LOX1.5 Antibody

What is LOX-1 and why is it an important research target?

LOX-1 (Lectin-like oxidized LDL receptor-1, also known as OLR1) is a 50 kDa transmembrane glycoprotein that functions as a class E scavenger receptor. It is primarily responsible for the recognition and uptake of oxidized low-density lipoprotein (oxLDL) . LOX-1 is expressed across multiple cell types, including vascular endothelial cells, macrophages, vascular smooth muscle cells, cardiomyocytes, platelets, and fibroblasts . Its importance as a research target stems from its critical roles in atherosclerosis development, endothelial dysfunction, immune responses, and cardiovascular disease pathogenesis .

Which cell types express LOX-1 and how does expression change in disease states?

LOX-1 is expressed in various cell types including:

• Vascular endothelial cells

• Macrophages

• Vascular smooth muscle cells

• Cardiomyocytes

• Platelets

• Fibroblasts

• Dendritic cells (particularly CD1c+ skin dermal DCs and blood myeloid DCs)

• Fractions of peripheral B cells and monocytes

What are the optimal applications for LOX-1 antibodies in research?

LOX-1 antibodies have demonstrated utility in several research applications:

LOX-1 antibodies are particularly valuable for studying dendritic cell-B cell interactions, atherosclerotic plaque development, and cardiovascular disease mechanisms .

How stable are LOX-1 antibodies and what are the optimal storage conditions?

According to the available data, reconstituted LOX-1 antibodies generally demonstrate the following stability profile:

• 12 months from date of receipt at -20°C to -70°C in manufacturer-supplied condition

• 1 month at 2-8°C under sterile conditions after reconstitution

• 6 months at -20°C to -70°C under sterile conditions after reconstitution

To maintain antibody integrity, it's recommended to:

• Use a manual defrost freezer

• Avoid repeated freeze-thaw cycles

• Aliquot antibodies after reconstitution to minimize freeze-thaw events

• Store according to manufacturer recommendations

How should I design experiments to study LOX-1's role in atherosclerosis progression?

When designing experiments to investigate LOX-1's role in atherosclerosis, consider the following comprehensive approach:

• Cell-based studies:

  • Use human endothelial cells (HUVECs or HAECs) treated with oxidized LDL

  • Monitor changes in cellular functions including adhesion molecule expression, endothelial activation, and apoptosis

  • Employ LOX-1 antibodies to block oxLDL binding and assess downstream effects

• Animal models:

  • Consider ApoE-/- or LDLR-/- mice on high-fat diets

  • Use LOX-1 knockout models to evaluate atherosclerosis progression

  • Implement anti-LOX-1 antibody treatment protocols to assess therapeutic potential

• Mechanistic investigation:

  • Examine MAPK and NF-κB signaling pathways activated downstream of LOX-1

  • Assess production of adhesion molecules and pro-inflammatory cytokines

  • Monitor foam cell formation through lipid uptake assays

When selecting antibodies, the clone DE15-4H4 has demonstrated efficacy in recognizing human LOX-1 and can be paired with clone DE17-4B9 for detection purposes in sandwich ELISA formats .

What controls should be included when using LOX-1 antibodies in flow cytometry?

For reliable flow cytometry results when using LOX-1 antibodies, incorporate the following controls:

• Isotype control: Use an appropriate isotype-matched control antibody (e.g., MAB0041 when using clone 331212) to determine nonspecific binding and set proper gating strategies

• Positive controls:

  • THP-1 cells treated with PMA (50 ng/mL for 72 hours) express detectable levels of LOX-1

  • Primary monocytes, particularly after activation

  • Endothelial cells stimulated with pro-inflammatory factors

• Negative controls:

  • Plasmacytoid DCs and Langerhans cells (which do not express LOX-1)

  • Unstimulated lymphocytes

• Secondary antibody control: Include a no-primary antibody control when using indirect detection methods with labeled secondary antibodies such as Allophycocyanin-conjugated Anti-Mouse IgG F(ab')2

• FMO (Fluorescence Minus One) controls: Especially important in multi-color panels to properly set gates

What factors affect LOX-1 expression in cell culture experiments?

Several factors can significantly influence LOX-1 expression in cell culture systems:

When designing experiments, it's crucial to standardize these conditions to ensure reproducibility. Additionally, LOX-1 expression levels should be verified by flow cytometry or Western blot prior to functional studies .

How can I design experiments to investigate LOX-1's role in immune responses?

LOX-1 plays significant roles in immune regulation, particularly in dendritic cell function and B cell responses. To investigate these roles:

• Dendritic cell-B cell interaction studies:

  • Isolate CD1c+ DCs from human blood or differentiate from monocytes

  • Treat DCs with anti-LOX-1 antibody (e.g., clone DE15-4H4)

  • Co-culture with B cells and assess:

    • B cell proliferation

    • Plasmablast differentiation

    • Antibody secretion

    • Class-switching responses

• Signaling pathway investigation:

  • Examine STAT3 and BLIMP1 expression in B cells following exposure to LOX-1-activated DCs

  • Measure BAFF and APRIL production by DCs after LOX-1 engagement

  • Investigate the specificity of LOX-1 in triggering these responses compared to other lectins (Dectin-1, DC-ASGPR, etc.)

• In vivo models:

  • Design antigen targeting studies using anti-LOX-1 antibodies conjugated to antigens

  • Assess the ability to elicit antigen-specific IFNγ-producing CD4+ T cell responses

When conducting these experiments, remember that LOX-1 is expressed at varying levels on different DC subsets - present on CD1c+ dermal DCs and blood myeloid DCs but absent on Langerhans cells and plasmacytoid DCs .

What are the optimal approaches for detecting soluble LOX-1 (sLOX-1) in patient samples?

Soluble LOX-1 (sLOX-1) results from proteolytic cleavage of membrane-bound LOX-1 and serves as a potential biomarker for cardiovascular disease. For optimal detection:

• Sandwich ELISA:

  • Use clone DE15-4H4 as capture antibody

  • Pair with biotinylated clone DE17-4B9 as detection antibody

  • Establish a standard curve using recombinant sLOX-1

  • Include appropriate quality controls and reference samples

• Sample handling considerations:

  • Collect samples in appropriate anticoagulant (EDTA preferred)

  • Process samples within 2 hours of collection

  • Centrifuge at 1000-2000×g for 10 minutes

  • Aliquot to avoid freeze-thaw cycles

  • Store at -80°C for long-term storage

• Patient stratification:

  • Consider pre-analytical variables (fasting status, time of collection)

  • Match cases and controls for age, sex, and cardiovascular risk factors

  • Document concurrent medications that might affect LOX-1 expression

Recent developments suggest sLOX-1 measurements may offer prognostic value in cardiovascular risk assessment, potentially complementing traditional lipid profiling .

How can LOX-1 antibodies be employed to study LOX-1's role in plaque instability?

Investigating LOX-1's role in plaque instability requires sophisticated experimental approaches:

• Histological analysis of human atherosclerotic plaques:

  • Obtain specimens from endarterectomy or autopsy

  • Perform immunohistochemistry using anti-LOX-1 antibodies

  • Co-stain for markers of plaque vulnerability (macrophages, MMPs, thin fibrous cap)

  • Quantify LOX-1 expression in stable versus unstable plaque regions

• Ex vivo plaque studies:

  • Treat fresh plaque specimens with LOX-1-blocking antibodies

  • Measure changes in inflammatory cytokine production

  • Assess matrix degradation enzymes and apoptotic markers

  • Analyze plaque structural integrity changes

• Animal models of plaque rupture:

  • Use ApoE-/- mice with tandem stenosis or collar placement

  • Administer anti-LOX-1 antibodies at different disease stages

  • Evaluate plaque composition, stability features, and rupture events

  • Analyze mechanistic pathways affected by LOX-1 blockade

By integrating these approaches, researchers can better understand how LOX-1 contributes to the transition from stable to vulnerable atherosclerotic plaques and potentially develop targeted therapies .

How can I optimize LOX-1 antibody-based ELISA methods for research applications?

Optimizing LOX-1 antibody-based ELISA requires careful consideration of several parameters:

• Antibody pair selection:

  • The combination of DE15-4H4 (capture) and biotinylated DE17-4B9 (detection) has been validated for human LOX-1 detection

  • These antibodies recognize distinct epitopes on LOX-1 protein, enhancing specificity

• Protocol optimization:

  • Coating concentration: Typically 1-5 μg/mL of capture antibody

  • Sample dilution: Optimize for expected concentration range

  • Detection antibody concentration: Usually 0.5-2 μg/mL

  • Substrate development time: Monitor to avoid saturation

• Troubleshooting common issues:

• Validation parameters:

  • Establish lower limit of detection (LLOD) and quantification (LLOQ)

  • Determine intra-assay and inter-assay coefficients of variation (aim for <15%)

  • Verify linearity, recovery, and specificity

What are the best approaches for characterizing newly developed LOX-1 antibodies?

When characterizing newly developed LOX-1 antibodies, implement this comprehensive validation strategy:

• Binding specificity assessment:

  • ELISA against recombinant LOX-1 protein

  • Western blot analysis on LOX-1-expressing cells

  • Cross-reactivity testing with related scavenger receptors

  • Immunoprecipitation followed by mass spectrometry

• Functional characterization:

  • Ability to block oxLDL binding to LOX-1

  • Effects on downstream signaling (MAPK, NF-κB pathways)

  • Influence on cellular functions (adhesion, migration, apoptosis)

  • Potential agonistic/antagonistic activity

• Epitope mapping:

  • Peptide array screening

  • Competition assays with known epitope-specific antibodies

  • Hydrogen/deuterium exchange mass spectrometry

  • X-ray crystallography of antibody-antigen complex

• Performance in multiple applications:

  • Flow cytometry on various cell types

  • Immunohistochemistry on fixed tissues

  • ELISA (direct, indirect, sandwich formats)

  • Immunofluorescence microscopy

This thorough characterization ensures reliable antibody performance across different experimental systems and applications.

How can I address technical challenges when using LOX-1 antibodies in flow cytometry?

Flow cytometry with LOX-1 antibodies presents several technical challenges that can be overcome with specific strategies:

• Low expression level detection:

  • Use signal amplification systems (e.g., biotin-streptavidin)

  • Implement fluorophores with higher quantum yield

  • Optimize cell stimulation conditions (e.g., PMA treatment for THP-1 cells)

  • Consider enzyme treatment to remove surface proteins potentially masking epitopes

• Non-specific binding reduction:

  • Optimize blocking with appropriate sera or protein solutions

  • Include human Fc receptor blocking reagents when using human samples

  • Carefully titrate antibody concentration

  • Perform fluorescence-minus-one (FMO) controls

• Distinguishing specific populations:

  • Implement a multi-marker panel to identify LOX-1-expressing cell subsets

  • For monocytes/macrophages: include CD14, CD16, HLA-DR

  • For dendritic cells: include CD1c, CD11c, HLA-DR

  • For endothelial cells: include CD31, CD144

• Sample preparation considerations:

  • Minimize time between collection and staining

  • Optimize fixation if required (paraformaldehyde concentration and time)

  • Evaluate detergent permeabilization if intracellular staining is needed

By addressing these challenges systematically, researchers can achieve more reliable and reproducible LOX-1 detection in flow cytometry applications .

How can LOX-1 antibodies be used to investigate the role of LOX-1 in immune cell function beyond atherosclerosis?

Recent research has revealed LOX-1's significant role in immune responses beyond vascular pathology:

• Dendritic cell-mediated T cell responses:

  • Use LOX-1 antibodies to target antigens to DCs

  • Investigate the generation of antigen-specific IFNγ-producing CD4+ T cells

  • Compare responses in both naïve and memory T cell populations

  • Analyze responses to both foreign and self-antigens

• B cell immunity enhancement:

  • Study how LOX-1 engagement on DCs affects:

    • BAFF and APRIL production

    • B cell class switching

    • Plasmablast generation

    • Antibody secretion profiles

  • Compare LOX-1 with other lectins that affect B cell responses differently

• Mucosal immunity:

  • Investigate LOX-1's role in promoting IgA1 and IgA2 class switching

  • Study the imprinting of CCR10 on plasmablasts by LOX-1-activated DCs

  • Develop vaccination strategies targeting mucosal immunity through LOX-1

• Autoimmunity connections:

  • Examine how endogenous LOX-1 ligands like oxidized-LDL can induce BAFF and APRIL secretion

  • Investigate potential connections to antibody-mediated autoimmune diseases like lupus

  • Develop blocking strategies to mitigate autoimmune responses

This emerging area represents an exciting frontier for immunology research using LOX-1 antibodies as both investigative and therapeutic tools.

What are the latest developments in therapeutic LOX-1 antibodies for cardiovascular disease?

The development of therapeutic LOX-1 antibodies has gained momentum as a promising approach for cardiovascular disease treatment:

• Current development status:

  • Several neutralizing antibodies have shown promising results in animal models

  • These antibodies function by blocking the interaction between LOX-1 and its ligands, including oxLDL

  • Preclinical studies demonstrate potential benefits in reducing atherosclerotic plaque formation and improving endothelial function

• Therapeutic mechanisms:

  • Inhibition of oxLDL uptake by vascular cells

  • Reduction of endothelial inflammation and dysfunction

  • Decreased foam cell formation

  • Inhibition of platelet activation and aggregation

  • Potential stabilization of vulnerable plaques

• Challenges and considerations:

  • Optimizing antibody pharmacokinetics and tissue penetration

  • Determining optimal dosing regimens

  • Identifying patient populations most likely to benefit

  • Developing companion diagnostics (e.g., sLOX-1 measurements)

  • Addressing potential immunogenicity of therapeutic antibodies

• Combination approaches:

  • Potential synergy with lipid-lowering therapies

  • Integration with anti-inflammatory treatments

  • Complementary use with antiplatelet agents

As research progresses, therapeutic LOX-1 antibodies may offer a novel approach to address residual cardiovascular risk in patients already receiving standard-of-care treatments .

How might LOX-1 antibodies contribute to developing novel diagnostic biomarkers for cardiovascular disease?

LOX-1 antibodies are instrumental in developing next-generation cardiovascular diagnostic approaches:

• Soluble LOX-1 (sLOX-1) as a biomarker:

  • High-sensitivity immunoassays using carefully selected antibody pairs can detect sLOX-1 in circulation

  • sLOX-1 levels may predict early vascular dysfunction before clinical manifestations

  • Potential for risk stratification in apparently healthy individuals and those with established cardiovascular disease

• Multi-marker panels:

  • Combining sLOX-1 with traditional markers (troponins, BNP) and other novel biomarkers

  • Development of algorithm-based risk scores incorporating sLOX-1 measurements

  • Personalized risk assessment based on biomarker profiles

• Imaging approaches:

  • LOX-1-targeted antibodies conjugated to contrast agents for molecular imaging

  • Potential applications in identifying vulnerable plaques

  • Non-invasive assessment of disease progression

• Point-of-care testing development:

  • Adaptation of laboratory-based LOX-1 immunoassays to rapid testing formats

  • Integration into emergency department workflows for acute coronary syndrome evaluation

  • Population screening in primary care settings

These diagnostic applications leverage the specificity of LOX-1 antibodies to provide clinically relevant information about disease status and prognosis, potentially enabling earlier intervention and more targeted treatment approaches .