SCARB2 Antibody

What is SCARB2 and why is it important in research?

SCARB2 (Scavenger Receptor Class B Member 2) is a type III transmembrane protein belonging to the scavenger receptor family, with significant roles in both normal cellular physiology and pathological conditions. In humans, the canonical protein consists of 478 amino acid residues with a molecular mass of approximately 54.3 kDa . SCARB2 is primarily localized in lysosomes and functions as a lysosomal receptor for glucosylceramidase (GBA1) targeting. It is widely expressed across numerous tissue types, making it an important subject for research in multiple biological systems. The protein undergoes post-translational modifications, notably glycosylation, which affects its functional properties. SCARB2 has gained research prominence because it serves as a functional receptor for Enterovirus 71 (EV71), mediating viral attachment, internalization, and uncoating . The gene has been associated with epilepsy, positioning SCARB2 as a relevant target in both infectious disease and neurological disorder research.

What are the known synonyms and orthologs of SCARB2?

Researchers should be aware of SCARB2's multiple synonyms when conducting literature searches or database queries. SCARB2 is also known as CD36L2, EPM4, HLGP85, LGP85, LIMP-2, LIMPII, SR-BII, and AMRF . This diversity in nomenclature reflects the protein's discovery in different research contexts and its multiple functional roles. SCARB2 is evolutionarily conserved, with orthologs identified in various species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This conservation across species suggests fundamental biological importance and provides researchers with multiple model systems for studying SCARB2 function. When designing experiments, it's essential to consider species-specific differences in SCARB2 structure and function, particularly when evaluating antibody specificity and cross-reactivity.

How do SCARB2 antibodies differ in their research applications?

SCARB2 antibodies are diverse in their applications, specificity, and methodology requirements. These antibodies are primarily used for immunodetection of SCARB2 protein across various experimental platforms. Western blot represents the most widely utilized application, allowing researchers to detect SCARB2 protein in cell or tissue lysates based on molecular weight separation . ELISA applications enable quantitative measurement of SCARB2 in solution, while immunohistochemistry provides insights into the spatial distribution of SCARB2 within tissues or cells . Some antibodies, such as the monoclonal JL2, demonstrate species specificity (binding to human but not mouse SCARB2) and functional activity (ability to block EV71 infection) . When selecting a SCARB2 antibody, researchers should consider the specific application requirements, target species, recognition epitope, and whether functional activity (like receptor blocking) is needed for their experimental design.

What is the structural basis for SCARB2's receptor function?

SCARB2's functionality as a receptor is determined by its complex three-dimensional structure. The protein possesses a twisted β-barrel core with strategically positioned α-helices that facilitate ligand interactions . Particularly important are α-helices 4, 5, and 7, which form a three-helix bundle that serves as a potential interaction site for ligands, including viral particles . The apical domain at the top of the SCARB2 molecule contains regions critical for EV71 binding and infection. Structural analysis has identified specific regions, including residues 142-204, as important for EV71 binding and infection, with residues 144-151 representing a highly variable region (HVR) across species that is critical for SCARB2-EV71 interaction . Additionally, residues 146-166 are essential for EV71 attachment . Understanding this structural organization is crucial when designing experiments to block or modify SCARB2-ligand interactions or when developing antibodies targeting specific functional domains.

How does SCARB2 mediate EV71 viral entry and infection?

SCARB2 facilitates EV71 infection through a multi-step process that exemplifies virus-receptor interaction mechanics. Initially, SCARB2 mediates viral attachment to the cell surface, as the protein is widely expressed on various cell types, including neurons . Upon attachment, SCARB2 facilitates virus internalization through the clathrin-mediated endocytic pathway . A critical feature of SCARB2's role in viral infection is its pH-dependent function during viral uncoating. SCARB2 is essential for EV71 uncoating at low pH, a necessary step for viral genome release into the cytoplasm and subsequent viral replication . The importance of SCARB2 in EV71 pathogenesis is highlighted by studies showing that transgenic mice with human SCARB2 overexpression become susceptible to EV71 infection . These mechanistic insights provide potential targets for therapeutic intervention and explain why SCARB2-specific antibodies like JL2, which can block the interaction between EV71 and SCARB2, may effectively inhibit viral infection.

What are the species-specific differences in SCARB2 structure and function?

Species-specific variations in SCARB2 structure significantly impact its functionality and antibody recognition. Human and mouse SCARB2 exhibit distinct differences, particularly in their ability to serve as receptors for EV71. Mouse SCARB2 is not an efficient EV71 receptor, which has enabled researchers to identify virus-binding sites using chimeras of mouse and human SCARB2 . Among the key species-specific differences is residue 77, which is arginine (R77) in human SCARB2 but glutamine (Q77) in mouse SCARB2 . This single amino acid difference appears to be a dominant contributor to the species-specific binding of antibodies like JL2, which binds to human but not mouse SCARB2 . The highly variable region (HVR) encompassing residues 144-151 also differs between species and affects EV71 binding capability . When designing experiments using SCARB2 antibodies, researchers must consider these species differences to ensure appropriate antibody selection, especially for cross-species studies or when using animal models to investigate human disease mechanisms.

What are the optimal protocols for using SCARB2 antibodies in Western blot applications?

Western blot represents the most common application for SCARB2 antibodies, requiring specific optimization for successful detection. When preparing samples, researchers should employ lysis buffers containing appropriate detergents (such as RIPA or NP-40) to efficiently extract SCARB2 from membrane compartments while preserving epitope integrity. Given SCARB2's molecular weight of 54.3 kDa , 10-12% polyacrylamide gels typically provide optimal resolution. Importantly, researchers should be aware that post-translational modifications, particularly glycosylation, may cause SCARB2 to migrate at an apparent molecular weight of approximately 85 kDa rather than the predicted 54.3 kDa . Primary antibody concentrations should be optimized through titration experiments, typically starting at 1:1000 dilution for most commercial antibodies. For blocking and antibody dilution, 5% non-fat milk in TBST generally provides low background, though BSA may be preferable for phospho-specific applications. Detection sensitivity can be enhanced using chemiluminescent substrates with appropriate signal development time. When interpreting results, researchers should consider the specific epitope recognized by their chosen antibody and the potential impact of different protein isoforms, as up to 2 different isoforms have been reported for SCARB2 .

How can SCARB2 antibodies be effectively used in immunohistochemistry and immunofluorescence?

Successful immunohistochemical and immunofluorescent detection of SCARB2 requires careful consideration of fixation, permeabilization, and retrieval techniques. For formalin-fixed paraffin-embedded tissues, antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically necessary to unmask epitopes. Since SCARB2 is primarily localized in lysosomes , sufficient permeabilization is essential for antibody access to intracellular compartments—0.1-0.3% Triton X-100 for immunofluorescence or proteolytic digestion for IHC are common approaches. When performing cellular immunofluorescence, co-staining with lysosomal markers (such as LAMP1) can provide confirmatory evidence of proper SCARB2 detection. Primary antibody concentrations typically range from 1-10 μg/mL, requiring optimization for each specific antibody and tissue/cell type. Since SCARB2 is widely expressed across many tissue types , appropriate positive and negative controls are crucial for validating staining specificity. Whenever possible, validation should include SCARB2 knockout samples or peptide competition assays to confirm antibody specificity. For quantitative applications, standardized image acquisition parameters and analysis protocols should be established to ensure consistent results across experiments.

What are the methodological considerations for using SCARB2 antibodies in virus neutralization assays?

SCARB2 antibodies can be employed in virus neutralization assays to investigate receptor-virus interactions and potential therapeutic approaches. Based on experimental approaches with the JL2 monoclonal antibody, researchers should consider a pre-treatment protocol where target cells are incubated with the anti-SCARB2 antibody (at varying concentrations, typically starting at 2 μg/mL) for approximately one hour before virus exposure . This allows the antibody to occupy receptor binding sites prior to viral challenge. For EV71 studies, GFP-expressing viruses provide a convenient readout system, with infection rates quantifiable by flow cytometry or fluorescence microscopy after 14-20 hours post-infection . When designing these experiments, important controls include isotype-matched control antibodies to rule out non-specific effects and dose-response studies to determine IC50 values. Researchers should be aware that neutralization may occur through multiple mechanisms: direct competition with virus for receptor binding, stabilization of SCARB2 structure at neutral pH (preventing conformational changes required for viral uncoating), or induction of receptor internalization . For comprehensive mechanistic understanding, time-of-addition experiments can help distinguish between effects on attachment versus post-entry events. Finally, when interpreting results, consider that different viral strains may exhibit varying dependence on SCARB2 for entry, potentially affecting neutralization efficiency.

What are the most effective approaches for producing monoclonal antibodies against SCARB2?

Generating high-quality monoclonal antibodies against SCARB2 requires strategic immunization and screening approaches. Based on successful methodologies, researchers should consider immunizing mice with SCARB2-transfected cells rather than purified protein to ensure the antigen maintains its native conformation . The immunization protocol might include initial injection of SCARB2-transfected cells (such as L929 cells) with 10 μg of mouse CpG as an adjuvant, followed by 3-4 boosters at monthly intervals . After the final boost, splenocytes should be harvested and fused with SP2/0 myeloma cells using 50% polyethylene glycol (PEG) 4000 to generate hybridomas . Critical to success is the screening strategy—flow cytometry with SCARB2-transfected cells provides a superior approach as it selects for antibodies recognizing native conformational epitopes . Positive hybridomas should undergo multiple rounds of subcloning until 100% positivity is achieved . For antibody production, either tissue culture supernatants or ascites from mice injected with hybridoma cells can be used, with subsequent purification via Protein G affinity chromatography . Throughout development, researchers should regularly confirm hybridoma stability and antibody specificity using techniques such as ELISA, flow cytometry, and Western blotting against both positive and negative control samples.

How can researchers effectively characterize the epitope specificity of SCARB2 antibodies?

Epitope mapping of SCARB2 antibodies provides crucial information about their potential applications and mechanisms. A systematic approach involves creating chimeric constructs between human and mouse SCARB2, particularly when the antibody shows species specificity . As demonstrated with the JL2 antibody, researchers constructed a series of human-mouse SCARB2 chimeras to identify that residues 77-113 of human SCARB2 contribute significantly to antibody binding . This approach can be complemented by site-directed mutagenesis of individual amino acids within the identified region to pinpoint specific critical residues—for instance, R77 was identified as potentially crucial for JL2's specificity for human over mouse SCARB2 . For structural characterization, X-ray crystallography of the antibody-SCARB2 complex provides the most definitive epitope information, revealing precise binding sites and interaction mechanisms . Alternative approaches include hydrogen-deuterium exchange mass spectrometry, which can identify protected regions upon antibody binding, or peptide array analysis using overlapping peptides spanning the SCARB2 sequence. Understanding epitope specificity is particularly valuable when developing antibodies for specific applications—for instance, antibodies targeting the EV71-binding region of SCARB2 may have potential therapeutic value by blocking viral infection .

What methods should be used to quantify SCARB2 antibody binding affinity and specificity?

Rigorous characterization of SCARB2 antibody binding properties is essential for research applications. Flow cytometry represents an effective method for determining binding efficiency, particularly using cell lines with stable expression of human SCARB2 (such as 293-hSCARB2) . Serial dilution of antibodies from 0.01 μg/mL to 10 μg/mL can generate binding curves from which EC50 values can be calculated . Surface plasmon resonance (SPR) provides a more precise measurement of binding kinetics, yielding association and dissociation rate constants and equilibrium dissociation constants (KD). For SPR analysis, recombinant SCARB2 can be produced using expression systems such as Sf9 insect cells with subsequent purification via affinity chromatography . Specificity assessment should include testing against SCARB2-knockout cell lines generated via CRISPR-Cas9 technology to confirm absence of non-specific binding . Cross-reactivity analysis against SCARB2 from different species helps determine species specificity and potential utility in comparative studies. Additionally, competitive binding assays with known ligands (such as EV71) can provide insight into whether the antibody interferes with physiological interactions. When quantifying binding, researchers should consider potential influences of post-translational modifications, particularly glycosylation, which might affect epitope accessibility or antibody recognition.

How can SCARB2 antibodies be employed to study virus-receptor interactions and develop antiviral strategies?

SCARB2 antibodies offer powerful tools for investigating the mechanistic details of virus-receptor interactions and developing potential therapeutic approaches. Researchers can use blocking antibodies like JL2 to dissect the specific steps of viral entry that depend on SCARB2 . By conducting time-of-addition experiments with SCARB2 antibodies added at different stages of viral infection, researchers can distinguish between effects on attachment, internalization, and uncoating. Confocal microscopy with fluorescently labeled viruses and antibodies enables real-time visualization of these interactions. For therapeutic development, SCARB2 antibodies can be evaluated for their ability to prevent infection in cellular and animal models. The protective efficacy of such antibodies would be particularly relevant for EV71, which causes hand, foot, and mouth disease (HFMD) and can lead to severe neurological complications . Using transgenic mice expressing human SCARB2 provides an in vivo model for testing antibody-based interventions . Additionally, structural studies of antibody-SCARB2 complexes can guide the rational design of small molecule inhibitors that target critical interaction interfaces. When designing such experiments, researchers should consider the specificity of different viral strains for SCARB2 and the potential for viruses to utilize alternative receptors, which might influence the effectiveness of SCARB2-targeted interventions.

What are the methodological approaches for using SCARB2 antibodies in the investigation of neurological disorders?

Given SCARB2's association with epilepsy and its expression in neurons, specialized approaches are needed when using SCARB2 antibodies to study neurological conditions. For immunohistochemical analysis of brain tissue, researchers should optimize fixation protocols to preserve both antigenicity and tissue architecture—4% paraformaldehyde fixation for 24-48 hours followed by sucrose cryoprotection is often suitable for frozen sections. Antigen retrieval methods may need modification for neural tissues, with careful temperature and pH optimization to avoid tissue damage while ensuring epitope accessibility. When examining SCARB2 in specific neural cell populations, dual immunofluorescence with cell-type-specific markers (NeuN for neurons, GFAP for astrocytes, Iba1 for microglia) helps establish cellular distribution patterns. For functional studies, researchers can employ SCARB2 antibodies in primary neuronal cultures to examine effects on neuronal excitability, synaptic transmission, or neurite outgrowth. In disease contexts, comparative immunoblotting or immunohistochemistry between control and epileptic tissue samples can reveal changes in SCARB2 expression or localization. Single-cell analysis approaches, such as imaging mass cytometry combined with SCARB2 antibodies, can provide insights into cell-type-specific alterations in heterogeneous brain tissue. When interpreting results from neurological studies, researchers should consider SCARB2's dual roles in lysosomal function and as a viral receptor, either of which might contribute to neurological manifestations.

How can researchers integrate SCARB2 antibodies with advanced imaging techniques for subcellular localization studies?

Combining SCARB2 antibodies with cutting-edge imaging approaches enables precise characterization of its subcellular distribution and trafficking dynamics. Super-resolution microscopy techniques, including structured illumination microscopy (SIM), stimulated emission depletion (STED), or stochastic optical reconstruction microscopy (STORM), can resolve SCARB2 localization beyond the diffraction limit, revealing detailed distribution patterns within lysosomes and other compartments. These techniques typically require additional optimization of fixation and antibody concentrations compared to conventional immunofluorescence. For live-cell imaging studies, researchers should consider using Fab fragments of SCARB2 antibodies conjugated to small fluorophores to minimize potential functional interference while tracking receptor dynamics. Multi-color imaging with markers for different endocytic compartments (early endosomes, late endosomes, lysosomes) can track SCARB2 trafficking pathways, particularly during viral infection processes. Correlative light and electron microscopy (CLEM) offers an integrative approach, combining immunofluorescence localization of SCARB2 with ultrastructural context from electron microscopy. For quantitative analysis of SCARB2 distribution, researchers should implement automated image analysis pipelines with appropriate controls for background subtraction and signal normalization. When designing these experiments, consideration should be given to potential epitope masking within certain subcellular compartments and the possible need for different fixation and permeabilization protocols to access all relevant pools of SCARB2.

What are the common challenges in SCARB2 antibody applications and how can they be addressed?

Researchers frequently encounter specific challenges when working with SCARB2 antibodies that require systematic troubleshooting approaches. One common issue is false-negative results in Western blots due to insufficient protein extraction, as SCARB2's transmembrane nature can make it difficult to solubilize. This can be addressed by using stronger lysis conditions with detergents like SDS or by incorporating membrane protein extraction kits. Another challenge is distinguishing between glycosylated forms of SCARB2, which may appear at different molecular weights. Treatment with glycosidases prior to electrophoresis can help clarify the contribution of glycosylation to apparent molecular weight variations. In immunostaining applications, high background is frequently reported and can be minimized by using specialized blocking reagents containing both serum and bovine serum albumin, or by including detergents like 0.3% Triton X-100 in washing steps. Epitope masking in fixed tissues is another common problem, potentially requiring extended antigen retrieval or alternative fixation methods. For functional blocking studies, insufficient neutralization may occur if antibody concentrations are too low—dose-response experiments with concentrations up to 20-50 μg/mL may be necessary. When encountering inconsistent results across different cell lines, researchers should verify endogenous SCARB2 expression levels, as variability in expression may affect detection sensitivity and functional outcomes.

How can researchers validate the specificity of SCARB2 antibodies in their experimental systems?

Rigorous validation of SCARB2 antibody specificity is essential for generating reliable research data. A comprehensive validation strategy includes multiple complementary approaches. SCARB2 knockout models generated via CRISPR-Cas9 technology provide definitive negative controls , with complete absence of signal confirming antibody specificity. Alternatively, siRNA-mediated knockdown of SCARB2 should result in proportional signal reduction. Peptide competition assays, where the immunizing peptide is pre-incubated with the antibody before application to samples, can confirm epitope-specific binding. Testing across multiple applications (Western blot, immunoprecipitation, immunofluorescence) helps ensure consistent recognition of the target. Species cross-reactivity should be systematically assessed using samples from different organisms, particularly when working with model systems. When possible, orthogonal detection methods using antibodies recognizing different SCARB2 epitopes should yield concordant results. Mass spectrometry analysis of immunoprecipitated proteins can confirm the identity of the recognized target. For monoclonal antibodies, isotype-matched control antibodies should be used at equivalent concentrations to distinguish specific from non-specific binding. Researchers should also be aware that certain experimental conditions, such as fixation methods or detergent treatments, may affect epitope accessibility or conformation, necessitating validation under the specific conditions used in their protocols.

What approaches should researchers use when encountering contradictory results with different SCARB2 antibodies?

When different SCARB2 antibodies yield conflicting results, a systematic analytical approach is required to resolve discrepancies. First, researchers should thoroughly characterize the epitopes recognized by each antibody—antibodies targeting different domains of SCARB2 may yield varying results depending on protein conformation, interaction partners, or post-translational modifications. Creating an epitope map of available antibodies helps identify whether discrepancies arise from recognition of different protein regions. Second, validation experiments using knockout controls should be performed with each antibody to confirm target specificity. Third, differences in results may reflect detection of different SCARB2 isoforms, as up to two isoforms have been reported —isoform-specific PCR can help determine which variants are expressed in the experimental system. Fourth, post-translational modifications, particularly glycosylation, may affect antibody recognition, warranting investigation with enzymatic deglycosylation experiments. Fifth, subcellular fractionation followed by immunoblotting with different antibodies can determine whether discrepancies reflect detection of SCARB2 in different cellular compartments. Sixth, for functional studies, researchers should consider that antibodies may have different effects on SCARB2 function depending on their epitopes—some may block ligand binding while others may have no functional impact despite recognizing the protein. Finally, when publishing results with SCARB2 antibodies, researchers should clearly report which antibody was used, including catalog numbers and epitope information, to facilitate interpretation and reproduction of findings.