LCR36 Antibody

What is CD36 and what are its main functions in human biology?

CD36 is a member of the class B scavenger receptor family expressed on multiple cell types including microvascular endothelium, adipocytes, skeletal muscle, epithelial cells of the retina, breast, and intestine, smooth muscle cells, erythroid precursors, platelets, megakaryocytes, dendritic cells, and monocytes/macrophages . Functionally, CD36 serves as a multiligand pattern recognition receptor that interacts with numerous structurally dissimilar ligands. It plays critical roles in lipid metabolism as a fatty acid translocase necessary for the binding and transport of long-chain fatty acids (LCFAs) . Additionally, CD36 contributes to the clearance of apoptotic cells and cell debris, mediates the anti-angiogenic effects of thrombospondin-1, and transduces signals leading to pro-inflammatory cellular responses upon ligand binding .

How do CD36 antibodies differ in their binding specificity?

CD36 antibodies vary in their binding specificity based on the epitope they recognize. For example, some antibodies like the monoclonal antibody eBioNL07 (NL07) recognize specific regions of human CD36 and are particularly suitable for flow cytometric analysis . The specificity of anti-CD36 antibodies can be determined through competition assays, where researchers can assess whether different antibodies compete for the same binding site. Recent research has identified novel anti-CD36 single-chain variable fragments (scFvs), such as D11, that compete with commercial anti-CD36 antibodies with proven efficacy in disease models . When selecting a CD36 antibody, researchers should consider which domain of CD36 they wish to target, as different domains mediate interactions with different ligands (e.g., the CLESH domain interacts with thrombospondin-1) .

What expression patterns of CD36 should researchers consider when designing experiments?

Researchers should account for the broad and varied expression pattern of CD36 across multiple cell types. CD36 is expressed on microvascular (but not large vessel) endothelium, adipocytes, skeletal muscle, dendritic cells, epithelia of the retina, breast, and intestine, smooth muscle cells, and hematopoietic cells including erythroid precursors, platelets, monocytes/macrophages, and megakaryocytes . Of particular note, expression on platelets is absent in Nakᵃ negative donors . This diverse expression pattern means that researchers must carefully select appropriate cell types and controls for their experiments, especially when studying CD36 in complex tissues or in vivo models. Additionally, CD36 expression levels may change under different pathological conditions, such as atherosclerosis or cancer, which should be considered when designing experiments to study disease mechanisms .

How should researchers optimize CD36 antibody use in flow cytometry?

For flow cytometric analysis of CD36 expression, researchers should follow these methodological guidelines:

• Titrate the antibody carefully to determine optimal concentration. For example, the eBioNL07 (NL07) antibody can be used at ≤0.5 μg per test, where a test is defined as the amount of antibody that will stain a cell sample in a final volume of 100 μL .

• Determine appropriate cell numbers empirically, typically ranging from 10⁵ to 10⁸ cells/test .

• Include proper controls, including isotype controls and known CD36-positive and CD36-negative samples.

• When analyzing platelets or monocytes/macrophages, be aware that CD36 expression levels may vary based on activation state.

• For multicolor flow cytometry, select fluorophores with minimal spectral overlap and perform compensation controls.

Optimal results require antibodies with high purity (>90% as determined by SDS-PAGE) and low aggregation (<10% as determined by HPLC) . Post-manufacturing filtration (0.2 μm) ensures consistency in results .

What are the recommended protocols for using CD36 antibodies in Western blot analysis?

For effective Western blot detection of CD36, researchers should consider the following protocol:

• Sample preparation: When working with tissue samples such as placenta or platelets, ensure proper lysis under reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 1) .

• Antibody concentration: Use approximately 1 μg/mL of anti-human CD36/SR-B3 antibody, followed by appropriate HRP-conjugated secondary antibody .

• Expected bands: Be aware that glycosylation affects the apparent molecular weight of CD36. Researchers should expect to detect bands at approximately:

  • 85-90 kDa in human placenta tissue and platelets under reducing conditions

  • 140 kDa in human placenta tissue using Simple Western system

  • 125 kDa in human adipose tissue using Simple Western system

• Loading control: Include appropriate loading controls based on your sample type.

The variation in molecular weight (85-140 kDa) is due to post-translational modifications, particularly glycosylation, which can differ between tissue types .

How can CD36 antibodies be effectively used to study lipid metabolism in cellular models?

CD36 antibodies can be powerful tools for studying lipid metabolism in cellular models through the following methodological approaches:

• Blocking experiments: Anti-CD36 antibodies can block the uptake of CD36 ligands. For example, the D11 scFv reduces lipid accumulation in macrophage-like THP-1 cells and HepG2 cells by blocking CD36-mediated lipid uptake .

• Phenotypic assays: After antibody treatment, researchers can assess:

  • Lipid droplet content via oil red staining

  • Expression of lipid metabolism genes through RT-qPCR

  • Foam cell formation in macrophages exposed to oxidized LDL

• Competitive binding assays: Determine if antibodies block specific ligand interactions (e.g., oxLDL, fatty acids) by pre-incubating cells with antibodies before adding labeled ligands.

• Comparison with genetic approaches: Compare antibody-mediated blockade with genetic knockdown/knockout of CD36 to distinguish between acute vs. chronic loss of CD36 function.

In THP-1 macrophage models, anti-CD36 antibodies have been shown to impair the acquisition of foam cell phenotype induced by oxLDL, decreasing both lipid droplet content and the expression of lipid metabolism genes . Similarly, in HepG2 cells, anti-CD36 antibodies reduce lipid accumulation and the enhanced clonogenicity stimulated by palmitate .

How does anti-CD36 interfere with red blood cell antibody screening and what methods can detect this interference?

Anti-CD36 has been confirmed to interfere with red blood cell (RBC) antibody screening, a phenomenon previously only suspected but recently confirmed through research presented at the AABB Plenary Oral Abstract Session . This interference presents a significant challenge in immunohematology testing, particularly in obstetrical settings. To address this interference:

• Detection method: When anti-CD36 interference is suspected, researchers should use recombinant CD36 protein (rCD36p) testing to confirm antibody specificity .

• Implementation strategy: The rCD36p test is described as inexpensive, easy-to-use, and effective not only for confirming anti-CD36 specificity but also for detecting potential underlying RBC alloantibodies of common specificity that might be masked by anti-CD36 .

• Patient populations: This testing is particularly important for pregnant individuals, as anti-CD36 has been associated with severe fetal/neonatal thrombocytopenia .

Research based on blood samples from 105 patients (99 women, 76 in obstetrical settings; and six men) with suspected anti-CD36 showed that patients' plasma reactivity could be fully neutralized with rCD36p, confirming the specificity of the antibody and its interference with standard screening methods .

What are the critical considerations when developing therapeutic anti-CD36 antibodies?

The development of therapeutic anti-CD36 antibodies requires careful attention to several key factors:

• Epitope selection: Target specific domains of CD36 based on the pathology being addressed. For example, targeting the domain responsible for oxLDL binding in atherosclerosis or the domain involved in fatty acid transport in metabolic disorders.

• Antibody format: Consider different formats beyond traditional antibodies. The development of single-chain variable fragments (scFvs) like D11 offers advantages in terms of tissue penetration and potential for further engineering .

• Competition with natural ligands: Assess whether the antibody effectively competes with disease-relevant CD36 ligands. For instance, D11 scFv competes with a commercial anti-CD36 antibody with proven efficacy in disease models .

• Functional validation: Evaluate the antibody's ability to block pathological processes in relevant cellular models. For example:

  • In macrophage-like THP-1 cells, effective anti-CD36 antibodies should impair foam cell formation induced by oxLDL

  • In HepG2 cells, they should reduce lipid accumulation and clonogenicity stimulated by palmitate

• Potential off-target effects: Given CD36's broad expression pattern, assess potential impacts on normal physiological functions in multiple tissues.

Recent research has identified human anti-CD36 scFvs with therapeutic potential that effectively block CD36 and reduce pathogenic features induced by CD36 ligands, suggesting promise for the development of therapeutic proteins targeting CD36 in diseases such as atherosclerosis and cancer .

How can researchers differentiate between the effects of different CD36 ligands when using anti-CD36 antibodies?

Differentiating between effects of various CD36 ligands requires sophisticated experimental designs:

• Domain-specific antibodies: Use antibodies that target specific domains of CD36 known to interact with particular ligands. For example, antibodies targeting the CLESH domain would specifically block thrombospondin-1 interactions without affecting fatty acid transport .

• Competitive binding assays: Perform assays where labeled and unlabeled ligands compete for CD36 binding in the presence/absence of specific antibodies to determine binding site overlap.

• Phenotypic readouts: Develop assays that measure ligand-specific cellular responses:

  • For fatty acids: measure lipid droplet formation and expression of lipid metabolism genes

  • For oxLDL: assess foam cell formation and inflammatory cytokine production

  • For thrombospondin-1: evaluate angiogenesis inhibition

• Combined approaches: Use antibodies in combination with specific ligand competitors or in cells with CD36 mutations affecting specific ligand-binding domains.

By carefully designing these experiments, researchers can determine whether an antibody blocks specific ligand interactions or has broader inhibitory effects across multiple CD36 functions .

What strategies can researchers employ to improve specificity when using CD36 antibodies in complex tissue samples?

Improving specificity when working with CD36 antibodies in complex tissues requires:

• Antibody validation: Confirm antibody specificity using positive controls (tissues known to express CD36) and negative controls (CD36-knockout tissues or cells) .

• Blocking peptides: Use recombinant CD36 proteins to pre-absorb antibodies and confirm binding specificity; non-specific binding will remain after pre-absorption .

• Multiple antibody approach: Use more than one antibody targeting different CD36 epitopes to confirm findings.

• Background reduction techniques:

  • For immunohistochemistry: Optimize blocking solutions (use sera from species different from both primary and secondary antibodies)

  • For Western blot: Increase washing steps and optimize blocking conditions

• Signal amplification methods: For tissues with low CD36 expression, consider tyramide signal amplification or other signal enhancement approaches.

• Antigen retrieval optimization: For fixed tissues, test multiple antigen retrieval methods to maximize epitope accessibility while maintaining tissue morphology.

When working with complex samples like human placenta tissue, using validated antibodies at optimized concentrations (e.g., 1 μg/mL for Western blot) helps ensure specific detection of CD36 bands at the expected molecular weights .

How should researchers address variability in CD36 glycosylation across different cell types?

CD36 is heavily glycosylated, and this glycosylation pattern varies across different cell types, affecting antibody binding and apparent molecular weight. Researchers should:

• Expect molecular weight variation: CD36 may appear at different molecular weights depending on the tissue source:

  • 85-90 kDa in human placenta tissue and platelets

  • 125-140 kDa in human adipose and placenta tissue using Simple Western systems

• Deglycosylation controls: Include enzymatic deglycosylation (PNGase F treatment) of some samples to confirm that bands of different molecular weights represent the same core protein.

• Selection of appropriate antibodies: Choose antibodies that recognize epitopes less affected by glycosylation, typically those binding to amino acid sequences rather than glycan structures.

• Cell-type specific protocols: Optimize extraction and detection protocols for each cell type or tissue being studied.

• Documentation: Clearly document the apparent molecular weight observed in each tissue type to facilitate comparison with published literature.

This variability in glycosylation not only affects detection but may also have functional implications, as glycosylation can influence CD36 ligand binding properties and cellular localization .

What are the common pitfalls when using anti-CD36 antibodies for flow cytometry, and how can they be avoided?

Common pitfalls in flow cytometry with anti-CD36 antibodies include:

• Suboptimal antibody concentration: Titrate antibodies carefully for each application. For example, eBioNL07 (NL07) antibody should be used at ≤0.5 μg per test, with empirical determination of optimal concentration .

• Inadequate controls: Always include:

  • Isotype controls to assess non-specific binding

  • FMO (fluorescence minus one) controls for multicolor panels

  • Known positive and negative cell populations

• Cell activation during preparation: CD36 expression can change with cell activation; use gentle cell preparation methods and keep cells cold to minimize activation.

• Interference from soluble CD36: In some samples, particularly plasma from patients with metabolic disorders, soluble CD36 may bind antibodies and reduce staining intensity.

• Autofluorescence: Macrophages and foam cells often exhibit high autofluorescence; use appropriate compensation and consider fluorophores with emission spectra distinct from cellular autofluorescence.

• CD36 internalization: Antibody binding may trigger CD36 internalization; perform kinetic studies to determine optimal staining times.

To avoid these issues, ensure high antibody purity (>90% as determined by SDS-PAGE) and low aggregation (<10% as determined by HPLC), and use post-manufacturing filtered (0.2 μm) antibodies for consistent results .

How might CD36 antibodies contribute to research on CD36 as a potential new erythroid blood group system?

Recent research has proposed that CD36 could become a novel erythroid blood group system, though more work is needed to support this classification . CD36 antibodies play a crucial role in this emerging research area:

• Detection of CD36 on erythroid cells: Antibodies help characterize CD36 expression patterns on erythroid precursors and mature red blood cells in different individuals.

• Interfering antibodies in clinical settings: Anti-CD36 antibodies have been shown to interfere with red blood cell antibody screening, particularly significant in obstetrical settings .

• Population studies: Antibodies enable large-scale studies of CD36 polymorphisms across different populations, essential for establishing a new blood group system.

• Neutralization assays: Research has demonstrated that patient plasma reactivity containing anti-CD36 can be fully neutralized with recombinant CD36 protein (rCD36p), confirming antibody specificity .

• Clinical implications: The association of anti-CD36 with severe fetal/neonatal thrombocytopenia underscores the importance of this research for maternal-fetal medicine .

Future research using CD36 antibodies will likely focus on characterizing additional CD36 polymorphisms, establishing standardized testing protocols, and further investigating the clinical significance of anti-CD36 in transfusion medicine and maternal-fetal compatibility .

What are the latest advances in using human anti-CD36 single-chain variable fragments (scFvs) for therapeutic applications?

Recent research has made significant progress in developing human anti-CD36 scFvs with therapeutic potential:

• Novel anti-CD36 scFv identification: Researchers have identified an anti-CD36 scFv called D11 that competes with commercial anti-CD36 antibodies with proven efficacy in disease models .

• Binding characteristics: D11 binds to CD36 expressed on cell membranes and effectively reduces the uptake of CD36 ligands in cellular models .

• Functional effects in disease models:

  • In macrophage-like THP-1 cells, D11 impairs the acquisition of foam cell phenotype induced by oxidized LDL, decreasing lipid droplet content and the expression of lipid metabolism genes

  • In HepG2 cells, D11 reduces lipid accumulation and enhanced clonogenicity stimulated by palmitate

• Therapeutic potential: By reducing the acquisition of pathogenic features induced by CD36 ligands, D11 could support the development of therapeutic proteins targeting CD36 in diseases such as atherosclerotic cardiovascular disease and cancer .

• Advantages of scFv format: The single-chain variable fragment format offers benefits including smaller size for better tissue penetration, potential for further engineering into various antibody formats, and possibly reduced immunogenicity compared to full antibodies .

These advances suggest promising directions for developing CD36-targeted therapeutics for conditions where CD36 overexpression contributes to disease pathology, including atherosclerosis, certain cancers, and metabolic disorders .

How can researchers leverage CD36 antibodies to investigate the role of CD36 in emerging research areas such as tumor metabolism and immune response?

CD36 antibodies provide valuable tools for investigating CD36's role in tumor metabolism and immune responses:

• Tumor metabolism studies:

  • Use blocking antibodies to inhibit fatty acid uptake in tumor cells and assess effects on growth, survival, and metastatic potential

  • Combine with metabolic tracers to quantify how CD36 blockade alters tumor metabolic profiles

  • Investigate clonogenicity assays, as recent research shows anti-CD36 antibodies can reduce the enhanced clonogenicity stimulated by palmitate in HepG2 cells

• Tumor microenvironment analysis:

  • Apply multicolor flow cytometry with CD36 antibodies to characterize CD36 expression on different cell populations within tumors

  • Use immunohistochemistry with anti-CD36 antibodies to map CD36 distribution in relation to hypoxic regions, metabolic gradients, and immune infiltrates

• Immune response investigations:

  • Employ CD36 antibodies to study how CD36-mediated uptake of oxidized lipids affects macrophage polarization and function

  • Investigate dendritic cell antigen presentation after CD36 blockade

  • Assess how CD36-dependent lipid uptake influences T cell activation and function in the tumor microenvironment

• Therapeutic combination approaches:

  • Test combinations of CD36 antibodies with immune checkpoint inhibitors

  • Evaluate CD36 blockade in combination with metabolic inhibitors targeting complementary pathways