CD21 Antibody, Biotin

What is CD21 and what are its main functions in the immune system?

CD21, also known as complement receptor 2 (CR2), is a 145 kDa cell surface glycoprotein that serves as a receptor for various ligands including complement component C3d, Epstein-Barr virus (EBV), and IFNalpha . When C3d is bound to antigens, CD21 attaches to these complexes on the B-cell surface, facilitating antigen recognition and uptake by B-cells . This interaction enhances B-cell activation and subsequent immune responses.

CD21 forms a complex with several partners on B-cell surfaces including CD19, FCRL5, and CD81 to form the B-cell coreceptor complex, which plays a crucial role in B-cell activation and signaling . It also induces specific intracellular signaling separately from the BCR and CD19 by activating tyrosine kinase SRC, which then phosphorylates nucleolin/NCL and triggers AKT and GSK3 kinase activities .

What cell types express CD21 and how does expression vary during B-cell development?

CD21 is expressed:

• Strongly on mature B cells

• On follicular dendritic cells

• Weakly on immature thymocytes

• Weakly on some T lymphocytes

In B-cell ontogeny, CD21 appears after the pre-B-stage, is maintained during peripheral B-cell development, and is lost upon terminal differentiation into plasma cells . CD21 expression is also gradually lost after stimulation of B cells in vitro . This dynamic regulation makes CD21 an important marker for tracking B-cell maturation and activation status.

What is a biotin-conjugated CD21 antibody and what are its applications?

A biotin-conjugated CD21 antibody is an antibody specific to CD21 that has been chemically linked to biotin molecules. This conjugation enables versatile detection methods through the strong and specific interaction between biotin and streptavidin/avidin proteins.

Applications include:

Biotin-conjugated CD21 antibodies are particularly valuable in multicolor flow cytometry panels and immunohistochemistry where signal amplification is beneficial .

What are the optimal conditions for using biotin-conjugated CD21 antibodies in flow cytometry?

For optimal results with biotin-conjugated CD21 antibodies in flow cytometry:

• Sample preparation:

  • Prepare single-cell suspensions from relevant tissues

  • Adjust cell concentration to approximately 1,000,000 cells per test

  • Use freshly isolated cells when possible

• Staining protocol:

  • For biotin-conjugated antibodies, use 5-10μl per 10^6 cells as a starting point

  • Incubate cells with antibody in staining buffer (PBS with 1-2% BSA) for 20-30 minutes at 4°C

  • Wash twice with staining buffer

  • Incubate with streptavidin-conjugated fluorochrome

  • Include proper compensation controls for multicolor panels

• Critical controls:

  • Isotype controls (mouse IgG2a for many CD21 antibodies)

  • Fluorescence-minus-one (FMO) controls

  • Unstained cells to establish autofluorescence baseline

  • Known positive (B cells) and negative (non-B cell) populations

The optimal concentration should be determined experimentally for each specific application and lot of antibody .

How should samples be prepared for CD21 immunohistochemistry using biotinylated antibodies?

For successful CD21 immunohistochemistry with biotinylated antibodies:

• Tissue fixation and processing:

  • Fix tissue in 10% neutral buffered formalin (24-48 hours)

  • Process and embed in paraffin

  • Cut 4-5μm sections and mount on positively charged slides

• Deparaffinization and antigen retrieval:

  • Deparaffinize completely in xylene

  • Rehydrate through graded alcohols

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking steps (crucial for biotinylated antibodies):

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Critical step: Block endogenous biotin using a commercial biotin blocking system

  • Block non-specific binding with serum or protein block

• Antibody incubation:

  • Apply biotin-conjugated CD21 antibody at 1:50-1:250 dilution

  • Incubate at 4°C overnight or room temperature for 1-2 hours

  • Use streptavidin-HRP for detection

  • Develop with DAB or other chromogen

• Counterstain, dehydrate, clear, and mount as per standard protocols

Always include positive control tissue (tonsil or lymph node) that contains follicular dendritic cells and mature B cells known to express CD21.

How can researchers validate the specificity of CD21 antibodies for confident experimental results?

Validating CD21 antibody specificity requires a multi-faceted approach:

• Multiple antibody validation:

  • Compare staining patterns from different CD21 antibody clones targeting distinct epitopes

  • Concordant results increase confidence in specificity

• Genetic validation techniques:

  • Use cells from CD21 knockout models as negative controls

  • Employ CD21 siRNA knockdown to confirm reduced staining correlates with reduced expression

• Biochemical validation:

  • Western blot analysis to confirm the antibody detects a protein of the expected molecular weight (145 kDa for CD21)

  • Immunoprecipitation followed by mass spectrometry to confirm identity

• Blocking experiments:

  • Pre-incubate with unconjugated antibody before adding biotin-conjugated version

  • Pre-absorb with immunizing peptide or recombinant CD21 protein

  • Specific staining should be competitively inhibited

• Pattern recognition:

  • Confirm membrane staining pattern on B cells

  • Verify expected expression pattern on follicular dendritic cells in lymphoid tissues

• Functional correlation:

  • Confirm that cells identified as CD21-positive display expected CD21-dependent functions

Combining multiple validation approaches provides the strongest evidence for antibody specificity and experimental reliability.

How can CD21 antibodies be used to study B cell anergy and autoimmunity?

CD21 antibodies provide valuable tools for investigating B cell anergy and autoimmunity:

• Identification of anergic B cells:

  • CD21−/lo B cells from rheumatoid arthritis and CVID patients predominantly express autoreactive antibodies

  • These cells are functionally unresponsive (anergic) - unable to induce calcium flux, become activated, or proliferate in response to BCR and/or CD40 triggering

• Isolation of anergic B cell populations:

  • Flow sorting using CD21 antibodies enables isolation of CD21−/lo B cells for functional and molecular studies

  • Comparative studies between CD21+ and CD21−/lo B cells reveal mechanisms of anergy

• Gene expression profiling:

  • Gene array analyses of CD21−/lo B cells have revealed molecules specifically expressed in these cells that likely induce their unresponsive state

  • These molecules represent potential biomarkers for identifying anergic B cells in humans

• Mechanistic studies:

  • CD21−/lo B cells can be studied to understand how autoreactive B cells are maintained in an anergic state

  • Investigation of factors that might break anergy and contribute to autoimmunity

• Clinical correlations:

  • Monitor frequencies of CD21−/lo B cells in autoimmune disease progression and treatment response

  • Explore relationships between CD21−/lo B cell frequencies and disease activity

The finding that CD21−/lo B cells contain mostly autoreactive unresponsive clones has significant implications for understanding B cell tolerance mechanisms and autoimmune disease pathogenesis .

What role does CD21 play in Epstein-Barr virus infection, and how can biotinylated CD21 antibodies contribute to this research?

CD21 serves as the primary receptor for Epstein-Barr virus (EBV), making CD21 antibodies crucial for studying EBV biology :

• Viral entry mechanisms:

  • Biotinylated CD21 antibodies can block EBV binding to B cells in competitive inhibition assays

  • Different antibody clones targeting distinct CD21 epitopes can identify regions critical for virus binding

  • Visualization of virus-receptor interactions using flow cytometry or confocal microscopy

• Receptor expression analysis:

  • Quantitative assessment of CD21 expression levels across different B cell subsets and correlation with EBV susceptibility

  • Identification of cells expressing CD21 in various tissues that may be potential EBV targets

• Receptor complex formation:

  • Investigation of how EBV binding to CD21 recruits co-receptors and triggers signaling

  • How viral proteins interact with CD21 and modify its signaling properties

• Therapeutic development:

  • Screening of antibodies that block EBV-CD21 interactions as potential therapeutic agents

  • Structure-function studies to identify critical binding domains

• EBV-associated disease research:

  • Study how EBV exploitation of CD21 contributes to various EBV-associated diseases

  • Investigate whether CD21 expression patterns correlate with disease progression

Biotinylated CD21 antibodies provide flexibility in detection methods, allowing for sensitive visualization of CD21-EBV interactions in various experimental settings.

How do CD21-negative B cells contribute to autoimmune disease pathogenesis?

Research using CD21 antibodies has revealed important connections between CD21-negative B cells and autoimmune disease:

• Enrichment in autoimmune conditions:

  • CD21−/lo B cells are found at increased frequencies in patients with autoimmune diseases such as rheumatoid arthritis and in CVID patients prone to autoimmunity

  • This suggests a potential role in disease pathogenesis

• Autoreactive antibody expression:

  • A majority of CD21−/lo B cells from RA and CVID patients express germline autoreactive antibodies that recognize nuclear and cytoplasmic structures

  • These cells may represent a reservoir of potentially pathogenic B cell specificities

• Anergic state characteristics:

  • Despite expressing autoreactive antibodies, these cells are functionally unresponsive (anergic)

  • Unable to induce calcium flux, become activated, or proliferate in response to BCR and/or CD40 triggering

  • This suggests they are under active tolerance mechanisms

• Potential failure of tolerance:

  • The presence of these cells may indicate a partial breakdown in tolerance mechanisms

  • Environmental factors or genetic predisposition may eventually allow these cells to escape anergy

• Molecular signatures:

  • Gene array analyses of CD21−/lo B cells have revealed specific molecular signatures that likely maintain their unresponsive state

  • Understanding these signatures could identify new therapeutic targets

• Biomarker potential:

  • CD21−/lo B cell frequencies or molecular signatures might serve as biomarkers for disease stratification or treatment response prediction

The study of CD21−/lo B cells using CD21 antibodies provides valuable insights into the complex relationship between autoreactive B cells, tolerance mechanisms, and autoimmune disease pathogenesis.

What are common challenges when working with biotin-conjugated CD21 antibodies and how can they be overcome?

Researchers frequently encounter several challenges when working with biotin-conjugated CD21 antibodies:

• Endogenous biotin interference:

  • Problem: Many tissues contain endogenous biotin that can cause high background

  • Solution: Use commercial biotin blocking kits before adding biotin-conjugated antibodies

  • Alternative: Consider using directly conjugated primary antibodies instead of biotin-conjugation

• Signal variability issues:

  • Problem: Variable CD21 expression levels across B cell subsets and activation states

  • Solution: Include appropriate positive controls and standardize analysis gates

  • Recommendation: Use quantitative beads to standardize fluorescence intensity measurements

• Epitope masking:

  • Problem: Natural ligands (C3d, EBV) may block antibody binding sites

  • Solution: Consider using antibodies targeting different epitopes

  • Approach: Acid washing can sometimes remove bound ligands without affecting cell viability

• Avidin/streptavidin detection system issues:

  • Problem: Non-specific binding of detection reagents

  • Solution: Optimize concentration of streptavidin conjugates

  • Alternative: Use streptavidin conjugates from different manufacturers to find optimal reagent

• Multicolor panel interference:

  • Problem: Spectral overlap between fluorochromes in multicolor panels

  • Solution: Proper compensation controls and careful panel design

  • Approach: Consider brightness of streptavidin-fluorochrome relative to CD21 expression level

• Fixation sensitivity:

  • Problem: Some CD21 epitopes may be sensitive to certain fixatives

  • Solution: Test different fixation protocols to optimize epitope preservation

  • Alternative: Consider live cell staining when possible

Systematic optimization approaches and appropriate controls are essential for successful experiments with biotin-conjugated CD21 antibodies.

How should researchers interpret changes in CD21 expression patterns in disease states?

Interpreting CD21 expression patterns requires understanding normal expression and disease-associated changes:

• Normal CD21 expression baseline:

  • Strong expression on mature B cells and follicular dendritic cells

  • Dynamic regulation during B cell development

  • Lost upon terminal differentiation into plasma cells

  • Gradually decreases after B cell activation

• Disease-associated changes to monitor:

  • Frequency of CD21−/lo B cells (increased in autoimmune conditions)

  • Mean fluorescence intensity (MFI) of CD21 on positive populations

  • Correlation with other markers (CD19, CD23, activation markers)

  • Distribution patterns in tissue sections (especially for follicular dendritic cell networks)

• Interpretation framework:

  • Increased CD21−/lo B cells in autoimmune diseases may represent anergic autoreactive cells

  • Altered distribution in tissues may reflect changes in lymphoid architecture

  • Correlation with disease activity may suggest prognostic value

  • Changes after treatment may indicate immunomodulatory effects

• Quantitative analysis approaches:

  • Compare percentages of CD21+ versus CD21−/lo within defined B cell subsets

  • Use MFI ratios rather than absolute values to control for instrument variation

  • Correlation analysis with clinical parameters or other biomarkers

  • Longitudinal monitoring of individual patients over time

• Combining with functional assays:

  • CD21 expression should be correlated with functional readouts (calcium flux, proliferation)

  • This connection provides mechanistic insights beyond descriptive changes

Understanding the biological significance of CD21 expression changes requires integration of phenotypic, functional, and clinical data for meaningful interpretation.

What is known about CD21's role in the complement system and B cell activation?

CD21 functions as a critical bridge between the complement system and adaptive immunity:

• Complement fragment recognition:

  • CD21 binds complement fragments C3d, C3dg, and iC3b that become attached to antigens during complement activation

  • When C3d is bound to antigens, CD21 attachment facilitates antigen recognition and uptake by B cells

• Co-receptor complex formation:

  • CD21 associates with CD19, CD81, and Leu13 to form a large signal-transduction complex

  • This complex lowers the threshold for B cell activation by engaging with the B cell receptor

• Signal transduction mechanisms:

  • CD21 induces specific intracellular signaling independently from BCR and CD19

  • Activates tyrosine kinase SRC, which phosphorylates nucleolin/NCL

  • Triggers AKT and GSK3 kinase activities in a SYK/CD19-independent manner

  • This provides additional activation signals to B cells

• Amplification of immune responses:

  • When antigens are tagged with C3d fragments, they become much more immunogenic

  • This mechanism ensures stronger responses to complement-activating pathogens

  • Serves as a natural adjuvant by lowering activation thresholds

• Follicular dendritic cell function:

  • CD21 on follicular dendritic cells captures and retains complement-tagged antigens

  • This retention is crucial for germinal center formation and maintenance

  • Contributes to affinity maturation and memory B cell development

CD21's role as a complement receptor makes it a crucial molecule for understanding how innate immune activation enhances adaptive immune responses, particularly in B cell-mediated immunity.

What experimental approaches can measure interactions between CD21 and its ligands?

Several experimental approaches can effectively measure interactions between CD21 and its ligands:

• Binding assays:

  • Surface Plasmon Resonance (SPR): Measures real-time binding kinetics between purified CD21 and ligands (C3d, EBV proteins)

  • ELISA-based binding assays: Plate-bound CD21 with titrated ligands to determine binding curves

  • Flow cytometry-based assays: Using fluorescently-labeled ligands to measure binding to CD21+ cells

• Cellular functional assays:

  • Calcium flux assays: Measure intracellular calcium mobilization after CD21 engagement

  • Proliferation assays: Compare B cell responses to antigens with and without complement tagging

  • Signaling studies: Phosphorylation of downstream targets (SRC, AKT, GSK3) after CD21 engagement

• Competitive inhibition approaches:

  • Use CD21 antibodies to block ligand binding

  • Compare different antibody clones to map binding domains

  • Use soluble CD21 fragments to compete with cellular CD21

• Microscopy techniques:

  • Confocal microscopy: Visualize co-localization of CD21 with ligands and co-receptors

  • FRET (Förster Resonance Energy Transfer): Measure proximity between CD21 and binding partners

  • Super-resolution microscopy: Analyze nanoscale organization of CD21 receptor complexes

• Structural studies:

  • X-ray crystallography of CD21-ligand complexes

  • Cryo-electron microscopy to visualize larger complexes

  • Molecular dynamics simulations based on structural data

• Genetic approaches:

  • CRISPR/Cas9 modification of CD21 binding domains to assess functional consequences

  • Domain swapping experiments to identify critical regions for ligand interactions

  • Mutagenesis studies of key amino acid residues

These complementary approaches provide comprehensive insights into the molecular mechanisms of CD21-ligand interactions and their functional consequences.