LOX1.6 Antibody

What is LOX-1 and what cellular functions does it regulate?

LOX-1 is a class E scavenger receptor encoded by the OLR1 gene on human chromosome 12. It functions as a type II transmembrane protein with four domains: an extracellular C-terminal lectin domain, a connecting neck domain, a single transcellular domain, and a short N-terminal cytoplasmic tail. LOX-1 forms a homodimer through a disulfide bond between monomers at cysteine 140 residues. While initially studied for its role in vascular disease pathogenesis through recognition of oxidized-LDL, C-reactive protein, and fibronectin, LOX-1 has emerged as a critical regulator of immune cell function, particularly in dendritic cells (DCs) where it promotes Th1-type immune responses and bridges DC-B cell interactions to enhance humoral immunity .

Which immune cell types express LOX-1?

LOX-1 demonstrates selective expression across immune cell populations. It is prominently expressed on CD1c+ skin dermal DCs and blood myeloid DCs but notably absent on Langerhans cells and plasmacytoid DCs in humans. Additionally, certain fractions of peripheral B cells and monocytes express LOX-1. Histological analysis of human spleens has revealed LOX-1+CD11c+ DCs interacting with IgD+ B cells in marginal zones, suggesting a role in B cell response coordination. Within the B cell compartment, LOX-1 is expressed on both naive and memory B cells but undergoes downregulation following activation .

How do I validate the specificity of an anti-LOX-1 antibody for research applications?

When validating anti-LOX-1 antibody specificity, implement a multi-step approach:

• Transfection validation: Express full-length human LOX-1 in a suitable cell line (e.g., 293F cells) and confirm antibody binding via flow cytometry

• Recombinant protein binding: Test antibody binding to recombinant LOX-1 ectodomain-Fc fusion protein with appropriate controls (e.g., human DCIR ectodomain-Fc fusion)

• Cell type specificity: Verify antibody binding to known LOX-1+ cell populations (CD11c+ DCs) and absence of binding to LOX-1− cells

• Functional validation: Confirm that antibody-mediated LOX-1 targeting elicits expected biological responses (e.g., DC activation, BAFF/APRIL production)

What are the recommended methodologies for LOX-1 detection in tissue samples?

For optimal LOX-1 detection in tissue samples, consider these methodological approaches:

• Immunohistochemistry: Utilize anti-LOX-1 monoclonal antibodies with detection systems such as Envision+ kit with hematoxylin counterstaining

• Serial section analysis: Perform comparative staining with macrophage markers (e.g., RAM-11) and smooth muscle cell markers (e.g., 1A4) to identify co-localization patterns

• Quantification: Determine LOX-1 expression density as a percentage of positively stained regions using digital microscopy

• Controls: Include subclass-matched irrelevant IgG as negative controls

• Complementary staining: Combine with Azan-Mallory and H&E staining to correlate LOX-1 expression with tissue morphology

How does anti-LOX-1 antibody treatment affect dendritic cell function in promoting B cell responses?

Anti-LOX-1 antibody treatment fundamentally reprograms DC function to enhance B cell responses through multiple mechanisms:

• Enhanced DC activation: Anti-LOX-1-treated DCs upregulate HLA-DR and CD86 expression while secreting chemokines including MCP-1, MIP-1α, and IL-8

• BAFF/APRIL production: Unlike other lectin receptors (Dectin-1, DC-ASGPR, DCIR, DC-SIGN, DC-SIGN/L, DEC-205, Langerin, CLEC6), LOX-1 ligation uniquely induces production of B cell-supporting factors BAFF and APRIL

• Plasmablast differentiation: Co-culture of naive B cells with anti-LOX-1-treated DCs significantly enhances B cell proliferation and differentiation into CD38+CD20− plasmablasts

• Transcription factor modulation: Anti-LOX-1-treated DCs induce increased expression of STAT3 and BLIMP1 in B cells, critical factors for plasma cell differentiation

• Antibody class switching: DCs activated via LOX-1 promote class-switching, particularly toward IgA1 and IgA2 production

These effects are concentration-dependent, with higher anti-LOX-1 antibody concentrations inducing more robust B cell responses .

What mechanisms underlie LOX-1-mediated immunoglobulin class switching?

LOX-1-mediated immunoglobulin class switching involves distinct molecular pathways:

• BAFF/APRIL signaling: Anti-LOX-1-activated DCs produce significantly elevated levels of BAFF and APRIL, which directly promote class-switching through binding to their receptors on B cells

• Differential roles of BAFF vs. APRIL: Neutralization experiments reveal APRIL predominantly influences IgA production, while BAFF more significantly affects IgG responses

• Receptor engagement: TACI-Fc and BCMA-Fc (which neutralize both APRIL and BAFF) significantly reduce IgM and IgA concentrations, with BCMA-Fc being more efficient, particularly for IgA2

• IgA mucosal homing: LOX-1 activation uniquely imprints CCR10 expression on plasmablasts, promoting mucosal homing of IgA-producing cells

• Synergy with TLR signaling: LOX-1 engagement amplifies class-switching when combined with TLR activation (e.g., CpG/TLR9)

How can LOX-1 antibodies be modified for in vivo imaging applications?

Modification of LOX-1 antibodies for in vivo imaging requires specific conjugation strategies:

• Radiolabeling approaches:

  • Direct labeling with 99mTc using hydrazinonicotinamide (HYNIC) as a bifunctional chelating agent

  • Conjugation ratio optimization to maintain antibody binding capacity

  • Purification via size-exclusion chromatography to remove unbound radioisotopes

• Validation of modified antibodies:

  • Flow cytometry comparison of native vs. modified antibodies

  • Cell binding assays using LOX-1-expressing cells

  • Competitive binding studies to confirm specificity

• In vivo application parameters:

  • Injection dose optimization (typically 50-100 μg per subject)

  • Imaging timepoint determination (optimal signal-to-background ratio)

  • Region of interest (ROI) analysis correlating tracer accumulation with LOX-1 expression density

These modifications allow visualization of LOX-1 expression in atherosclerotic plaques and other tissues where LOX-1 is upregulated .

What experimental considerations are important when studying the role of LOX-1 in disease models?

When investigating LOX-1 in disease models, consider these critical experimental parameters:

• Cell type-specific effects:

  • LOX-1 expression varies significantly between cell types (endothelial cells, DCs, B cells)

  • Cell-specific knockout or conditional expression systems provide clearer insights than global manipulation

• Ligand complexity:

  • Multiple endogenous ligands (oxidized-LDL, CRP, fibronectin) may compete for LOX-1 binding

  • Oxidized-LDL itself can induce DCs to secrete BAFF and APRIL, complicating interpretation of antibody effects

• Disease context considerations:

  • In atherosclerosis: LOX-1 expression correlates with plaque instability

  • In immunity: LOX-1 may promote beneficial anti-cancer/anti-viral immunity but exacerbate autoimmunity

  • In mucosal immunity: LOX-1's role in IgA class-switching suggests importance in mucosal defense

• Temporal dynamics:

  • LOX-1 expression can be dynamically regulated (e.g., downregulated on B cells following activation)

  • Time-course studies are essential to capture the full biological impact

How do different anti-LOX-1 antibody clones compare in research applications?

When selecting an anti-LOX-1 antibody clone, researchers should consider the specific application requirements, target cell populations, and whether activating or neutralizing function is desired .

What are the implications of LOX-1 targeting for vaccine development?

LOX-1 targeting represents a promising strategy for next-generation vaccines based on several key properties:

• Th1 response promotion: LOX-1 targeting efficiently elicits antigen-specific IFNγ-producing CD4+ T cell responses both in human in vitro systems and in nonhuman primate models in vivo

• IgA class-switching: LOX-1-activated DCs uniquely promote IgA1 and IgA2 production, making LOX-1 an attractive target for mucosal vaccines

• CCR10 imprinting: LOX-1 engagement leads to CCR10 expression on plasmablasts, potentially enhancing mucosal homing of antibody-producing cells

• Vaccine carrier potential: Studies using influenza HA1 antigen targeted to LOX-1 demonstrated protective antibody responses in rhesus macaques

• Foreign and self-antigen responses: LOX-1 targeting can elicit responses to both foreign antigens and self-antigens, suggesting applications in cancer immunotherapy

How might LOX-1 engagement contribute to autoimmune pathology?

LOX-1 engagement may contribute to autoimmune pathology through several mechanisms:

• Enhanced humoral immunity: LOX-1's capacity to promote B cell responses and antibody production could potentially contribute to autoantibody generation in predisposed individuals

• Endogenous ligand activation: Oxidized-LDL, an endogenous ligand of LOX-1, can induce DCs to secrete BAFF and APRIL, potentially creating a feed-forward loop of B cell activation in inflammatory conditions

• Th1 polarization: LOX-1-mediated promotion of Th1 responses could exacerbate Th1-mediated inflammatory diseases

• Breaking of tolerance: The ability of LOX-1 targeting to enhance responses to self-antigens suggests a potential role in breaking immunological tolerance

• Disease-specific considerations: In systemic lupus erythematosus, the LOX-1 pathway could contribute to pathogenic autoantibody production and immune complex formation

What methodological approaches best evaluate LOX-1 expression in clinical samples?

For optimal evaluation of LOX-1 expression in clinical samples, implement this comprehensive workflow:

• Multi-marker immunohistochemistry:

  • LOX-1 staining coupled with cell-type markers (CD11c, RAM-11, 1A4)

  • Digital quantification of expression density as percentage of positively stained region

• Flow cytometry protocol:

  • Fresh tissue digestion or blood sample preparation

  • Staining with fluorochrome-conjugated anti-LOX-1 antibodies

  • Multi-parameter panels including lineage markers (CD11c, CD1c, IgD, CD27)

• Expression correlation analysis:

  • Division of samples into regions of interest (ROIs)

  • Correlation of LOX-1 expression with disease parameters

  • Classification of lesions based on histological features (adaptive thickening, atheromatous, fibroatheromatous, collagen-rich)

• Statistical considerations:

  • Appropriate sample size (e.g., n>20 per group)

  • Multiple ROIs per sample to account for heterogeneity

  • Correlation coefficient calculation between expression density and clinical parameters

What are the optimal storage and handling conditions for LOX-1 antibodies?

For maintaining LOX-1 antibody integrity and performance, observe these handling guidelines:

• Storage temperature: Store at -20°C for long-term storage; 4°C is acceptable for short-term (1-2 weeks)

• Aliquoting: Prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Buffer conditions: Store in PBS with 0.09% sodium azide and carrier protein (e.g., 1% BSA)

• Concentration: For research applications, maintain stock concentrations of 0.5-2 mg/ml

• Stability monitoring: Periodically validate functionality using positive control cells/tissues

• Working dilution preparation: Dilute immediately before use and maintain at 4°C during experiments

How should researchers troubleshoot inconsistent results with LOX-1 antibodies?

When encountering inconsistent results with LOX-1 antibodies, implement this systematic troubleshooting approach:

• Antibody validation:

  • Confirm antibody specificity using positive and negative control cells/tissues

  • Verify antibody functionality with recombinant LOX-1 binding tests

• Experimental conditions review:

  • Cell activation status: LOX-1 expression varies with cellular activation

  • Buffer composition: Ensure compatibility with antibody binding

  • Incubation times and temperatures: Standardize across experiments

• Technical considerations:

  • For flow cytometry: Optimize antibody concentration and fluorochrome choice

  • For immunohistochemistry: Review fixation methods and antigen retrieval protocols

  • For functional studies: Standardize cell numbers and culture conditions

• Biological variability assessment:

  • Donor-to-donor variation in primary cells

  • Disease state influence on LOX-1 expression

  • Influence of concurrent medications or treatments

What experimental controls are essential when studying LOX-1 function?

Robust research on LOX-1 function requires these essential controls:

• Antibody specificity controls:

  • Isotype-matched control antibodies (e.g., mouse IgG2a at equivalent concentration)

  • LOX-1 knockout or silenced cells as negative controls

  • Blocking with recombinant LOX-1 to confirm specificity

• Functional assay controls:

  • When studying DC-B cell interactions: B cells alone, DCs alone, and co-culture without anti-LOX-1

  • For cytokine production: Multiple timepoints to capture optimal secretion

  • For class-switching: Positive controls using known class-switch inducers (e.g., CD40L+IL-4)

• Mechanistic investigation controls:

  • Neutralizing antibodies (e.g., TACI-Fc, BCMA-Fc, anti-BAFF)

  • Recombinant BAFF/APRIL to mimic LOX-1 effects

  • Receptor blockade on target cells

• Validation across systems:

  • Multiple cell sources (primary cells, cell lines)

  • Cross-species validation where applicable

  • Multiple anti-LOX-1 antibody clones to rule out clone-specific effects

How might LOX-1 function in anti-tumor immunity?

LOX-1's potential role in anti-tumor immunity warrants investigation based on several key properties:

• Th1 polarization capacity: LOX-1 engagement promotes Th1-type responses that are generally beneficial for anti-tumor immunity through IFNγ production

• DC activation: Anti-LOX-1 antibody treatment enhances DC activation and maturation, potentially improving tumor antigen presentation

• Humoral response augmentation: LOX-1-mediated enhancement of antibody production could potentially boost anti-tumor antibody responses

• Target potential: LOX-1 expression in the tumor microenvironment could provide a means to deliver immunomodulatory payloads

• Research approaches: Studies combining anti-LOX-1 antibodies with tumor antigen targeting constructs could evaluate therapeutic potential in cancer models

What is the relationship between LOX-1 and other pattern recognition receptors in immune regulation?

The complex interplay between LOX-1 and other pattern recognition receptors (PRRs) shapes immune outcomes:

• Functional divergence:

  • LOX-1 promotes Th1 responses

  • Dectin-1 promotes Th17 responses

  • DC-ASGPR promotes regulatory T cell responses

• Cooperative signaling possibilities:

  • LOX-1 may synergize with TLR signaling (evidenced by enhanced effects with CpG co-stimulation)

  • Potential cross-talk with inflammasome activation pathways

  • Integration with cytosolic PRR signals

• Cell-specific contexts:

  • In DCs: LOX-1 uniquely induces BAFF/APRIL production versus other lectins tested

  • In endothelial cells: LOX-1 functions primarily in oxidized-LDL recognition

  • In other immune cells: Varied and context-dependent functions

• Hierarchical relationships:

  • Temporal sequence of engagement may determine dominant outcomes

  • Competition for downstream signaling components

  • Potential physical associations between receptor complexes

How does post-translational modification of LOX-1 affect antibody recognition and function?

Researchers should consider these modifications when selecting antibodies for specific applications and when interpreting experimental results across different cellular contexts .