CPRD49 Antibody

What is CD49d and what are its biological functions?

CD49d (also known as α4 integrin, VLA-4 α chain, integrin α4, or ITGA4) is a 150 kD glycoprotein and member of the integrin family. It associates noncovalently with either β1 (CD29) to form the VLA-4 (α4β1) heterodimer or with β7 to form the α4β7 heterodimer. These complexes play critical roles in:

• Cell adhesion and T cell costimulation

• Leukocyte migration from blood to tissue at sites of inflammation

• Binding to ligands including VCAM-1, MAdCAM-1, and fibronectin

CD49d is expressed on T and B cells, monocytes, eosinophils, basophils, mast cells, thymocytes, NK cells, and dendritic cells, with notable absence on neutrophils .

How do different CD49d antibody clones vary in their applications?

Different antibody clones recognize distinct epitopes and demonstrate varying functionalities:

• R1-2 clone (anti-mouse CD49d): Partially blocks CD49d-mediated interactions and can be used in combination with 9C10 (MFR4.B) antibody to completely block VCAM-1 binding to VLA-4

• 7.2 clone (anti-human CD49d): Widely used for flow cytometry and other applications with human samples

• DX5 clone: Reacts with CD49b (different subunit) expressed on mouse NK cells and T cell subsets

Each clone has been validated for specific applications including flow cytometry, blocking assays, immunoprecipitation, and immunohistochemistry depending on the clone.

What are the optimal protocols for using CD49d antibodies in flow cytometry?

For effective flow cytometry applications:

• Titrate antibodies for optimal performance (typically ≤0.25 μg per test for 10^5-10^8 cells)

• Use appropriate buffer (PBS with <0.1% sodium azide and stabilizer)

• Include proper controls:

  • Isotype controls (Rat IgG2b, κ for R1-2; Mouse IgG1κ for 7.2)

  • Unstained and single-color controls for compensation

Storage recommendations include keeping antibodies at 2-8°C, avoiding exposure to light (especially for fluorochrome-conjugated antibodies), and not freezing the preparations .

How can CD49d antibodies be effectively utilized in cell adhesion blocking experiments?

For blocking experiments:

• Ultra-LEAF™ purified antibody formulations (Endotoxin < 0.01 EU/μg, Azide-Free, 0.2 μm filtered) are recommended for functional assays

• The R1-2 antibody provides partial blocking of CD49d-mediated interactions

• For complete blocking of VCAM-1 binding to VLA-4, combine R1-2 with 9C10 (MFR4.B) antibody

• Establish appropriate concentration through titration experiments

• Include proper controls to validate blocking specificity

What are the best practices for immunohistochemistry applications with CD49d antibodies?

When using CD49d antibodies for immunohistochemistry:

• Frozen sections are recommended over paraffin-embedded tissues

• Fixation protocols should be optimized to preserve epitope integrity

• Signal amplification systems may be necessary for detecting low expression levels

• Include appropriate positive controls (tissues known to express CD49d)

• Validate staining patterns with alternative detection methods

How can CD49d antibodies be integrated into immunophenotyping panels for disease research?

CD49d antibodies serve as valuable markers in multiparameter immunophenotyping:

• Include in panels targeting lymphocyte and leukocyte subsets

• Combine with lineage markers (CD3, CD4, CD8, CD19) and activation markers

• Use with computational analysis methods like machine learning for identifying distinct immunotypes

• Apply standardized z-scores for cross-cohort comparisons

In COVID-19 research, CD49d has been used alongside cytokine measurements and other antibodies to identify three distinct immunotypes: balanced response immunotype (BRI), excessive inflammation immunotype (EXI), and low antibody immunotype (LAI) .

What considerations are important when analyzing CD49d expression data using computational approaches?

When applying computational analysis to CD49d expression data:

• Normalize data appropriately (e.g., using log transformation and z-scores)

• For hierarchical clustering:

  • Use appropriate distance metrics (e.g., Euclidean distance)

  • Apply Ward's Hierarchical Agglomerative Clustering Method (ward.d2)

  • Determine optimal cluster numbers using multiple indices

• For network analysis:

  • Consider relationships between CD49d expression and other relevant markers

  • Apply appropriate transformations for edge weighting

  • Filter connections to focus on the most significant relationships

Table: Computational Analysis Methods for CD49d Expression Data

How does CD49d expression correlate with disease phenotypes in immune-mediated conditions?

CD49d expression analysis provides insights into disease mechanisms:

• Expression patterns differ between health and disease states

• Can be used to monitor therapeutic responses, particularly for treatments targeting adhesion molecules

• Correlates with inflammatory activity in autoimmune conditions

• Serves as a potential biomarker for predicting disease progression

In COVID-19 patients, analysis of CD49d alongside other markers helped identify distinct immunotypes that correlated with disease severity and clinical outcomes, demonstrating the utility of this approach for patient stratification .

What are common technical challenges when working with CD49d antibodies and how can they be addressed?

Researchers may encounter several challenges:

• Background staining issues:

  • Solution: Optimize blocking protocols and use proper isotype controls

  • Ensure filter settings are appropriate for the fluorochrome

• Poor signal intensity:

  • Solution: Titrate antibody concentration, adjust incubation times and temperatures

  • Verify target expression levels in positive control samples

• Cross-reactivity concerns:

  • Solution: Validate antibody specificity using knockout or silenced cell controls

  • Refer to validated strain reactivity data (e.g., DX5 clone has been tested with BALB/c, C57BL/6, C3H, CBA, DBA, AKR, SJL and 129 strains)

How can researchers validate CD49d antibody performance in their specific experimental systems?

Validation approaches include:

• Comparing results across multiple antibody clones targeting different epitopes

• Using positive and negative control samples with known CD49d expression levels

• Confirming expression through complementary techniques (flow cytometry, Western blot, qPCR)

• Testing blocking efficiency using functional assays that measure adhesion

• Analyzing co-expression with known interaction partners (CD29, CD106)

How are CD49d antibodies being utilized in novel immunotherapy research?

CD49d antibodies are increasingly important in therapeutic development:

• As tools for identifying patient subgroups likely to respond to integrin-targeting therapies

• For monitoring therapy-induced changes in immune cell trafficking

• In developing blocking strategies that could reduce inflammatory cell recruitment

• For understanding resistance mechanisms to existing therapies

Research combining CD49d assessment with machine learning approaches helps identify patient immunotypes that might benefit from personalized therapeutic strategies .

What role does CD49d play in immunological memory and long-term immune responses?

Understanding CD49d in memory responses involves:

• Assessing differential expression on naive versus memory T and B cell populations

• Examining the role of CD49d in bone marrow homing of memory cells

• Investigating CD49d-dependent retention of memory cells in specific tissue niches

• Studying how CD49d-dependent interactions influence recall responses

These investigations can benefit from comprehensive immunophenotyping approaches that incorporate CD49d antibodies within broader panels examining memory marker expression.