CD36 Recombinant Monoclonal Antibody

What is a CD36 recombinant monoclonal antibody and how is it produced?

CD36 recombinant monoclonal antibodies are laboratory-engineered antibodies that specifically target the CD36 protein, which is expressed on various cell types including macrophages, platelets, and adipocytes. Unlike traditional monoclonal antibodies produced in animals, recombinant antibodies are created using in vitro expression systems through a multi-step process.

The production typically begins with the isolation of CD36 antibody genes from B cells derived from immunoreactive rabbits. These genes undergo amplification and cloning into phage vectors, which are subsequently introduced into mammalian cell lines to facilitate the generation of functional antibodies in substantial quantities. The resulting antibodies are then purified from the culture supernatant through affinity chromatography methods like protein A purification .

This in vitro approach allows for precise control over the antibody's characteristics and eliminates batch-to-batch variability associated with traditional antibody production methods. The recombinant process also enables the synthesis of antibodies with improved specificity and sensitivity compared to conventional methods .

What are the main advantages of using recombinant monoclonal antibodies for CD36 detection?

Recombinant monoclonal antibodies offer several significant advantages over conventional antibodies for CD36 detection in research applications:

• Enhanced specificity and sensitivity: The recombinant production process allows for selection of optimal antibody sequences, resulting in higher target specificity and improved signal-to-noise ratios in experimental applications .

• Lot-to-lot consistency: By using defined genetic sequences and controlled expression systems, recombinant antibodies demonstrate minimal batch-to-batch variation, ensuring experimental reproducibility over time and across different studies .

• Animal origin-free formulations: This approach reduces ethical concerns related to animal use and eliminates potential contaminants from animal sources, making them more suitable for certain applications .

• Broader immunoreactivity: Particularly with rabbit-derived sequences, recombinant antibodies can recognize a wider range of epitopes due to the larger rabbit immune repertoire, enhancing their utility across different experimental systems .

• Molecular-level identity confirmation: Advanced biophysical quality control methods verify antibody identity at the molecular level, further ensuring consistency between production batches .

These advantages make recombinant CD36 antibodies particularly valuable for longitudinal studies where consistent reagent performance is critical to experimental outcomes .

Where is CD36 expressed and what cellular functions does it serve?

CD36 has a broad expression pattern across multiple cell types and tissues, making it an important target for various research applications:

CD36 functions as a scavenger receptor that recognizes multiple ligands including oxidized low-density lipoproteins, long-chain fatty acids, apoptotic cells, thrombospondin, collagen, and malaria-infected erythrocytes. It plays critical roles in lipid metabolism, inflammation, atherosclerosis, angiogenesis, and innate immunity .

In pathological contexts, CD36 has been implicated in various diseases including atherosclerosis, metabolic disorders, malaria pathogenesis, and cancer progression, making it an important target for biomedical research .

How can CD36 recombinant monoclonal antibodies be used to study lipid metabolism pathways?

CD36 recombinant monoclonal antibodies provide valuable tools for investigating lipid metabolism due to CD36's central role as a fatty acid translocase. Several methodological approaches can be employed:

Cellular Lipid Uptake Analysis:
Researchers can use fluorescently-labeled fatty acids (such as Nile Red) in combination with CD36 immunostaining to evaluate co-localization patterns in various cell types. This approach allows visualization of CD36's role in fatty acid transport at the subcellular level . Additionally, comparing lipid uptake in normal versus CD36 antibody-blocked cells can quantify the proportion of fatty acid transport that is CD36-dependent.

Mechanistic Pathway Investigations:
Co-immunoprecipitation experiments using CD36 antibodies can identify protein interaction partners in lipid metabolism pathways. Time-course analyses tracking CD36 localization after lipid exposure via immunofluorescence can reveal trafficking dynamics related to fatty acid transport. Different epitope-targeting antibodies can distinguish between functional domains of CD36 involved in lipid binding versus other functions .

Translational Research Applications:
CD36 expression levels can be correlated with lipid accumulation in tissues from models of metabolic disease using immunohistochemistry or Western blot techniques. Changes in CD36 expression in response to high-fat diets or lipid-modulating drugs can be monitored to understand regulatory mechanisms . Comparing wild-type and CD36-deficient models helps establish causality in lipid metabolism defects.

The functional blocking capability of some CD36 antibodies makes them particularly valuable for dissecting CD36's multiple roles in lipid metabolism pathways and distinguishing between its various physiological functions .

What are the considerations for using CD36 antibodies in atherosclerosis and cardiovascular disease research?

When employing CD36 antibodies in atherosclerosis and cardiovascular disease research, several methodological considerations are essential for generating reliable and interpretable results:

Model Selection and Validation:
Researchers must determine whether human or animal models are being studied, as epitope recognition can vary across species . It's crucial to validate antibody reactivity in the specific cardiovascular tissues of interest, as expression patterns may differ from standard test tissues. Considering multiple antibody clones targeting different epitopes can provide confirmation of findings and more comprehensive insights.

Experimental Design Factors:
For atherosclerotic plaque studies, optimizing immunohistochemistry protocols for lipid-rich environments is essential, often requiring specialized fixation and antigen retrieval methods . When studying oxidized LDL (oxLDL) uptake via CD36, researchers should ensure that the antibody epitope doesn't overlap with the oxLDL binding domain to avoid competitive inhibition that could confound results. In flow cytometry applications, careful gating strategies are necessary to distinguish CD36-positive foam cells from other macrophage populations.

CD36 antibodies are particularly valuable in cardiovascular research for tracking macrophage infiltration, foam cell formation in vessel walls, and assessing the impact of therapeutic interventions on these processes .

How do researchers distinguish between different functional domains of CD36 using monoclonal antibodies?

Distinguishing between CD36's functional domains is crucial for understanding its diverse roles. Recombinant monoclonal antibodies serve as valuable tools for this purpose through several approaches:

Epitope Mapping Strategies:
Researchers can utilize panels of antibodies with known epitope recognition sites to identify specific functional regions of the CD36 protein . Cross-blocking experiments, where two antibodies compete for binding, help determine if they recognize overlapping epitopes, providing structural insights . Creating domain deletion mutants of CD36 and testing antibody binding allows precise mapping of recognition sites that can be correlated with functional domains.

Structure-Function Analysis:
The region defined by amino acids 155-183 has been identified as an important functional and immunogenic domain, as demonstrated by antibodies like OKM5, FA6-152, L103, and 10/5 that bind this region . Antibodies recognizing distinct domains can be used in parallel experiments to correlate domain accessibility with functional outputs in various biological processes. Comparing the effects of domain-specific blocking antibodies on different CD36 functions (e.g., thrombospondin binding versus fatty acid uptake) helps dissect the protein's multifunctional nature.

Applied Research Examples:
In malaria research, antibodies targeting specific CD36 domains can differentiate between regions involved in Plasmodium falciparum binding and those involved in other functions . For lipid metabolism studies, antibodies recognizing the fatty acid binding domain provide insights into transport mechanisms that other domain-specific antibodies cannot .

This approach has revealed that the 155-183 sequence is a surface-exposed, immunogenic region important for various ligand interactions, including the binding of anti-Nak(a) serum to CD36, demonstrating how antibodies can uncover functional architecture of complex receptors .

What are the common issues encountered in Western blot applications and how can they be resolved?

Western blotting with CD36 antibodies presents several technical challenges due to CD36's complex post-translational modifications and membrane localization:

Band Size Variability:
While CD36 has a theoretical molecular weight of 53 kDa, observed bands typically range from 74-88 kDa due to extensive glycosylation . Multiple bands may appear due to different glycosylation states, proteolytic processing, or splice variants. This variability can be addressed by including positive control lysates (e.g., platelets or adipose tissue) to establish expected band patterns for comparison .

Sample Preparation Challenges:
As a membrane protein, CD36 may aggregate during sample preparation, causing smeared bands that complicate interpretation. To avoid this, researchers should avoid boiling samples and instead heat at 37°C or 70°C in sample buffer containing adequate SDS (1-2%). Including reducing agents like β-mercaptoethanol is essential to break disulfide bonds and achieve consistent results.

Optimization Parameters for CD36 Western Blotting:

Detection Sensitivity Issues:
Low expression in some cell types may lead to weak signals that are difficult to interpret. Enhanced chemiluminescence substrates can improve detection, as can increasing protein loading (50-100 μg total protein). Longer primary antibody incubation (overnight at 4°C) typically improves signal quality for CD36 detection.

These optimizations have been demonstrated to produce clear bands at expected molecular weights when using validated CD36 recombinant monoclonal antibodies in various experimental systems .

How can researchers validate the specificity of CD36 recombinant monoclonal antibodies?

Validating antibody specificity is critical for ensuring reliable experimental results. For CD36 recombinant monoclonal antibodies, several complementary approaches are recommended:

Positive and Negative Control Samples:
Researchers should use tissues/cells known to express high levels of CD36 (platelets, adipocytes, macrophages) as positive controls . CD36-deficient cell lines or tissues from CD36 knockout animals serve as essential negative controls to confirm specificity. Testing multiple antibody clones targeting different CD36 epitopes can help confirm consistent detection patterns and increase confidence in specificity.

Genetic Manipulation Approaches:
Overexpression validation involves testing the antibody on cells transfected with CD36 expression vectors, which should show enhanced signal compared to non-transfected cells . Knockdown validation using CD36-targeting siRNA/shRNA should demonstrate proportional decreases in signal intensity. CRISPR/Cas9 knockout cells provide definitive negative controls that should show complete absence of specific signal.

Advanced Validation Methods:
Immunoprecipitation followed by mass spectrometry can confirm the identity of the captured protein as CD36. Correlation of protein detection with mRNA expression across different cell types provides additional validation of specificity. Multi-method confirmation using different detection techniques (Western blot, immunohistochemistry, flow cytometry) strengthens confidence in antibody specificity.

Cross-Species Reactivity Testing:
If the antibody claims multi-species reactivity, it should be validated in each species separately using appropriate controls. Some antibodies, like clone 002, have species-specific reactivity (e.g., rat-specific) that must be considered when designing experiments .

These validation approaches ensure that experimental findings truly reflect CD36 biology rather than non-specific interactions or artifacts, which is essential for generating reproducible and reliable research data .

What are the optimal fixation and antigen retrieval methods for immunohistochemistry?

Detecting CD36 in tissue sections requires careful consideration of fixation and processing methods to preserve both antigenicity and membrane localization:

Fixation Options and Considerations:

Effective Antigen Retrieval Protocols:
Heat-mediated retrieval using Tris-EDTA buffer (pH 9.0) has been successfully validated with CD36 antibodies and is often preferred over citrate buffer . Pressure cooker methods (20 minutes) typically yield better results than water bath methods for consistent epitope exposure. The retrieval conditions should be optimized for each tissue type and antibody clone to balance antigen recovery with tissue morphology preservation.

Tissue-Specific Considerations:
Adipose tissue may require shorter fixation times due to lipid content that can interfere with antibody penetration. Cardiac tissue often requires robust antigen retrieval for optimal CD36 detection due to its density . Vascular tissue requires attention to elastic lamina autofluorescence in fluorescent applications, which may require specific quenching steps.

Antibody Incubation Parameters:
For paraffin sections, primary antibody dilutions of 1:50-1:200 are typically effective, with higher dilutions (1:10,000) possible for highly sensitive recombinant antibodies . Overnight incubation at 4°C often improves signal-to-noise ratio compared to shorter incubations at room temperature. Secondary detection should be optimized based on the expression level, with ready-to-use polymer detection systems offering good sensitivity for most applications.

These protocols have been validated in studies using CD36 antibodies on various tissues including cardiac muscle, hepatocellular cancer, and adipose tissue, demonstrating consistent and specific staining patterns .

How should researchers choose between different clones of CD36 recombinant monoclonal antibodies?

Selecting the optimal CD36 antibody clone is critical for experimental success. Consider these factors when choosing between different recombinant monoclonal antibodies:

Application-Specific Performance Characteristics:

Epitope Considerations for Specific Research Questions:
Extracellular domain epitopes (aa 30-439) are accessible in flow cytometry and live-cell applications, making them suitable for cell surface studies . The 155-183 region is important for multiple ligand interactions; antibodies to this region may have blocking activity useful for functional studies . C-terminal epitopes may better detect all splice variants in expression studies.

Species Cross-Reactivity Requirements:
Researchers working across species should verify that the antibody recognizes conserved epitopes to ensure consistent results. Some clones like EPR6573 are human-specific, while others recognize multiple species . Clone 002 is specifically optimized for rat CD36 detection, which is important for rodent model studies .

Technical Validation Status:
Priority should be given to antibodies validated through multiple techniques and independent studies for greater reliability. Clones validated on tissue microarrays provide broader application confirmation . Widely used clones like EPR6573 offer the advantage of extensive literature support and optimization protocols .

This systematic approach to antibody selection helps ensure reliable results across different experimental systems and research questions, particularly important for studies focused on CD36's multiple functional roles .

What are the considerations for quantifying CD36 expression levels?

Accurate quantification of CD36 expression requires careful attention to methodology and controls to ensure reliable and reproducible results:

Sample Preparation and Normalization Strategies:
For Western blot quantification, normalize CD36 signal to stable reference proteins (β-actin, GAPDH) with caution, as CD36 expression can vary widely across conditions . Flow cytometry analysis requires proper isotype controls and fluorescence-minus-one (FMO) controls to set accurate gates and account for background. RT-qPCR validation using multiple reference genes helps correlate mRNA with protein expression for more comprehensive analysis.

Quantification Techniques by Method:

Dynamic Range and Expression Variability:
CD36 expression can change dramatically (>10-fold) in response to stimuli like fatty acids, requiring methods with sufficient dynamic range . Ensure detection methods can capture these wide expression differences without saturation at high levels or loss of sensitivity at low levels. Consider log-scale reporting for wide expression differences to accurately represent the full range of biological responses.

Physiological and Pathological Variables:
CD36 expression increases with macrophage foam cell formation and adipocyte differentiation, requiring appropriate time points for meaningful comparisons. Expression is modulated by dietary conditions (e.g., high-fat diets increase CD36 in multiple tissues), necessitating careful control of experimental variables . Different functional pools of CD36 (surface vs. intracellular) may require distinct quantification approaches to fully characterize the protein's distribution.

Technical Validation Requirements:
Confirm antibody linearity across the expected expression range through dilution series. Use multiple antibody clones or detection methods to validate expression changes and avoid clone-specific artifacts. Consider absolute quantification methods for more precise comparisons between independent studies.

These approaches enable reliable quantification of CD36 under various experimental conditions, from baseline expression to pathological states, providing meaningful insights into its biological regulation .

How can CD36 recombinant monoclonal antibodies be used in multiplex imaging applications?

Multiplex imaging with CD36 antibodies allows simultaneous visualization of CD36 with other markers, providing context for its function in complex tissues and cellular systems:

Antibody Selection and Conjugation Strategies:
Direct conjugation options include labeling CD36 antibodies with fluorophores, enzymes, or metal isotopes depending on the imaging platform. Secondary detection systems using species-specific or isotype-specific secondary antibodies with minimal cross-reactivity provide flexibility. Signal amplification systems like tyramide signal amplification can enhance detection of low-abundance targets in multiplex contexts.

Panel Design Considerations:
Ensure antibodies in the panel recognize distinct, non-overlapping epitopes to avoid competitive binding issues. Combine antibodies from different host species (rabbit CD36 antibodies pair well with mouse antibodies for other targets) to simplify detection schemes. Select fluorophores with minimal spectral overlap and consider brightness relative to target abundance for balanced signal intensity across markers.

Research Applications for Multiplex Imaging:

Technical Optimization and Quality Control:
The order of antibody application significantly impacts results; typically apply rare target antibodies first to maximize detection. Consider signal-to-noise ratio for each marker when designing acquisition settings to achieve balanced visualization. Automated image analysis algorithms can help quantify co-localization and expression patterns across multiple markers.

Validation Requirements:
Single-stain controls are essential to confirm specificity and optimize exposure settings in the multiplex context. Include biological controls (tissues known to express or lack CD36) to verify detection sensitivity and specificity. Compare results with alternative methods (e.g., flow cytometry or sequential sections) to confirm multiplex findings.

These multiplex approaches enable researchers to study CD36 in its cellular and molecular context, revealing functional relationships not apparent with single-marker approaches and providing deeper insights into its role in complex biological processes .