AICDA Antibody, HRP conjugated

What is AICDA and what are its primary functions in B cells?

AICDA (also known as AID) is a 24 kDa single-stranded DNA-specific cytidine deaminase that plays essential roles in antibody diversification. It functions by:

• Deaminating cytidines to uracils in single-stranded DNA within transcription bubbles

• Initiating somatic hypermutation (SHM) by introducing point mutations in the variable regions of immunoglobulin genes

• Facilitating class switch recombination (CSR) by generating double-strand breaks in switch regions

• Potentially participating in DNA demethylation processes through deamination of 5-methylcytosine

AICDA is primarily expressed in germinal center B cells, where it drives the affinity maturation process essential for generating high-affinity antibodies during immune responses .

Why are HRP-conjugated antibodies particularly useful for AICDA detection?

HRP (horseradish peroxidase)-conjugated antibodies provide several methodological advantages:

• Enhanced sensitivity: The enzymatic amplification of signal improves detection of low-abundance AICDA protein

• Versatility across multiple applications: Particularly valuable for IHC-P, IHC-F, and Western blotting applications

• Quantitative measurement: Enables precise measurement through colorimetric or chemiluminescent detection

• Relatively stable under proper storage conditions (generally at -20°C with glycerol and appropriate buffers)

• Elimination of secondary antibody step, reducing background and cross-reactivity issues

The HRP conjugation particularly benefits AICDA detection in tissue sections where signal amplification is essential for visualizing expression patterns in germinal centers .

How can AICDA antibodies be used to investigate aberrant somatic hypermutation in B-cell malignancies?

AICDA antibodies, including HRP-conjugated variants, can be employed to study aberrant SHM in B-cell malignancies through:

• Immunohistochemical profiling: Comparing AICDA expression patterns between normal germinal centers and malignant B cells to identify dysregulation.

• Correlation with genetic alterations: Using AICDA staining alongside analysis of:

  • c-MYC/IGH translocations in Burkitt lymphoma

  • BCL6 mutations in diffuse large B-cell lymphoma

  • Increased intraclonal heterogeneity in various B-cell neoplasms

• Prognostic assessment: Higher AICDA expression correlates with more aggressive disease and poorer prognosis in several lymphoma subtypes.

Research has shown that AID expression is highly specific to, and abundantly expressed in B-cell-derived cancers. Notably, studies have revealed that AID expression significantly impacts genomic stability, proliferation, migration, and drug resistance in lymphoma models .

How does AICDA-mediated DNA demethylation contribute to epigenetic regulation in normal and pathological states?

AICDA's role in DNA demethylation represents a cutting-edge area of investigation with emerging mechanistic insights:

AICDA participates in DNA demethylation through:

• Deamination of 5-methylcytosine (5mC) to thymidine

• Subsequent recruitment of base excision repair (BER) machinery

• Replacement with unmethylated cytosine through DNA repair processes

This activity has been implicated in:

• Epigenetic reprogramming during B cell development

• Modulation of enhancer accessibility during antibody diversification

• Potential contribution to aberrant DNA methylation patterns in malignancy

Recent research indicates that AID-driven DNA demethylation can contribute to epigenetic heterogeneity, affecting prognosis and treatment response in diffuse large B-cell lymphoma (DLBCL) .

What role does AICDA play in establishing B-cell tolerance, and how can HRP-conjugated antibodies help elucidate this function?

AICDA has a surprising role in central B-cell tolerance that can be investigated using HRP-conjugated antibodies:

• Detection in early B-cell development:

  • Transient expression in immature B cells co-expressing RAG2

  • Association with cells lacking MCL-1 and expressing active caspase-3

• Evaluating tolerance checkpoint defects:

  • AICDA-deficient immature B cells show resistance to tolerization

  • Impaired central B-cell tolerance in AICDA-/- mice

  • Increased frequency of polyreactive B cell clones in AID-deficient patients

• Mechanistic investigations:

  • HRP-conjugated AICDA antibodies can be used to identify B cells undergoing tolerance-induced mutation

  • Co-localization studies with BCR and TLR signaling components

  • Correlation with apoptotic markers in developing B cells

This research area is particularly important for understanding autoimmunity development, as studies have shown that AID-deficient patients and AICDA-KO mice display defective central B-cell tolerance checkpoints .

What are the optimal tissue preparation and antigen retrieval protocols for AICDA immunohistochemistry using HRP-conjugated antibodies?

Effective AICDA detection in tissues requires optimized protocols:

Tissue preparation:

• Fixation: 10% neutral-buffered formalin for 24-48 hours

• Processing: Standard paraffin embedding

• Sectioning: 4-5 μm thickness optimal for IHC-P applications

Antigen retrieval options:

• Heat-induced epitope retrieval (HIER):

  • Citrate buffer (pH 6.0): 95-98°C for 20 minutes

  • EDTA buffer (pH 9.0): Often provides superior results for AICDA detection

Immunostaining parameters:

• Blocking: 3-5% BSA or normal serum for 1 hour at room temperature

• Primary antibody (HRP-conjugated anti-AICDA): 1:50-1:200 dilution, overnight at 4°C

• Development: DAB substrate for 5-10 minutes (optimize for signal-to-noise ratio)

• Counterstaining: Hematoxylin (light) to visualize nuclei

When evaluating germinal centers, it's crucial to compare AICDA staining with other germinal center markers (CD10, BCL6) to accurately identify B-cell subpopulations .

What controls should be included when using AICDA antibody, HRP conjugated, and how should results be validated?

Comprehensive experimental validation requires multiple controls:

Positive controls:

• Reactive tonsil tissue (germinal centers show strong AICDA expression)

• Ramos human Burkitt's lymphoma cell line (known to express AICDA)

• AICDA-transfected cell lines with verified expression

Negative controls:

• Isotype-matched irrelevant antibody (same host species and concentration)

• Tissues known to lack AICDA expression (non-lymphoid tissues)

• AICDA-knockout/knockdown samples when available

Validation strategies:

• Correlation with mRNA expression (RT-PCR or RNA-seq)

• Orthogonal detection methods (alternative antibody clones, non-HRP conjugated)

• Functional assays (mutation analysis in SHM targets)

• siRNA knockdown confirmation to demonstrate specificity

Importantly, researchers should note that AICDA expression is primarily restricted to germinal center B cells, with occasional detection in early immature B cells during tolerance establishment .

What are the key differences between using monoclonal versus polyclonal HRP-conjugated AICDA antibodies?

The choice between monoclonal and polyclonal antibodies has important methodological implications:

When studying AICDA, monoclonal antibodies like clone EPR23436-45 offer superior specificity for applications requiring precise localization, while polyclonal antibodies may be preferred for detecting low-abundance AICDA expression or when epitope accessibility is limited by fixation .

What are common causes of false positive or false negative results when using HRP-conjugated AICDA antibodies?

False positive causes:

• Endogenous peroxidase activity: Inadequate quenching of tissue peroxidases

• Non-specific binding: Insufficient blocking or high antibody concentration

• Cross-reactivity: Other APOBEC family members share homology with AICDA

• Edge effects: Drying artifacts during incubation steps

• Overly sensitive detection systems: Excessive substrate development time

False negative causes:

• Epitope masking: Improper fixation or inadequate antigen retrieval

• Low expression levels: AICDA expression is restricted and may be transient

• Antibody degradation: Improper storage or repeated freeze-thaw cycles

• Suboptimal incubation conditions: Temperature, time, or pH issues

• Competitive inhibition: Excessive blocking or presence of interfering substances

Methodological solution: A systematic approach to antibody validation including appropriate controls, titration experiments, and comparison with known AICDA expression patterns in germinal centers can minimize these issues .

How can researchers troubleshoot inconsistent AICDA staining patterns in germinal center B cells?

When experiencing variable staining of germinal center B cells:

Systematic troubleshooting approach:

• Tissue quality assessment:

  • Evaluate fixation timing and conditions

  • Determine tissue age and storage conditions

  • Check for processing artifacts or autolysis

• Protocol optimization:

  • Systematic testing of antigen retrieval methods

  • Titration of antibody concentration (1:50, 1:100, 1:200, etc.)

  • Extension of incubation times (overnight at 4°C often improves signal)

• Technical considerations:

  • Use fresh substrate solutions

  • Ensure consistent temperature during incubations

  • Implement humidity chambers to prevent edge effects

• Biological variables:

  • AICDA expression varies with germinal center stage and activation

  • Compare dark zone (higher AICDA) vs. light zone (lower AICDA) patterns

  • Consider biological heterogeneity based on activation state

The dark and light zone distribution of AICDA in germinal centers should be systematically documented, as this pattern can provide insights into B cell differentiation dynamics .

How does AICDA contribute to autoimmunity, and what experimental models can be used to study this relationship?

AICDA plays complex roles in autoimmunity that can be investigated through multiple experimental approaches:

AICDA in autoimmunity mechanisms:

• Defective central B-cell tolerance in AICDA-deficient individuals

• Increased frequency of polyreactive B cells in circulation

• Impaired peripheral tolerance checkpoint function

• Altered germinal center reactions with prolonged antigen presentation

• Increased Tfh cell production and cytokine secretion (IL-4, IL-10, IL-21)

Experimental models:

• BXD2 autoimmune mouse model:

  • Expressing dominant negative AICDA (Aicda-DN) suppresses autoantibody production

  • Results in decreased proliferation and increased apoptosis in germinal centers

  • Leads to lower IgG-containing immune complexes

• Human patient-derived samples:

  • Patients with AICDA mutations show impaired peripheral B-cell tolerance

  • Systemic sclerosis and SLE patients show defective B-cell stimulation via TLR9

  • Analysis of autoantibody profiles in AICDA-deficient patients

These studies have revealed that the "double-edged sword" of AICDA function includes roles in both preventing autoreactivity during B cell development and potentially contributing to it through aberrant activity .

What is the relationship between AICDA expression and oncogenesis, particularly in B-cell malignancies?

AICDA has significant oncogenic potential through multiple mechanisms:

Oncogenic mechanisms:

• Genomic instability:

  • Off-target DNA damage in actively transcribed genes

  • Generation of chromosomal translocations (e.g., c-MYC/IGH in Burkitt lymphoma)

  • Introduction of point mutations in tumor suppressor genes and oncogenes

• Epigenetic dysregulation:

  • Aberrant DNA demethylation

  • Altered gene expression patterns

  • Epigenetic heterogeneity affecting treatment response

Research findings:

• Overexpression of AID leads to rapid cell death in experimental models

• AID expression significantly impacts genomic stability, proliferation, migration, and drug resistance

• Knock-down models reveal AID as an important driver of lymphoma pathogenesis

• AICDA is upregulated by NF-κB, STAT6, and Smad transcription pathways and Th2/Treg cytokines (IL-4, IL-13, TGF-β)

Interestingly, researchers have shown that inflammatory conditions can induce aberrant AID expression in non-lymphoid tissues, potentially connecting chronic inflammation to carcinogenesis in multiple tissue types .

What are the immunological consequences of AICDA deficiency, and how can they be studied using HRP-conjugated antibodies?

AICDA deficiency leads to profound immunological alterations:

Clinical and immunological manifestations:

• Hyper-IgM syndrome type 2 (autosomal recessive form)

• Elevated serum IgM with absence of IgG, IgA, and IgE

• Recurrent sinopulmonary infections and opportunistic infections

• Absence of class-switch recombination and somatic hypermutation

• Lymph node hyperplasia with intense apoptosis

Research applications of HRP-conjugated AICDA antibodies:

• Confirmation of absent/mutated AICDA expression in patient samples

• Evaluation of germinal center architecture in AICDA-deficient models

• Analysis of B cell subpopulations in primary and secondary lymphoid tissues

• Assessment of remaining AICDA function in hypomorphic mutations

• Characterization of compensatory mechanisms in AICDA deficiency

Comparative studies between wild-type and AICDA-deficient tissues can provide insights into the precise roles of AICDA in lymphoid organization and normal immune responses. The absence of AICDA expression in germinal centers of affected patients can be definitively confirmed using properly validated HRP-conjugated antibodies .

How might AICDA targeting be developed as a therapeutic strategy for autoimmune diseases or B-cell malignancies?

Emerging research suggests several potential therapeutic approaches:

Therapeutic targeting strategies:

• Small molecule inhibitors:

  • Direct catalytic domain inhibitors

  • Disruption of AICDA-DNA interaction

  • Inhibition of nuclear localization

• Dominant negative approaches:

  • Expression of catalytically inactive AICDA (H56R/E58Q mutations)

  • Interference with AICDA multimerization

  • Prevention of PKA-mediated phosphorylation (S38A mutation)

• Expression modulation:

  • miRNA-based approaches (miR-155, miR-181b, miR-361)

  • Targeting transcription factors (NF-κB, STAT6)

  • Epigenetic modification of AICDA locus

Proof-of-concept evidence:
The BXD2-Aicda-DN transgenic mouse model demonstrates that targeted inhibition of the catalytic domain of AID results in decreased autoantibody production and smaller germinal centers through both decreased proliferation and increased apoptosis, providing evidence that normalization of AID catalytic function could be a novel therapeutic target .

What novel methods are being developed to study the genome-wide off-target effects of AICDA activity?

Cutting-edge approaches for mapping AICDA activity include:

Advanced methodologies:

• High-throughput sequencing approaches:

  • HTGTS (High-Throughput Genome-wide Translocation Sequencing)

  • TC-Seq (Translocation Capture Sequencing)

  • AICDA ChIP-seq with catalytic site mutants

• DNA damage detection techniques:

  • γH2AX ChIP-seq for DSB mapping

  • BLESS (Direct in situ breaks labeling, enrichment on streptavidin, and sequencing)

  • END-seq for DSB detection

• Single-cell approaches:

  • scRNA-seq with AICDA expression correlation

  • scATAC-seq for chromatin accessibility at AICDA targets

  • CITE-seq to correlate AICDA protein levels with transcriptome

These methodologies can help identify genome-wide off-target activities of AICDA that contribute to lymphomagenesis and potentially to other cancers, providing deeper understanding of both physiological and pathological roles of this enzyme .

How can HRP-conjugated AICDA antibodies be effectively used in multiplexed immunofluorescence studies?

For researchers pursuing multiplexed detection strategies:

Methodological considerations:

• Sequential tyramide signal amplification (TSA):

  • Use HRP-conjugated AICDA antibody as first primary

  • Develop with TSA-fluorophore (e.g., TSA-FITC)

  • Quench peroxidase activity (3% H₂O₂, 10 min)

  • Continue with subsequent antibodies

• Optimized panel design:

  • Pair AICDA with germinal center markers (CD20, CD10, BCL6)

  • Include proliferation markers (Ki-67) to correlate with AICDA activity

  • Add DNA damage markers (γH2AX) to assess AICDA-induced damage

• Signal separation strategies:

  • Spectral unmixing for overlapping fluorophores

  • Carefully selected fluorophore combinations

  • Nuclear vs. cytoplasmic localization for signal discrimination

Technical validation:
Single-stain controls, fluorescence minus one (FMO) controls, and spectral controls are essential for accurate signal interpretation in multiplexed studies with AICDA detection .

What are the considerations for using AICDA antibodies in ChIP assays to study AICDA's DNA binding properties?

ChIP applications require specialized considerations:

Protocol optimization for AICDA ChIP:

• Crosslinking optimization:

  • Dual crosslinking (1% formaldehyde followed by protein crosslinkers)

  • Reduced crosslinking time (5-10 minutes) to prevent epitope masking

  • Sonication parameters adjusted for optimal chromatin shearing

• Antibody selection considerations:

  • Non-conjugated antibodies preferred over HRP-conjugated for ChIP

  • Monoclonal antibodies targeting DNA-binding domain may interfere with target binding

  • C-terminal targeting antibodies may better preserve DNA-protein interaction

• Controls and validation:

  • Input normalization critical for quantification

  • IgG control to establish background

  • AICDA knockout/knockdown for specificity validation

  • qPCR confirmation of known AICDA targets (Ig switch regions)

Emerging applications:
ChIP-seq analysis of AICDA can reveal genome-wide binding patterns and potential off-target activities that contribute to genomic instability in normal and malignant B cells .

How should researchers approach epitope mapping of AICDA to select the most appropriate antibody for specific applications?

Strategic epitope selection requires understanding AICDA's functional domains:

AICDA domain structure and functional implications:

• N-terminal domain (1-54):

  • Contains nuclear export signal

  • Important for protein-protein interactions

  • Antibodies targeting this region may detect SHM-deficient variants

• Catalytic domain (55-94):

  • Contains H56, E58, C87, C90 catalytic residues

  • Critical for deamination activity

  • Antibodies targeting this region may interfere with enzyme activity

• C-terminal domain (140-198):

  • Important for class switch recombination

  • Contains nuclear localization signal

  • Antibodies targeting this region may preferentially detect CSR-competent AID

Antibody selection guidance:

• For detection of all AICDA forms: Target preserved regions like aa 140-190

• For functional studies: Avoid catalytic domain recognition

• For subcellular localization studies: Consider epitope accessibility in different compartments