CDF4 Antibody

What is CD4 and where is it expressed in human tissues?

CD4 is a glycoprotein containing four immunoglobulin-like domains (D1-D4) that functions as a co-receptor during antigen presentation and associates with the T cell receptor upon MHC binding . It is primarily expressed on:

• Helper/inducer T cells (high expression)

• Monocytes, tissue macrophages, and granulocytes (lower expression)

Research has documented CD4 expression in multiple tissues:

• Blood (PubMed ID: 1425921)

• Brain (PubMed ID: 9074930)

• Liver (PubMed ID: 19159218)

• Pancreas (PubMed ID: 15489334)

• Thymus (PubMed ID: 14702039)

This tissue distribution has significant implications for experimental design when targeting CD4+ cells in different anatomical contexts.

What applications are most suitable for CD4 antibodies in research settings?

CD4 antibodies have been validated for multiple experimental applications:

When transitioning between applications, researchers should validate the antibody for each specific use case, as performance can vary significantly between techniques .

How do structural properties of CD4 antibodies influence their experimental utility?

The interaction between CD4 antibodies and their target involves specific structural domains that influence their research applications:

• Many CD4-directed antibodies target the D1 domain, which is also the binding site for HIV gp120

• Comparative studies of 225 different CD4-directed antibodies revealed substantial variations in binding properties and kinetics

• Some antibodies, like MAX.16H5 IgG1, share fine specificities with HIV gp120 in recognizing mutated CD4 variants

• Specific peptide sequences (e.g., TbYICbEbVEDQKAcEE) can inhibit both gp120 and certain antibody binding to CD4

Understanding these structural relationships is critical for selecting appropriate CD4 antibodies for specialized applications, particularly in HIV research or T-cell modulation studies.

How do broadly neutralizing CD4-binding site antibodies overcome viral resistance mechanisms?

Broadly neutralizing antibodies (bNAbs) targeting the CD4-binding site have evolved sophisticated mechanisms to counter viral resistance:

• The N6 antibody exemplifies evolutionary adaptations, neutralizing 98% of HIV-1 isolates, including 16 of 20 that were resistant to other CD4-binding site antibodies

• Unlike other antibodies in its class, N6 evolved to tolerate the absence of individual CD4bs contacts across the immunoglobulin heavy chain

• Structural analysis revealed that N6's orientation allows it to avoid steric clashes with glycans, a common mechanism of resistance

• When the conserved residue Asp was introduced at position 279, most pseudoviruses became sensitive to N6 neutralization

These insights into antibody evolution provide valuable information for designing HIV therapies, prophylaxis, and vaccines that can overcome viral escape mechanisms .

What distinguishes the epitope specificity of different CD4 antibodies?

CD4 antibodies exhibit diverse epitope specificities with significant experimental implications:

• Different antibody clones recognize distinct epitopes across the four domains (D1-D4) of CD4

• Some antibodies partially block binding of gp120 to CD4 but fail to inhibit syncytia formation

• Other antibodies inhibit syncytia formation while only weakly blocking gp120 binding

• Antibodies targeting the same general region can have markedly different functional outcomes based on the precise epitope recognized

When selecting CD4 antibodies for research, epitope specificity should be carefully considered in relation to the biological function being studied. This is particularly important for functional studies where blocking specific CD4 interactions may be desired.

How has antibody engineering improved CD4-targeted therapeutics?

Antibody engineering has significantly advanced CD4-directed therapeutics:

• The murine MAX.16H5 IgG1 antibody was successfully applied in clinical trials for autoimmune diseases and transplantation rejection with notable response rates

• To reduce potential immune reactions against the murine Fc part, researchers developed a chimeric version combining:

  • CD4-directed variable domains from the original MAX.16H5 IgG1

  • Human IgG4 backbone to minimize effector functions

• Both antibody variants were subsequently tested in vitro and in humanized mouse transplantation models

These engineering approaches demonstrate how modification of antibody structures can maintain target specificity while improving safety profiles and reducing immunogenicity for therapeutic applications.

What controls the variation in CD4 antibody performance across different tissue types?

Several factors contribute to variable CD4 antibody performance across tissues:

• Tissue-specific CD4 expression levels affect detection sensitivity

• Different fixation methods can alter epitope accessibility in various tissues

• Glycosylation patterns may vary between tissues, potentially masking epitopes

• Background staining characteristics differ between highly vascularized tissues like liver versus other tissues

Researchers have reported positive staining of CD4 in liver cell membranes, initially questioning if this represented specific binding, but literature confirmed CD4 expression in hepatic tissue (PubMed ID: 19159218) . This highlights the importance of tissue-specific validation when expanding CD4 antibody applications to new contexts.

What fixation protocols optimize CD4 antibody performance in immunohistochemistry?

Fixation significantly impacts CD4 antibody performance in tissue sections:

• Paraformaldehyde (PFA) is generally recommended due to its superior tissue penetration properties

• PFA must be prepared fresh before use, as long-term stored PFA converts to formalin by molecular congregation

• For paraffin-embedded tissues, antigen retrieval methods may be necessary to expose CD4 epitopes

• Both frozen and paraffin-embedded sections can be used for CD4 detection, but protocol optimization differs

The choice between fixation methods should be guided by the specific antibody clone and experimental goals, with pilot experiments recommended when establishing new protocols .

How should researchers validate CD4 antibodies for cross-species applications?

Cross-species validation requires systematic assessment:

• Sequence analysis:

  • Perform BLAST comparison between the immunogen sequence and target species to evaluate potential cross-reactivity

  • Assess sequence homology, particularly in the regions containing critical epitopes

• Experimental validation:

  • Conduct pilot tests when sequence homology suggests potential cross-reactivity

  • Include appropriate controls from the target species

  • Compare staining patterns with species-specific antibodies when available

Some manufacturers offer innovator award programs for researchers who validate antibodies in new species applications, potentially offsetting the costs associated with such validation efforts .

What troubleshooting strategies address inconsistent CD4 antibody staining?

When encountering variable CD4 antibody staining results, consider these systematic approaches:

• Antibody factors:

  • Verify storage conditions and minimize freeze-thaw cycles

  • Perform titration experiments to determine optimal concentration

  • Consider lot-to-lot variations that may affect performance

• Sample preparation:

  • Standardize fixation duration and conditions

  • Ensure consistent processing times between sample collection and fixation

  • Implement quality control for key reagents

• Protocol optimization:

  • Adjust incubation times and temperatures

  • Modify blocking reagents to reduce background

  • Test alternative detection systems

• Tissue-specific factors:

  • Consider the reported variable expression of CD4 across different tissues

  • Adjust protocols based on tissue density and antigen accessibility

  • Include tissue-specific positive and negative controls

These approaches help distinguish technical issues from true biological variation in CD4 expression patterns.

How can researchers optimize CD4 antibody protocols for flow cytometry?

Flow cytometry with CD4 antibodies requires specific optimizations:

• Sample preparation:

  • Fresh samples typically yield better results than frozen material

  • Consistent processing minimizes variability

  • Gentle handling preserves surface CD4 expression

• Staining protocol:

  • Determine optimal antibody concentration through titration

  • Adjust incubation time based on sample type (15-30 minutes for blood cells)

  • Consider potential internalization during extended incubations

• Panel design:

  • Select fluorochromes based on expected CD4 expression level

  • Include appropriate compensation controls

  • Implement Fluorescence Minus One (FMO) controls for accurate gating

• Validation:

  • Confirm CD4 detection in known positive populations (T helper cells)

  • Use consistent gating strategies between experiments

  • Consider quantitative approaches using standardized beads

These considerations help ensure reliable and reproducible flow cytometry results when phenotyping CD4+ populations .

How are CD4 antibodies utilized in HIV research?

CD4 antibodies play critical roles in HIV research:

• Mechanistic studies:

  • Investigation of viral entry mechanisms via CD4 binding

  • Analysis of CD4-gp120 interactions and structural requirements

  • Characterization of epitopes shared between antibodies and viral proteins

• Broadly neutralizing antibody development:

  • Identification of antibodies with exceptional breadth and potency, like N6

  • Structural analysis of antibody-HIV Env interactions

  • Evaluation of resistance mechanisms and escape mutations

• Therapeutic development:

  • Design of antibodies that can neutralize diverse viral strains

  • Evolution studies tracking how antibodies develop breadth

  • Structure-guided immunogen design for vaccines

The N6 antibody represents a significant advancement in this field, neutralizing 98% of HIV-1 isolates through a unique mode of recognition that evolved to circumvent common resistance mechanisms .

What considerations are important when developing CD4 antibodies for therapeutic applications?

Therapeutic CD4 antibody development involves specialized considerations:

• Antibody engineering:

  • Humanization to reduce immunogenicity (e.g., chimerization of the murine MAX.16H5)

  • Fc modification to modulate effector functions (e.g., human IgG4 backbone)

  • Optimizing binding affinity and specificity

• Functional assessment:

  • T cell modulation effects in autoimmune disease models

  • Impact on allogeneic transplantation outcomes

  • Preservation of pathogen defense capabilities

• Clinical development:

  • Pharmacodynamic and pharmacokinetic data collection

  • Monitoring for allergic reactions or side effects

  • Assessment of response rates in target conditions

The MAX.16H5 antibody exemplifies this development pathway, progressing from a murine antibody used in clinical trials to a chimeric version with improved properties for therapeutic applications .

How do CD4 antibodies contribute to understanding T cell functionality?

CD4 antibodies provide valuable tools for investigating T cell biology:

• T cell subtype identification:

  • Phenotypic characterization of helper T cell populations

  • Quantification of CD4+ subsets in different tissues

  • Analysis of CD4 expression levels in disease states

• Functional studies:

  • Investigation of CD4's role as a co-receptor during antigen presentation

  • Analysis of T cell modulation in autoimmune diseases

  • Evaluation of CD4-dependent signaling pathways

• Therapeutic mechanism elucidation:

  • Understanding how anti-CD4 antibodies affect T cell responses in autoimmunity

  • Investigating the preservation of immunological functions during treatment

  • Analyzing effects on allogeneic cell and organ transplantation outcomes

These applications contribute to the broader goal of selectively modulating T cell responses while maintaining essential immune functions, a critical balance in both research and therapeutic contexts .

What emerging technologies are enhancing CD4 antibody applications in research?

The field of CD4 antibody research continues to evolve with several technological advances:

• Structural biology approaches:

  • High-resolution imaging of antibody-antigen complexes

  • Hydrogen-deuterium exchange for epitope mapping

  • Cryo-electron microscopy for visualizing binding conformations

• Next-generation sequencing:

  • Analysis of antibody lineage development

  • Tracking co-evolution of virus and antibody responses

  • Identification of key somatic mutations that confer breadth and potency

• Humanized model systems:

  • Specialized humanized mouse transplantation models

  • Ex vivo human tissue systems

  • Organoid technology for tissue-specific studies