CDA4 Antibody

What is CD4 and why are CD4 antibodies important research tools?

CD4 is a 51 kDa surface glycoprotein expressed primarily on T helper cells, but also found on macrophages, dendritic cells, and NK cells. It functions as a critical coreceptor for the T-cell receptor (TCR) in interactions with MHC class II complexes. CD4 antibodies are essential tools for identifying and characterizing these cell populations, studying signal transduction pathways, and understanding HIV pathogenesis since CD4 serves as the primary receptor for HIV-1 .

The methodological importance of CD4 antibodies lies in their ability to specifically bind to CD4 molecules, allowing researchers to detect, quantify, isolate, or manipulate CD4+ cells in various experimental contexts. Applications range from flow cytometry and immunohistochemistry to functional assays investigating T cell activation and therapeutic development.

How do I select the appropriate CD4 antibody clone for my flow cytometry experiments?

Selection of the optimal CD4 antibody clone depends on several experimental factors:

• Epitope specificity: Different clones (e.g., clone #11830, clone #1068647) recognize distinct epitopes on CD4, which may affect binding under certain experimental conditions .

• Species compatibility: Confirm the antibody recognizes your species of interest. Human CD4 antibodies may not cross-react with murine CD4.

• Application compatibility: Verify the clone has been validated for flow cytometry. For example, the clone #11830 has demonstrated efficacy in detecting CD4 in human peripheral blood lymphocytes by flow cytometry when used with appropriate secondary antibodies .

• Fluorophore selection: Consider your cytometer configuration and other markers in your panel to avoid spectral overlap.

• Titration: Always perform antibody titration to determine optimal concentration, as noted in product guidance: "Optimal dilutions should be determined by each laboratory for each application" .

For multicolor panels, test for potential interference with other antibodies, particularly those targeting nearby epitopes on the CD4 molecule.

What are the fundamental differences between monoclonal and polyclonal CD4 antibodies in research applications?

When designing experiments investigating specific CD4 domains, monoclonal antibodies are preferred due to their epitope precision. For example, in studies examining the differential effects of CD4 binding in rat adjuvant arthritis, researchers specifically utilized distinct monoclonal antibodies (W3/25, OX35, and RIB5/2) with known epitope specificities to examine their varied effects on T-cell function .

How can I optimize CD4 antibody staining to detect low expression populations in flow cytometry?

Detecting CD4dim populations requires methodological optimization:

• Antibody titration: Determine the optimal antibody concentration that maximizes separation between positive and negative populations while minimizing background. This is critical as noted in multiple antibody specifications .

• Bright fluorophores: For dim populations, select brighter fluorophores (PE, APC) rather than FITC or Pacific Blue.

• Blocking protocol: Implement robust Fc receptor blocking to reduce non-specific binding, particularly important when analyzing macrophages or dendritic cells expressing CD4.

• Advanced compensation: Perform stringent compensation using single-stained controls with the same cells as your experimental sample.

• Signal amplification: Consider biotin-streptavidin systems or sequential staining approaches for signal enhancement.

• Instrument optimization: Adjust PMT voltages for optimal signal resolution of dim populations.

• Data analysis strategies: Use visualization techniques like biexponential scaling and consider dimensionality reduction techniques (tSNE, UMAP) to better resolve population clusters.

The scientific data provided for the Human CD4 Monoclonal Antibody (Catalog # MAB37911) demonstrates successful staining of human peripheral blood lymphocytes when used with PE-conjugated anti-Mouse IgG secondary antibody and an appropriate isotype control .

What considerations are important when using CD4 antibodies for functional studies versus phenotypic characterization?

Research has demonstrated that different anti-CD4 mAbs can induce varied functional effects. For example, in studies of rat adjuvant arthritis, differential effects on T-cell function and TNF-α secretion were observed depending on which anti-CD4 mAb was used (RIB5/2 vs. W3/25 or OX35) . When designing functional studies, researchers should carefully select antibody clones based on their known functional impacts.

How do broadly neutralizing CD4-binding-site antibodies differ from standard anti-CD4 antibodies in HIV research?

CD4-binding-site (CD4bs) antibodies represent a specialized class of antibodies targeting the site on HIV envelope glycoprotein (Env) that interacts with CD4, rather than directly targeting CD4 itself. Their distinction from standard anti-CD4 antibodies is crucial for HIV research:

• Target specificity: Standard anti-CD4 antibodies bind directly to the CD4 receptor on host cells, while CD4bs antibodies target the corresponding binding site on HIV Env.

• Neutralization mechanism: CD4bs antibodies (like N6) achieve HIV neutralization by preventing viral engagement with CD4, demonstrating extraordinary breadth against diverse HIV-1 isolates. For example, N6 potently neutralized 98% of HIV-1 isolates tested, including 16 of 20 that were resistant to other antibodies in its class .

• Structural recognition patterns: Advanced CD4bs antibodies like N6 have evolved unique modes of recognition. N6 specifically "evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain" and avoids "steric clashes with glycans, which is a common mechanism of resistance" .

• Immunotherapeutic potential: While standard anti-CD4 antibodies may be immunosuppressive (targeting host immune cells), CD4bs antibodies specifically target the virus, making them attractive candidates for therapeutic and prophylactic applications against HIV-1 .

• Vaccine design applications: CD4bs antibodies inform immunogen design strategies for vaccines, as demonstrated by efforts to design antigens that elicit broadly neutralizing antibodies targeting the CD4 binding site .

Understanding these differences is essential for researchers designing HIV immunotherapies or vaccines targeting the CD4-HIV interaction interface.

What methodological approaches can resolve contradictory results when using different CD4 antibody clones in the same experiment?

Contradictory results with different CD4 antibody clones are not uncommon and require systematic troubleshooting:

• Epitope mapping analysis: Different antibody clones recognize distinct epitopes on CD4. For example, clone #11830 targets the extracellular domain , while others may target different regions. Map the epitopes recognized by each clone using epitope prediction tools or experimental approaches.

• Differential epitope accessibility: Consider whether your experimental conditions (fixation, permeabilization) differentially affect epitope accessibility for different clones.

• Cross-validation approaches:

  • Compare results using multiple detection methods (flow cytometry, Western blot, ELISA)

  • Use genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • Implement orthogonal detection strategies (mRNA analysis, functional assays)

• Systematic comparison design: When comparing clones, use a standardized matrix approach:

• Biological context consideration: Research has demonstrated that different anti-CD4 mAbs can have contradictory effects in the same model. For instance, in rat adjuvant arthritis studies, the anti-CD4 mAb RIB5/2 led to significantly higher TNF-α secretion compared to other anti-CD4 mAbs (W3/25 and OX35) . These findings suggest that biological responses to different CD4 antibody clones may genuinely differ based on their specific epitope targets and functional properties.

How can CD4 antibodies be integrated into advanced multiparameter single-cell analysis workflows?

Integration of CD4 antibodies into advanced single-cell analysis requires strategic considerations:

• Panel design strategy:

  • Position CD4 in the appropriate fluorophore channel based on expected expression level

  • For high-dimensional cytometry (CyTOF, spectral), consider metal-conjugated CD4 antibodies

  • When using transcriptomics approaches, integrate with antibody-derived tags for CITE-seq

• Multimodal analysis approaches:

  • Combine CD4 antibody staining with functional readouts (cytokine production, phospho-flow)

  • Integrate with transcription factor staining for comprehensive T cell subset characterization

  • Consider CD4 as part of larger lineage-defining marker panels

• Advanced analytical frameworks:

  • Implement unsupervised clustering algorithms to identify novel CD4+ subpopulations

  • Use trajectory analysis to map developmental relationships between CD4+ populations

  • Apply machine learning approaches to correlate CD4 expression patterns with functional outcomes

• Technical validation procedures:

  • Verify CD4 antibody performance in the context of full panel through spike-in controls

  • Assess potential fluorophore interactions that may affect CD4 detection

  • Validate findings across multiple donors/samples to ensure reproducibility

• Single-cell sequencing integration:

  • Use index sorting to correlate CD4 antibody signal with transcriptomic profiles

  • For CITE-seq/REAP-seq approaches, carefully titrate CD4 antibody-oligo conjugates

  • Ensure computational pipelines correctly integrate protein (CD4) and transcript data

Researchers have successfully utilized CD4 antibodies in conjunction with other markers (such as CD3 epsilon) for precise identification of T cell subsets, as demonstrated in flow cytometry protocols using anti-CD4 monoclonal antibodies alongside CD3 epsilon APC-conjugated antibodies .

What are the most common sources of variability in CD4 antibody performance across different experimental systems?

Variability in CD4 antibody performance can stem from multiple sources:

• Sample preparation factors:

  • Cell isolation method (enzymatic vs. mechanical) affecting epitope integrity

  • Fixation/permeabilization protocols altering conformational epitopes

  • Freeze-thaw cycles potentially degrading CD4 surface expression

• Antibody-specific factors:

  • Lot-to-lot variability in antibody production

  • Storage conditions impacting antibody stability

  • Conjugation efficiency differences between lots

• Technical variables:

  • Instrument calibration inconsistencies

  • Operator technique variations in staining protocols

  • Data analysis gating strategy differences

• Biological variables:

  • Donor-to-donor variation in CD4 expression levels

  • Activation state altering CD4 surface density

  • Disease conditions modifying CD4 epitope accessibility

• Protocol standardization issues:

  • Inconsistent adherence to manufacturer recommendations regarding "optimal dilutions should be determined by each laboratory for each application"

  • Variable blocking protocols affecting background staining

  • Inconsistent secondary antibody selection when using unconjugated primaries

Quality control measures should include routine titration of new antibody lots, inclusion of biological controls (known positive and negative samples), and implementation of standardized protocols with detailed documentation of all variables.

How can researchers distinguish between true CD4 signal and non-specific binding in challenging tissue samples?

Distinguishing specific from non-specific CD4 antibody binding requires rigorous controls and optimization:

• Essential control framework:

  • Isotype controls matched to antibody class and conjugate (e.g., Mouse IgG2A for clone #11830)

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

  • Biological negative controls (CD4-knockout or CD4-negative cell lines)

  • Blocking peptide controls using soluble CD4 to compete for antibody binding

• Signal validation approaches:

  • Parallel staining with two different CD4 antibody clones recognizing distinct epitopes

  • Correlation of protein detection with mRNA expression (RNA-FISH or single-cell RNA-seq)

  • Titration series to identify optimal signal-to-noise concentration

• Advanced tissue-specific considerations:

  • Autofluorescence reduction protocols (e.g., Sudan Black B treatment)

  • Antigen retrieval optimization for fixed tissues

  • Implementation of spectral unmixing for challenging tissue autofluorescence

• Analytical validation strategies:

  • Quantitative assessment of staining index (SI) across multiple conditions

  • Background subtraction based on appropriate negative controls

  • Signal-to-noise ratio calculation for objective quality assessment

• Multiparameter verification:

  • Co-staining with lineage-specific markers to confirm expected distribution

  • Functional correlation (e.g., cytokine production in putative CD4+ T cells)

  • Spatial distribution analysis in tissue consistent with known CD4+ cell localization

Scientific data reported for validated CD4 antibodies typically demonstrates clear discrimination between positive and negative populations, as shown in flow cytometry experiments comparing staining with anti-CD4 antibodies versus isotype controls .

How are CD4 antibodies being utilized in emerging single-cell spatial analysis platforms?

CD4 antibodies are being integrated into cutting-edge spatial analysis techniques:

• Imaging Mass Cytometry (IMC):

  • Metal-conjugated CD4 antibodies enable high-dimensional spatial analysis

  • Multiplexed with 40+ other markers for comprehensive immune microenvironment mapping

  • Resolution at subcellular level allows precise localization of CD4 relative to other markers

• Multiplexed Ion Beam Imaging (MIBI):

  • Similar to IMC but utilizing different detection technology

  • CD4 antibodies incorporated into panels for tumor microenvironment analysis

  • Allows quantification of CD4+ T cell infiltration patterns in spatial context

• Cyclic Immunofluorescence (CyCIF):

  • Sequential staining/bleaching approaches incorporating CD4 antibodies

  • Enables co-detection of CD4 with dozens of other markers on the same tissue section

  • Particularly valuable for analyzing T cell-APC interactions in lymphoid tissues

• In situ Sequencing with Antibody Detection:

  • Combining CD4 protein detection with transcriptomic analysis in tissue context

  • Links CD4+ cell phenotype with functional gene expression in intact tissues

  • Enables discovery of tissue-specific CD4+ T cell states

• Spatial Transcriptomics with Antibody Integration:

  • CD4 antibodies used alongside spatial transcriptomics platforms

  • Correlates CD4 protein expression with gene expression profiles in spatial context

  • Particularly valuable for understanding tissue-resident memory CD4+ T cell populations

These emerging technologies are revealing previously unappreciated spatial relationships between CD4+ cells and other immune cells, providing new insights into immune response coordination in tissues.

What methodological approaches are being developed to utilize CD4 antibodies in HIV vaccine research and immunogen design?

CD4 antibodies and CD4-binding site (CD4bs) antibodies are playing crucial roles in HIV vaccine development:

• Structure-guided immunogen design strategies:

  • CD4bs antibodies like N6 inform the design of immunogens that can elicit broadly neutralizing antibodies against HIV

  • Computational frameworks leverage CD4bs antibody structural information to design antigen panels

  • These designed antigens are tested for stability and recognition by known HIV antibodies

• Sequential immunization approaches:

  • Multi-stage vaccination strategies informed by CD4bs antibody development:
    a) Initial activation of appropriate precursor B cells
    b) Guided affinity maturation along pathways toward broadly neutralizing antibodies
    c) Final "polishing" stage using mixtures of optimized Env variants

• Fitness landscape-based antigen design:

  • Using the "gp160 fitness landscape, which measures the ability of the virus to tolerate mutations"

  • Design of antigen panels that specifically target the CD4 binding site while accounting for viral escape mechanisms

• Antibody lineage-based vaccine strategies:

  • Analysis of how potent CD4bs antibodies like N6 evolve from precursors

  • N6 specifically "evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain"

  • Design of immunization regimens to recapitulate this evolutionary pathway

• Germline-targeting immunogens:

  • Design of antigens specifically targeting the germline precursors of CD4bs antibodies

  • Sequential boosting strategies to guide antibody maturation toward broadly neutralizing variants

Research on the CD4bs antibody N6 has been particularly informative, as it "potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class" and evolved structural features that "permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance" .

How do anti-CD4 antibodies perform in different animal models, and what cross-reactivity considerations are important?

Cross-species application of CD4 antibodies requires careful consideration:

Species-specific methodological considerations:

• Humanized mouse models:

  • Human CD4 antibodies can be used to track human T cells

  • Special blocking required to reduce background in chimeric systems

  • CD4 expression levels may differ from normal human samples

• Non-human primate studies:

  • Epitope conservation verification needed before study

  • Clone-dependent cross-reactivity requires validation

  • May require different secondary detection systems

• Transgenic systems:

  • Murine CD4-knockout/human CD4-transgenic systems allow testing of anti-human-CD4 mAbs in various disease models

  • Expression levels may differ from natural systems

  • Background strain considerations impact experimental design

Research in rat models has demonstrated the value of species-specific approaches, with studies using rat-specific anti-CD4 mAbs (W3/25, OX35, and RIB5/2) showing differential effects in adjuvant arthritis models .

What are the current limitations and advances in using CD4 antibodies for therapeutic applications versus research applications?

Current limitations in therapeutic applications:

• Immunogenicity concerns: Anti-drug antibody formation against mouse-derived sequences

• Broad immunosuppressive effects: Anti-CD4 therapies may cause general immunosuppression

• Variable clinical efficacy: Different anti-CD4 mAbs show "variability in the clinical efficacy in the treatment of rheumatoid arthritis"

• Epitope-dependent outcomes: "Clinical efficacy (and its time course) may depend on the actual immunological constellation"

Recent advances:

• Humanized antibody development: "New, humanized anti-CD4 mAbs" show promise in clinical applications

• Differential epitope targeting: Understanding that "the individual features and effects of a particular anti-CD4 mAb have to be assessed before treatment trials"

• CD4bs antibodies for HIV: Novel antibodies like N6 achieving "potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis"

• Preclinical testing systems: Development of "huCD4-transgenic systems to assess the immunological effects of a particular anti-human-CD4 mAb in various disease models"

These advances continue to bridge the gap between research and therapeutic applications of CD4 antibodies, with ongoing refinement of humanized antibodies showing particular promise.