CD21 Antibody

What is CD21 and what is its role in the immune system?

CD21, also known as complement receptor type 2 (CR2), is a type I integral membrane glycoprotein of approximately 140-145 kDa that functions as a receptor for the C3d fragment of the third complement component. It plays crucial roles in:

• Facilitating B cell activation and proliferation by enhancing B cell antigen receptor (BCR) signaling strength

• Serving as a receptor for Epstein-Barr virus (EBV)

• Modulating the activation threshold of B cells

• Participating in immunocomplex uptake and retention

• Supporting memory B cell survival

• Contributing to humoral response development to T-dependent antigens

CD21 undergoes phosphorylation following B cell activation, influencing downstream signaling pathways and cellular outcomes. It functions in a multimolecular complex with CD19 and CD81, creating a co-receptor complex that significantly amplifies B cell responses to antigens .

What cell types express CD21?

CD21 expression has been definitively established in:

• Mature B cells (high expression)

• Follicular dendritic cells

• Subsets of epithelial cells

• B cell chronic lymphocytic leukemias

• Follicular and mantle cell lymphomas

Recent research has revealed controversial findings regarding CD21 expression on T cells:

• Some studies detect CD21 on mature peripheral T cells (using anti-CD21 clone HB5)

• Other studies report no CD21 expression on T cells (using anti-CD21 clone Bly4)

• When detected, CD21 expression appears on naive, memory, and effector CD4+ and CD8+ T cells, with highest expression in naive T-cell populations

What are the primary types of CD21 antibodies available for research?

What is the optimal method for detecting CD21 expression on different lymphocyte populations?

• Antibody clone selection is critical:

  • For comprehensive detection of CD21 on both B and T cells, the HB5 clone is recommended

  • The Bly4 clone detects CD21 only on B cells and will generate false negatives for T cell expression

  • Research questions focused on potential T cell CD21 expression should use multiple antibody clones for validation

• Recommended flow cytometry protocol:

  • Isolate peripheral blood mononuclear cells using density gradient centrifugation

  • Use 5 μL (0.125 μg) of PE-conjugated anti-CD21 per test of 10^5-10^8 cells

  • Include appropriate lineage markers (CD19 for B cells, CD3/CD4/CD8 for T cells)

  • Use additional markers (CCR7, CD45RA) to distinguish naive (CD45RA+CCR7+), memory (CD45RA-CCR7+/-), and effector (CD45RA+CCR7-) T cell subsets for detailed analysis

• Data interpretation considerations:

  • CD21 expression is nearly ubiquitous on peripheral B cells (~95-100%)

  • When detected on T cells, expression is lower (~34.4% CD21+ on average)

  • Geometric mean fluorescence intensity (gMFI) is significantly higher in CD8+ compared to CD4+ T cells (p=0.0005)

How should Western blot experiments be optimized for CD21 detection?

For successful Western blot detection of CD21:

• Sample preparation:

  • Human spleen tissue or purified B cells provide high CD21 expression

  • Use non-reducing conditions as some CD21 antibodies (like MAB4909) require intact disulfide bonds for recognition

  • Include protease inhibitors during sample preparation to prevent degradation

• Electrophoresis and membrane transfer:

  • Use PVDF membrane for optimal protein retention

  • Expected molecular weight: approximately 145 kDa under non-reducing conditions

• Antibody incubation:

  • Primary antibody concentration: 2 μg/mL of Mouse Anti-Human CD21 Monoclonal Antibody

  • Use appropriate HRP-conjugated secondary antibody

  • Implement Immunoblot Buffer Group 1 for optimal results

• Controls:

  • Positive control: human spleen tissue lysate

  • Negative control: tissues known not to express CD21

  • Loading control: housekeeping protein (like β-actin)

What are the methodological considerations for studying CD21 in the context of EBV infection?

When investigating CD21's role in EBV infection:

• Neutralizing antibody assays:

  • Pre-incubate EBV with anti-gp350 antibodies to block viral attachment

  • Pre-treat target cells with anti-CD21 antibodies (HB5 clone) to block receptor accessibility

  • Measure infection efficiency through viral DNA quantification by qPCR at 1 and 3 days post-infection

• CD21 expression manipulation approaches:

  • CRISPR-Cas9 knockout of CD21 in cell lines (e.g., Jurkat T cells) provides definitive evidence for CD21's necessity in EBV entry

  • Cell sorting based on CD21 expression levels allows comparison of infection susceptibility between CD21hi and CD21lo populations

  • Confirm CD21 knockout/knockdown by both protein (flow cytometry) and mRNA (qPCR) analysis

• Viral entry analysis:

  • Sort cells based on CD21 expression (CD21hi vs. CD21lo)

  • Infect with standardized viral preparations (5 EBV genome copies/cell recommended)

  • Extract DNA at defined timepoints (1 and 3 days post-infection)

  • Quantify viral DNA by qPCR to assess infection efficiency

How does CD21 interact with the B cell co-receptor complex and what methods best study this interaction?

CD21 forms a critical multimolecular complex with CD19 and CD81 on B cells, significantly impacting B cell activation thresholds:

• Biochemical approaches to study the complex:

  • Co-immunoprecipitation using anti-CD21 antibodies to pull down associated proteins

  • Immunoblotting for CD19, CD81, and other putative partners

  • Cross-linking experiments to stabilize transient interactions

• Functional analysis methods:

  • Calcium flux assays following B cell receptor stimulation with/without CD21 engagement

  • Phosphorylation studies of downstream signaling molecules (particularly focusing on CD21 phosphorylation following activation)

  • Confocal microscopy using differentially labeled antibodies to visualize co-localization

  • Proximity ligation assays to confirm direct protein-protein interactions in situ

• Key experimental consideration:

  • Use complement component C3d conjugated to model antigens to specifically engage CD21 during BCR stimulation

  • Implement CD21 knockout/knockdown systems to evaluate co-receptor complex dependency

  • Utilize super-resolution microscopy to define spatial organization of the complex at nanoscale resolution

What is the current understanding of CD21 expression on T cells and appropriate methods to resolve the controversy?

The controversy regarding CD21 expression on T cells requires careful methodological approaches:

• Recommended multi-validation approaches:

  • Flow cytometry using multiple anti-CD21 antibody clones (particularly HB5 and Bly4)

  • Quantitative RT-PCR for CD21 mRNA in purified T cell populations

  • Single-cell RNA sequencing to identify potential CD21-expressing T cell subsets

  • Immunofluorescence microscopy with co-staining for T cell markers

• Sorting strategy for purifying T cells for CD21 analysis:

  • Negative selection of CD3+ T cells to avoid potential activation

  • Staining with anti-CD21 (HB5 clone) and sorting into CD21hi and CD21lo populations

  • Confirming purity by re-analysis of sorted populations (≥97% purity target)

  • Validating CD21 expression at mRNA level by qPCR (expect approximately 21-fold higher expression in CD21hi vs. CD21lo fractions)

• Functional validation experiments:

  • EBV infection susceptibility comparisons between CD21hi and CD21lo T cell populations

  • Blocking experiments using anti-CD21 antibodies prior to EBV exposure

  • CRISPR-Cas9 knockout of CD21 in T cell lines to evaluate functional consequences

What methods should be employed to study CD21's role in autoimmune conditions?

CD21 deficiencies or mutations have been linked to defective immune responses and increased susceptibility to autoimmune conditions:

• Patient sample analysis approaches:

  • Flow cytometric quantification of CD21 expression levels on B cells from patients with autoimmune conditions compared to healthy controls

  • Genetic screening for CD21 mutations or polymorphisms in patient cohorts

  • Analysis of soluble CD21 (sCD21) levels in serum by ELISA as a potential biomarker

• Animal model methodologies:

  • CD21 knockout mouse models to evaluate autoimmune phenotypes

  • Chimeric models with selective CD21 deficiency in specific cell types

  • Adoptive transfer experiments to determine cell-intrinsic versus cell-extrinsic effects

• Molecular and cellular techniques:

  • B cell tolerance assays with/without functional CD21

  • Analysis of BCR signaling thresholds in the context of CD21 deficiency

  • Immune complex handling and complement interaction studies

  • Investigation of CD21-CD19 complex integrity in autoimmune conditions

What are common challenges in CD21 detection and how can they be addressed?

How can researchers optimize antibody selection for specific CD21 research applications?

• For T cell CD21 studies:

  • Primary recommendation: HB5 clone - demonstrated ability to detect CD21 on T cell subsets

  • Avoid: Bly4 clone - fails to detect CD21 on T cells despite detecting on B cells

  • Validation approach: Compare results from multiple clones side-by-side

• For Western blot applications:

  • Recommended: MAB4909 under non-reducing conditions only

  • Expected molecular weight: ~145 kDa

  • Sample source: Human spleen tissue provides reliable expression

• For multi-parameter flow cytometry:

  • Consider spectral overlap when selecting fluorophore conjugates

  • PE-conjugated anti-CD21 provides good signal-to-noise ratio

  • Use 5 μL (0.125 μg) per test of 10^5-10^8 cells for optimal staining

What controls should be included in experiments investigating CD21 function or expression?

• Flow cytometry controls:

  • Isotype control antibodies matched to anti-CD21 antibody class and fluorophore

  • Fluorescence-minus-one (FMO) controls to establish gating boundaries

  • B cell samples as positive controls for CD21 expression

  • Known CD21-negative cells as negative controls

• Western blot controls:

  • Human spleen tissue as positive control

  • Isotype-matched antibody control

  • Loading control (β-actin or similar housekeeping protein)

  • Non-reducing vs. reducing condition comparisons

• Functional assay controls:

  • CD21 blocking with specific antibodies

  • CRISPR knockout validation

  • Rescue experiments with CD21 re-expression

  • Comparison of CD21hi vs. CD21lo sorted populations