CD226 Antibody

What is CD226 and what are its key structural features?

CD226, also known as DNAM-1 (DNAX accessory molecule-1), is a transmembrane glycoprotein with a molecular weight of approximately 38.6 kDa (canonical form consists of 336 amino acid residues). It is primarily localized in the cell membrane and undergoes post-translational modifications, particularly glycosylation, which can increase its apparent molecular weight to approximately 67 kDa when detected in Western blots . CD226 has two extracellular domains, both of which are necessary for optimal functional interaction with its ligands, although one domain can bind with lower affinity in solid binding assays .

What cell types express CD226 and what are its primary functions?

CD226 is predominantly expressed on:

• Peripheral blood T-lymphocytes

• Natural killer (NK) cells

• B cells

• Platelets

Functionally, CD226 plays crucial roles in:

• Intercellular adhesion

• Lymphocyte signaling

• Cytotoxicity and lymphokine secretion by CTLs and NK cells

• Regulating the proinflammatory (Th1/Th17) and anti-inflammatory (Th2) balance in immune responses

What alternative names and synonyms are used for CD226 in the scientific literature?

When searching the literature, researchers should be aware of these alternative designations for CD226:

• DNAM-1 (DNAX accessory molecule-1)

• PTA1

• TLiSA1

• CD226 antigen

What are the principal research applications for CD226 antibodies?

CD226 antibodies are employed in multiple research techniques, with the most common applications being:

• Flow cytometry - for characterizing CD226 expression on various immune cell populations

• Western blotting - for detecting CD226 protein in cell or tissue lysates

• Immunohistochemistry - for examining CD226 expression in tissue sections

• ELISA - for detecting soluble CD226 in biological fluids

• Functional studies - including blocking CD226 signaling in immunological assays

What methodological considerations are important for flow cytometry with CD226 antibodies?

When using CD226 antibodies for flow cytometry:

• Fresh samples are preferable as CD226 expression may be affected by extended storage

• CD226 antibodies can detect both exogenous transfected CD226 on cell surfaces and natural CD226 on immune cells like platelets and lymphocytes

• Multi-parameter panels should include markers for specific immune cell subsets (e.g., CD3, CD4, CD19) to properly identify CD226-expressing populations

• Consider fixation protocols carefully as some may affect CD226 epitope accessibility

How can researchers establish a reliable ELISA system for soluble CD226 detection?

Based on published methodologies, a sandwich ELISA system for soluble CD226 can be established by:

• Coating ELISA plates with purified anti-CD226 mAbs (e.g., mA1.3 in mouse studies)

• Using a second anti-CD226 mAb (e.g., mA1.1) labeled with horseradish peroxidase or biotin as the detection antibody

• Optimizing antibody concentrations to achieve detection sensitivities of 0.25-3.0 ng/mL

This approach has been successfully used to measure soluble CD226 levels in plasma from mouse models of disease, revealing that soluble CD226 levels decreased in LPS-induced sepsis compared to normal controls .

How are CD226 antibodies utilized in autoimmune disease research, particularly SLE?

CD226 antibodies have become valuable tools in studying autoimmune diseases, particularly systemic lupus erythematosus (SLE). Key applications include:

• Quantifying CD226+ B cell populations, which are significantly elevated in SLE patients compared to healthy controls

• Correlating CD226+ B cell percentages with disease activity metrics:

  • SLEDAI-2K scores (ρ = 0.39, P = 0.006)

  • Anti-dsDNA antibody titers (ρ = 0.45, P = 0.001)

  • Complement levels (negatively correlated with C3: ρ = -0.35, P = 0.015 and C4: ρ = -0.38, P = 0.008)

• Monitoring CD226+ B cells as potential biomarkers for disease prognosis, as lower baseline percentages of CD226+ B cells correlate with better outcomes (Lupus Low Disease Activity State) after 12 months of treatment

What is the role of CD226 antibodies in transplantation research?

In transplantation research, CD226 antibodies are used both as analytical tools and potential therapeutic agents:

• As analytical tools:

  • Monitoring CD226 expression on lymphocyte populations during transplant rejection

  • Analyzing the balance between effector and regulatory T cells

• As experimental therapeutic agents:

  • Anti-CD226 monoclonal antibodies promote expansion of regulatory T cells (Tregs) in mixed lymphocyte cultures

  • Treatment with CD226 mAbs inhibits the cytotoxicity of effector cells

  • In allogeneic skin transplant mouse models, administration of CD226 mAbs reduces inflammation and prolongs allograft survival by increasing Treg frequency

How does CD226 contribute to T cell differentiation and function?

CD226 antibodies have revealed differential expression patterns across T cell subsets:

• Th1 cells:

  • CD226 strongly promotes Th1 differentiation

  • Enhances IFN-γ production in naive T cells

  • CD226 knockdown results in decreased T-bet and IFN-γ expression

• Th2 cells:

  • CD226 and its ligand CD155 are downregulated on Th2-polarized naive T cells

  • IL-13 production does not correlate with CD226 expression

• Th17 cells:

  • CD226 and CD155 are highly expressed under Th17-polarizing conditions

  • Most IL-17-producing cells express high levels of CD226

These findings establish CD226 as an important regulator of the proinflammatory (Th1/Th17) versus anti-inflammatory (Th2) balance in immune responses .

What controls should be included when validating CD226 antibodies for research applications?

When validating CD226 antibodies, researchers should include:

• Positive controls:

  • Cell lines with confirmed high CD226 expression (e.g., activated T cells, NK cells)

  • Recombinant CD226 protein for Western blots and ELISA

• Negative controls:

  • CD226 knockout (CD226 KO) cells or tissues

  • Isotype control antibodies matched to the CD226 antibody class and species

  • Primary antibody omission controls

• Validation approaches:

  • Competitive blocking with soluble CD226 protein

  • siRNA or shRNA knockdown of CD226 to confirm antibody specificity

  • Testing across multiple applications (flow cytometry, Western blotting) to confirm consistent results

How should researchers address potential cross-reactivity issues with CD226 antibodies?

To address potential cross-reactivity:

• Verification strategies:

  • Test antibodies on CD226 knockout samples

  • Compare results across multiple antibody clones targeting different CD226 epitopes

  • Perform peptide competition assays with specific CD226 peptides

• Species considerations:

  • Verify specificity when working across species, as CD226 gene orthologs have been reported in mouse, rat, bovine, zebrafish, chimpanzee, and chicken

  • Be aware that anti-human CD226 antibodies may not recognize mouse CD226 and vice versa

• Experimental design:

  • Include comprehensive panel of surface markers to exclude potential binding to other adhesion molecules

  • Validate antibody performance in your specific experimental system before conducting full studies

What approaches can resolve contradictory data in CD226 expression analysis?

When faced with contradictory data regarding CD226 expression:

• Technical considerations:

  • Evaluate antibody clone differences (some epitopes may be masked in certain contexts)

  • Review cell preparation techniques (enzymatic dissociation may cleave surface CD226)

  • Consider fixation and permeabilization effects on epitope accessibility

• Biological variables:

  • Account for activation-dependent changes in CD226 expression

  • Consider splice variants or post-translational modifications that may affect antibody binding

  • Evaluate the impact of the microenvironment on CD226 expression

• Complementary approaches:

  • Combine protein detection (flow cytometry, Western blot) with mRNA analysis (qRT-PCR)

  • Use CD226 reporter systems in model organisms

  • Employ multiple antibody clones targeting different epitopes

How are CD226 antibodies being used to study the CD226-TIGIT axis?

The CD226-TIGIT axis represents an important immune checkpoint pathway:

• Interaction dynamics:

  • CD226 and TIGIT both bind to CD112 and CD155 ligands

  • TIGIT inhibits the interaction between CD155 and CD226

  • CD226 delivers activating signals while TIGIT provides inhibitory signals

• Research applications:

  • Blocking antibodies against CD226 can be used to study how disrupting CD226-CD155 interactions affects T cell and NK cell functions

  • Dual staining with anti-CD226 and anti-TIGIT antibodies helps analyze the balance between activating and inhibitory signals

  • CD226 antibodies can be used alongside TIGIT-Ig fusion proteins to study their therapeutic potential in autoimmune disease models

What are potential biomarker applications of CD226 antibodies in clinical research?

CD226 antibodies show promise for developing clinical biomarkers:

• In SLE:

  • CD226+ B cells correlate with disease activity metrics (SLEDAI-2K scores, anti-dsDNA antibody titers)

  • Baseline percentages of CD226+ B cells predict disease outcomes at 12 months

  • CD226+ B cells may serve as markers for renal involvement and predict complete renal remission

• In transplantation:

  • Monitoring CD226 expression patterns may help predict rejection risk

  • CD226 expression on specific lymphocyte subsets might serve as a biomarker for immunosuppression efficacy

• Methodological considerations:

  • Flow cytometry panels using CD226 antibodies combined with subset markers can identify clinically relevant immune populations

  • Standardized protocols are needed for potential translation to clinical applications

What strategies can improve the specificity of CD226 detection in complex samples?

Enhancing specificity for CD226 detection requires:

• Sample preparation optimization:

  • Fresh isolation of cells to preserve CD226 epitopes

  • Careful selection of enzymatic dissociation methods to avoid cleaving surface CD226

  • Optimized fixation protocols that maintain epitope integrity

• Advanced detection approaches:

  • Dual staining with antibodies targeting different CD226 epitopes

  • Combining surface and intracellular staining to detect total CD226 pools

  • Using proximity ligation assays to verify CD226-ligand interactions in situ

• Data analysis refinement:

  • Implementing strict gating strategies that exclude potential false positives

  • Using fluorescence-minus-one (FMO) controls for accurate gating

  • Applying computational approaches to resolve complex expression patterns

How can researchers effectively block CD226 function in experimental systems?

For functional blocking of CD226:

• Antibody-based approaches:

  • Select anti-CD226 mAbs demonstrated to block CD226-CD155 interactions

  • Determine optimal antibody concentrations through titration experiments

  • Consider using F(ab')2 fragments to avoid Fc-mediated effects

• Alternative blocking strategies:

  • Recombinant soluble CD155 to compete with cell-bound CD155 for CD226 binding

  • Both extracellular domains of CD226 are required for optimal functional interaction with CD155, so truncated recombinant proteins containing both domains may be effective competitors

  • siRNA or CRISPR-based knockdown/knockout of CD226 for long-term functional studies

• Validation of blocking efficacy:

  • Measure downstream signaling events known to be CD226-dependent

  • Assess functional outcomes like cytokine production or cytotoxicity

  • Confirm specificity by testing effects on CD226-deficient cells