CD4-like protein Antibody

What are CD4-like protein antibodies and how do they function in research?

CD4-like protein antibodies encompass both antibodies that recognize the CD4 receptor and engineered proteins that mimic CD4 binding properties. Traditional anti-CD4 antibodies like the monoclonal CA-4 (MA1016) bind to the CD4 receptor on cell surfaces and can be used to identify CD4-positive cells or block CD4-dependent interactions . Meanwhile, CD4 mimics such as CD4M33 are engineered 27-amino acid peptides designed to present optimal interactions with gp120 and bind to viral particles with CD4-like affinity .

In research, these molecules serve multiple functions including:

• Identification and isolation of CD4-expressing cells through flow cytometry

• Visualization of CD4 expression patterns in tissues via immunohistochemistry

• Blocking CD4-dependent interactions to study downstream signaling effects

• Inhibiting HIV-1 entry by competing with cellular CD4 for binding to viral envelope proteins

• Unmasking conserved neutralization epitopes of gp120 that are normally cryptic on unbound glycoprotein

What tissues express CD4 proteins that can be targeted by CD4 antibodies?

CD4 expression is not limited to T cells but is present across multiple tissue types. According to experimental data and literature references, CD4 is expressed in the following tissues:

This diverse expression pattern necessitates careful interpretation of experimental results when using anti-CD4 antibodies across different tissue contexts. Researchers should validate staining patterns through appropriate controls, especially when examining non-lymphoid tissues where CD4 expression might be unexpected but legitimate .

What experimental applications are suitable for anti-CD4 antibodies?

Anti-CD4 antibodies have been validated for multiple experimental applications, with specific considerations for each:

• Flow Cytometry: Anti-CD4 antibodies like MA1016 are effective for identifying CD4+ cells in blood and isolated cell populations. This application requires proper single-cell suspensions and appropriate compensation controls .

• Immunohistochemistry (IHC): Both frozen and paraffin-embedded tissue sections can be analyzed using anti-CD4 antibodies. The antibody MA1016 has been validated for human tissues in IHC applications .

• Immunocytochemistry (ICC): Anti-CD4 antibodies work well for cellular localization studies in cultured cells and cytological preparations .

• Western Blotting: While not explicitly mentioned in the search results, some anti-CD4 antibodies have been reported to work for this application based on user feedback .

When switching between applications (e.g., from ICC to IHC), researchers should conduct validation studies to ensure antibody performance, as noted in user questions regarding transitioning from ICC to IHC with leukocyte samples .

How can researchers differentiate between specific CD4 epitope binding with antibodies versus gp120 interactions?

Distinguishing between antibody-CD4 interactions and gp120-CD4 interactions requires multiple analytical approaches:

• Peptide inhibition studies: The peptide T bYIC bE bVEDQK AcEE has been reported to inhibit CD4 binding of both gp120 and the MAX.16H5 IgG1 antibody, suggesting overlapping binding sites . Researchers can use competitive binding assays with specific peptides to determine if an antibody targets the same epitope as gp120.

• Mutational analysis: Creating point mutations in CD4, particularly at the D368R position which abrogates CD4 binding to gp120, can help determine if an antibody binds to the same region. For example, the D368R mutation prevents sCD4-SNAP-A488 binding while not affecting binding of antibodies targeting other epitopes like mAb 2G12 .

• Comparative binding studies: Direct comparison of 225 different CD4-directed antibodies regarding their CD4 binding properties and kinetics showed that MAX.16H5 IgG1 shared some fine specificities with gp120 in recognition of different mutated CD4 versions .

• Structural analysis: Crystallographic or cryo-EM studies of antibody-CD4 complexes compared to gp120-CD4 complexes provide the most definitive evidence of binding similarities or differences. The CD4 surface binding to gp120 centers on the CDR2-like loop, forming a specific antiparallel β-sheet interaction with the gp120 β15 strand .

By combining these approaches, researchers can precisely characterize which CD4 epitopes are targeted by specific antibodies and how they relate to natural ligand binding sites.

What methodologies are most effective for analyzing stoichiometric relationships between soluble CD4 and HIV-1 envelope proteins?

For precise stoichiometric analysis of CD4-envelope protein interactions, single-molecule detection (SMD) approaches have proven particularly effective:

• Fluorescently labeled components: Utilizing sCD4-SNAP-A488 (soluble CD4 labeled with a fluorescent tag) allows visualization and quantification of binding events at the single-molecule level .

• Immobilized antigen assays: Immobilizing HIV-1 envelope proteins on glass coverslips and probing with fluorescently labeled sCD4 enables direct visualization of binding stoichiometry .

• Mutational controls: The D368R mutation in gp120 that abrogates CD4 binding serves as an excellent negative control. Parallel binding studies with antibodies like mAb 2G12 (which targets carbohydrate clusters unaffected by the D368R mutation) provide positive controls for protein integrity .

• Cryo-EM structural analysis: For higher-resolution stoichiometric analysis, cryo-EM studies of CD4-binding site antibodies in complex with envelope trimers (such as HmAb64 with CNE40 SOSIP trimer) reveal detailed binding configurations .

These methodologies collectively enable researchers to determine not only binding affinity but also the precise stoichiometry and orientation of CD4-envelope interactions, which is crucial for understanding HIV entry mechanisms and designing effective inhibitors.

What are the structural considerations when designing CD4 mimics for HIV-1 inhibition studies?

The rational design of CD4 mimics for HIV-1 inhibition requires detailed structural considerations:

• Focus on critical binding interfaces: The CD4M33 mimic was designed as a 27-amino acid peptide that presents optimal interactions with gp120, based on structural information from CD4-gp120-17b antibody complex crystallography .

• Targeting conserved surfaces: Effective CD4 mimics must target the conserved surfaces of HIV-1 envelope involved in receptor binding, which represent potential targets for entry inhibitors .

• Functional properties preservation: Beyond mere binding, CD4 mimics should preserve key functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein .

• β-sheet interaction modeling: The interaction between CD4 and gp120 centers on the CDR2-like loop, a protruding β-hairpin with its C′′ β-strand forming an antiparallel β-sheet with the gp120 β15 strand. This specific structural arrangement must be preserved in mimics .

• Broad spectrum activity: Effective mimics must bind to diverse HIV-1 envelopes with CD4-like affinity, including primary patient isolates that are generally resistant to inhibition by soluble CD4 .

These structural considerations have led to prototype inhibitors of HIV-1 entry that, in complex with envelope proteins, also represent potential components of vaccine formulations or molecular targets for developing broad-spectrum neutralizing antibodies .

How do researchers validate the specificity of CD4-binding site antibodies in neutralization assays?

Validating CD4-binding site (CD4bs) antibody specificity in neutralization assays involves a multi-faceted approach:

• Cross-clade neutralization panels: Testing against large, diverse panels of HIV-1 pseudovirus strains is essential. For example, HmAb64 was validated against a cross-clade panel of 208 HIV-1 pseudo-virus strains, demonstrating neutralization of 20 (10%), including tier-2 strains from clades B, BC, C, and G .

• Mutational analysis: Introducing specific mutations in the CD4 binding site of gp120 (such as D368R) helps confirm that neutralization is specifically mediated through CD4bs targeting .

• Competitive binding assays: Demonstrating that the antibody competes with soluble CD4 for binding to envelope proteins confirms specificity for the CD4 binding site region.

• Structural characterization: Cryo-EM or crystallographic studies of the antibody-envelope complex provide definitive evidence of binding location. For HmAb64, cryo-EM structure analysis revealed that it uses both heavy and light CDR3s to recognize the CD4-binding loop, confirming its classification as a CD4bs antibody .

• Germline gene analysis: Characterization of antibody lineage can provide supporting evidence. HmAb64 was derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39, with a CDR H3 region of 15 amino acids—typical characteristics of CD4bs antibodies .

This comprehensive validation ensures that neutralization activity is specifically attributable to CD4 binding site targeting rather than alternative mechanisms.

What are the optimal storage and handling conditions for anti-CD4 antibodies?

Proper storage and handling of anti-CD4 antibodies is critical for maintaining their activity and specificity:

• Long-term storage: Store lyophilized antibodies at -20°C for up to one year from the date of receipt. This preserves antibody integrity and prevents degradation .

• Post-reconstitution storage: After reconstitution, antibodies like MA1016 can be stored at 4°C for one month or aliquoted and frozen at -20°C for up to six months .

• Avoiding freeze-thaw cycles: Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of antibody activity .

• BSA considerations: For certain applications, BSA-free formulations may be required. Some manufacturers can provide BSA-free versions of antibodies upon request, with preparation taking approximately 3 extra days .

• Reconstitution medium: The choice of reconstitution buffer can impact antibody performance. For most applications, sterile PBS or manufacturer-recommended buffers should be used.

These storage and handling guidelines ensure optimal antibody performance across experimental applications and extend the useful life of these research reagents.

How do murine versus chimerized anti-CD4 antibodies differ in experimental applications?

Murine and chimerized anti-CD4 antibodies exhibit important differences that influence their experimental applications:

Both antibody formats have been studied extensively in vitro and in specific humanized mouse transplantation models in vivo, providing complementary insights based on their distinct properties .

What fixation methods are recommended for optimal CD4 detection in immunohistochemistry?

Effective CD4 detection in immunohistochemistry requires appropriate fixation techniques:

• Paraformaldehyde (PFA) fixation: Commonly used for preserving cell surface antigens like CD4, typically at 4% concentration. The exact protocol depends on tissue type and section thickness .

• Frozen versus paraffin-embedded sections: Anti-CD4 antibodies like MA1016 have been validated for both frozen and paraffin-embedded sections. For frozen sections, brief fixation (10-20 minutes) with 4% PFA is typically sufficient, while paraffin embedding requires more extensive fixation protocols .

• Antigen retrieval considerations: For paraffin-embedded tissues, heat-induced epitope retrieval methods may be necessary to expose CD4 epitopes masked during fixation and embedding. Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) are commonly used.

• Tissue-specific optimization: Different tissues may require modified fixation protocols:

  • Lymphoid tissues generally work well with standard PFA fixation

  • Liver tissues may benefit from shorter fixation times to prevent excessive crosslinking

  • Brain tissues often require specialized fixation approaches due to lipid content

• Balanced approach: Over-fixation can mask CD4 epitopes, while under-fixation can compromise tissue morphology. Researchers should optimize fixation duration based on section thickness and tissue type .

Researchers are advised to validate any new fixation method with appropriate positive and negative controls before proceeding with experimental samples.

How should researchers interpret unexpected CD4 staining patterns in non-lymphoid tissues?

When encountering unexpected CD4 staining in non-lymphoid tissues, researchers should follow a systematic approach to interpretation:

• Validate with literature: CD4 expression has been documented in multiple non-lymphoid tissues including liver, brain, and pancreas. Unexpected staining may represent legitimate CD4 expression rather than non-specific binding .

• Employ multiple antibody clones: Testing with different anti-CD4 antibody clones targeting distinct epitopes can help confirm whether staining represents true CD4 expression or potential cross-reactivity.

• Perform appropriate controls:

  • Isotype controls to rule out non-specific binding

  • Blocking peptide controls to confirm epitope specificity

  • CD4-knockout or CD4-negative tissues as negative controls

  • Known CD4-positive samples as positive controls

• Correlate with gene expression data: RNA-seq or qPCR data showing CD4 mRNA expression in the tissue of interest supports protein-level findings.

• Consider cell subpopulations: Positive staining in tissues like liver may represent resident or infiltrating immune cells rather than expression by parenchymal cells. Co-staining with cell-type specific markers can resolve this ambiguity .

For example, when researchers observed positive staining in liver cell membranes using anti-CD4 antibody (Monoclonal, CA-4), Boster Scientific Support confirmed this finding was consistent with literature documenting CD4 expression in liver (PubMed ID: 19159218) .

What approaches can resolve issues with CD4 antibody performance in flow cytometry applications?

When troubleshooting CD4 antibody performance in flow cytometry, researchers should consider:

• Titration optimization: Determining the optimal antibody concentration through titration experiments ensures sufficient signal without background staining. This is particularly important when transitioning between applications (e.g., from IHC to flow cytometry).

• Buffer composition: The presence of certain buffer components like BSA can affect antibody performance. For sensitive applications, BSA-free antibody formulations may be requested from manufacturers .

• Sample preparation considerations:

  • Fresh versus frozen samples: Fresh samples typically yield better results

  • Red blood cell lysis protocols: Incomplete lysis can interfere with detection

  • Fixation effects: Some fixatives can mask CD4 epitopes

  • Permeabilization: Only needed if detecting intracellular CD4 pools

• Multi-parameter panel design: When incorporating anti-CD4 antibodies into multi-parameter panels, spectral overlap must be considered. Proper compensation controls are essential for accurate interpretation.

• Instrument settings optimization: PMT voltages should be optimized to place negative and positive populations appropriately on scale. Resolution of CD4-bright and CD4-dim populations may require careful adjustment of detector settings.

Based on user questions about flow cytometry applications with blood samples, manufacturers have confirmed that anti-CD4 antibodies like MA1016 are suitable for flow cytometry with blood samples due to high CD4 expression in this tissue type .

How can researchers effectively use CD4-like proteins in HIV vaccine development strategies?

CD4-like proteins offer several strategic approaches in HIV vaccine development:

• Epitope unmasking: CD4-like proteins such as CD4M33 can unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein, potentially serving as components of vaccine formulations .

• Stabilized envelope complexes: CD4-binding site antibodies or mimics can be used to stabilize envelope proteins in conformations that expose conserved neutralization epitopes, creating more effective immunogens.

• Molecular targets for antibody development: CD4 mimics in complex with envelope proteins can serve as molecular targets in phage display technology to develop broad-spectrum neutralizing antibodies .

• Polyvalent vaccine strategies: Recent research demonstrates that polyvalent DNA prime-protein boost HIV vaccines can elicit CD4-binding site antibodies like HmAb64 capable of neutralizing tier-2 HIV strains .

• Structure-guided immunogen design: Cryo-EM structural analysis of CD4-binding site antibodies like HmAb64 in complex with CNE40 SOSIP trimer reveals critical binding details. HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, providing templates for rational immunogen design .

The elicitation of HmAb64 through vaccination demonstrates that gp120-based vaccines can induce antibodies capable of tier-2 HIV neutralization, a significant advancement in HIV vaccine research that had previously been challenging to achieve .

What are the emerging applications of CD4-like protein antibodies beyond HIV research?

While HIV research has driven much of the development around CD4-like protein antibodies, several emerging applications show promise:

• Autoimmune disease therapies: Anti-human CD4 antibodies like MAX.16H5 have been applied intravenously in clinical trials for treating autoimmune diseases such as rheumatoid arthritis, with detailed studies of effects on lymphocytes, cytokines, and clinical parameters .

• Transplantation immunology: Chimerized versions of anti-CD4 antibodies have been studied in humanized mouse transplantation models, suggesting applications in preventing graft rejection through selective modulation of CD4+ T cell responses .

• Cancer immunotherapy: Modulating CD4+ T cell functions has implications for enhancing anti-tumor immunity. CD4-targeting strategies could complement existing checkpoint inhibitor approaches.

• Neurodegenerative disease research: Given the documented expression of CD4 in brain tissue (PubMed ID: 9074930), anti-CD4 antibodies may help elucidate neuroimmune interactions in conditions like multiple sclerosis and Alzheimer's disease .

• Liver immunobiology: The confirmed expression of CD4 in liver cells opens avenues for investigating CD4's role in liver pathologies and potential therapeutic interventions .

As our understanding of CD4 biology expands beyond its classical role in helper T cell function, these applications represent fertile ground for translational research involving CD4-like protein antibodies.

How might advances in antibody engineering impact next-generation CD4-targeting therapeutics?

Emerging antibody engineering technologies are poised to revolutionize CD4-targeting therapeutics:

• Bispecific antibody platforms: Combining CD4 targeting with other functionalities (e.g., CD3 engagement or checkpoint inhibition) could enable precise modulation of immune responses in various disease contexts.

• Antibody-drug conjugates: Selective delivery of payloads to CD4-expressing cells could provide new therapeutic approaches for conditions ranging from T cell malignancies to autoimmune disorders.

• Enhanced Fc engineering: Modification of Fc regions to fine-tune effector functions could allow precise control over whether a CD4-targeting antibody depletes cells or simply blocks function. The chimerization of MAX.16H5 demonstrates this principle by replacing a murine IgG1 Fc with a human IgG4 backbone to alter effector properties .

• Domain-specific targeting: Rather than targeting the whole CD4 molecule, next-generation antibodies might target specific domains (D1-D4) to modulate distinct CD4 functions while preserving others.

• Intrabody approaches: Engineered antibodies expressed intracellularly could target intracellular pools of CD4 or specific CD4 signaling complexes, offering unprecedented specificity in modulating CD4 functions.

These advances build upon foundational work with antibodies like MAX.16H5, which demonstrated high affinity to CD4 and was selected for therapeutic development after comparative evaluation of multiple CD4-directed antibodies .