) and short-lived plasma cells (SLPC) express CD28, CD28-driven enhanced survival occurs specifically in bone marrow LLPC but not splenic SLPC .

• CD28 Ligand Interaction: CD28 engagement with CD80/CD86 triggers survival signaling pathways in LLPC. In vivo studies demonstrate that loss of CD28 or its ligands causes significant reduction in LLPC half-life from 426 to 63 days .

• Maintenance of Antibody Titers: CD28 signaling is essential for maintaining long-term antigen-specific antibody titers. Even with sufficient T cell help, loss of CD28 on plasma cells compromises the ability to sustain long-term humoral immunity .

• Bone Marrow Microenvironment: CD80/CD86-expressing cells in the bone marrow niche likely provide critical survival signals to LLPC through CD28 engagement.

• Clinical Correlation: In multiple myeloma (malignant plasma cells), CD28 expression correlates with poorer prognosis and disease progression, suggesting CD28 provides survival advantages to plasma cells .

These findings establish LLPC as a distinct subset necessary for sustaining antibody titers and highlight CD28's central role in the longevity of plasma cells and durable humoral immunity.

How can researchers address non-specific binding when using biotinylated CD28 antibodies?

Non-specific binding with biotinylated CD28 antibodies can be addressed through several approaches:

• Block endogenous biotin: Pretreat samples with avidin/biotin blocking kits, particularly important for cells with high metabolic activity.

• Optimize antibody concentration: Carefully titrate both the biotinylated antibody and streptavidin conjugate—excess of either increases background. For CD28.2 clone, start at ≤1 μg per test and optimize from there .

• Improve blocking strategy: Include 5-10% serum from the same species as the cells to block Fc receptors. Consider using specialized blocking buffers containing irrelevant immunoglobulins.

• Refine staining protocol:

  • Extend washing steps after primary antibody incubation

  • Maintain consistent 4°C temperature during staining

  • Use sequential rather than simultaneous staining

• Control for biotin-specific effects: When studying functional responses, include non-specific biotinylated antibodies as controls, especially with CD28-containing CAR constructs which can be activated by biotin alone .

• Gating strategy optimization: In flow cytometry analysis, implement stringent gating to exclude doublets, dead cells, and non-specific binding populations.

If problems persist, consider alternative detection strategies or different CD28 antibody clones with direct fluorophore conjugation.

How should researchers interpret differences in CD28 expression versus functional outcomes?

Discrepancies between CD28 expression and functional outcomes require careful consideration of several factors:

• Epitope accessibility: Different anti-CD28 antibody clones may recognize distinct epitopes with varying accessibility in different cellular contexts.

• Signaling pathway integrity: Expression of CD28 does not guarantee functional signaling pathways—examine downstream components (PI3K, Akt, mTOR) when expression and function don't correlate.

• Cell-type specific responses: CD28 produces different outcomes in:

  • Naive vs. memory T cells

  • CD4+ vs. CD8+ T cells

  • T cells vs. plasma cells

• Context-dependent functionality: LLPC and SLPC both express CD28, but CD28-driven enhanced survival occurs only in LLPC , indicating context-dependent functioning of the same receptor.

• Receptor modulation: Recent activation can cause CD28 downregulation while maintaining downstream signaling through already-initiated pathways.

• Experimental conditions: Consider whether differences reflect true biological phenomena or technical variables:

  • Antibody presentation format (soluble vs. immobilized)

  • Timing of measurements relative to stimulation

  • Presence of other costimulatory signals

When analyzing such discrepancies, comprehensive assessment of both surface expression and multiple functional readouts provides the most complete picture.

What factors affect the stability and performance of biotinylated CD28 antibodies in long-term storage?

Several factors influence the stability and performance of biotinylated CD28 antibodies during storage:

• Temperature: Store at -20°C for long-term stability; avoid repeated freeze-thaw cycles by preparing single-use aliquots.

• Buffer composition:

  • Optimal pH (7.2-7.4)

  • Protein stabilizers (0.1-1% BSA or gelatin)

  • Preservatives (0.05-0.1% sodium azide prevents microbial growth)

• Light exposure: Protect from light to preserve both antibody function and biotin conjugation.

• Concentration: More concentrated stocks generally maintain stability better than diluted preparations.

• Aggregation control: Centrifuge briefly before use to remove potential aggregates; consider filtration before initial storage.

• Biotinylation chemistry: NHS-ester biotinylation can hydrolyze over time, affecting binding efficiency.

• Quality control: Periodically validate stored antibodies against fresh lots by comparing:

  • Staining index in flow cytometry

  • Functional performance in activation assays

  • Biotin availability using streptavidin binding tests

The CD28.2 antibody is typically filtered (0.2 μm post-manufacturing) , but additional quality control measures may be necessary for long-term storage of working dilutions.

What future directions are emerging for CD28 antibody applications in immunotherapy?

Recent developments in CD28 antibody research point to several promising future directions:

• Non-superagonistic CD28 targeting: The development of antibodies like E1P2 demonstrates the feasibility of creating CD28-targeting therapies without superagonistic properties, potentially revitalizing this approach after the TGN1412 setback .

• Combination therapies: Enhancing the efficacy of CD3 bispecific T-cell engagers by providing CD28 co-stimulation represents an attractive strategy to boost T-cell activity against cancer. The combination of E1P2 with CD3 bispecific antibodies has shown enhanced tumor cell killing and T-cell proliferation in preclinical studies .

• Third-generation CAR designs: CAR-T cells incorporating both CD28 and 4-1BB co-stimulatory domains (third-generation design) show promising specificity and anti-tumor activity profiles, potentially addressing limitations of previous CAR-T cell technologies .

• Modulating plasma cell longevity: The discovery of CD28's role in long-lived plasma cell survival opens new avenues for interventions aimed at either enhancing vaccine responses or targeting pathogenic plasma cells in autoimmune diseases .

• Universal CAR platforms: Biotin-streptavidin based universal CAR systems allow flexible targeting and potentially safer control of engineered T cells, with CD28 signaling domains playing a crucial role in their design and function .

These emerging applications highlight the continued importance of CD28 antibodies in both basic research and translational medicine, with increasing focus on their therapeutic potential in cancer and infectious disease immunotherapy.