LILRA2 Antibody

What is LILRA2 and what is its role in the immune system?

LILRA2 (Leukocyte Immunoglobulin-Like Receptor A2, also known as ILT1, CD85h, or LIR7) is a 51 kDa type I transmembrane glycoprotein belonging to the LILR family of immunoreceptors. It functions primarily as an activating receptor on myeloid cells, containing four Ig-like C2-type domains in its extracellular region and signaling through association with ITAM-containing adaptor molecules such as the FcRγ chain .

LILRA2 plays dual roles in immunity:

• Pathogen sensing: It recognizes microbially cleaved immunoglobulins (N-truncated Igs) generated by bacterial proteases from species like Legionella pneumophila, Streptococcus pneumoniae, and Mycoplasma .

• Inflammation regulation: It recognizes solid-phase fibrinogen (but not soluble fibrinogen) as an endogenous ligand, activating inflammatory pathways in monocytes .

This receptor represents an elegant immune surveillance mechanism that detects both microbial evasion tactics and tissue damage signals.

How does LILRA2 differ from other LILR family members?

While LILRA2 is classified as a group 1 LILR member, it demonstrates unique ligand recognition properties compared to other family members:

Unlike most group 1 LILRs, LILRA2 does not bind to HLA class I molecules due to structural differences in its binding domains . Rather, LILRA2's unique binding properties enable it to function as a sensor for both microbial activity and tissue damage .

What applications are LILRA2 antibodies most commonly used for in research?

LILRA2 antibodies are valuable tools for multiple research applications:

• Flow cytometry: Detecting LILRA2 expression on immune cell populations, particularly neutrophils and monocytes .

• Blocking experiments: Neutralizing LILRA2 function in cellular assays to assess its role in immune responses .

• Immunohistochemistry (IHC): Analyzing LILRA2 expression in tissue sections, particularly in inflammatory conditions .

• Western blotting: Detecting LILRA2 protein expression in cell lysates .

• Immunoprecipitation: Isolating LILRA2 and associated proteins for downstream analysis .

Research has demonstrated that anti-LILRA2 antibodies (particularly clone 600007) effectively block the interaction between LILRA2 and its ligands at concentrations of 10 μg/mL, making them valuable for mechanistic studies of LILRA2-mediated immune activation .

What controls should be included when using LILRA2 antibodies in flow cytometry?

When performing flow cytometry with LILRA2 antibodies, include the following controls to ensure valid and interpretable results:

• Isotype controls: Match the isotype, host species, and conjugate of your LILRA2 antibody to control for non-specific binding.

• Negative cell population: Include LILRA2-negative cells (e.g., lymphocytes) alongside LILRA2-positive myeloid cells.

• Blocking controls: For functional assays, include both:

  • IgG isotype control antibody at matching concentration

  • Anti-LILRA2 antibody (10 μg/mL of clone 600007 has been validated)

• Compensations: For multi-color panels, proper fluorophore compensation is essential as LILRA2 is often co-expressed with other myeloid markers.

• Activation state controls: Since LILRA2 surface expression can change upon cell activation, include both resting and activated cell populations (e.g., fMLP, LPS, or TNFα-stimulated) .

Multiple studies have validated anti-LILRA2 antibodies for flow cytometry using both conventional and mass cytometry approaches , making this a reliable application for studying LILRA2 biology.

How can LILRA2 antibodies be used to investigate microbially cleaved immunoglobulin recognition?

Investigating LILRA2's recognition of microbially cleaved immunoglobulins requires sophisticated experimental approaches:

Methodological approach:

• Generation of N-truncated Igs: Prepare N-truncated IgG or IgM by exposure to bacterial proteases (e.g., from L. pneumophila, S. pneumoniae, or M. hyorhinis) or by enzymatic digestion .

• Binding assays:

  • Surface Plasmon Resonance (SPR) analysis measuring the interaction between immobilized N-truncated Igs and LILRA2-Fc fusion proteins (Kd ≈ 4.8 μM)

  • ELISA-based binding assays with coated N-truncated Igs

• Competitive inhibition assays:

  • Pre-incubate cells with anti-LILRA2 antibodies (10 μg/mL) before exposure to N-truncated Igs

  • Compare with other potential LILRA2 ligands (e.g., fibrinogen) to establish binding specificity

• Reporter cell assays:

  • Use NFAT-GFP reporter cells expressing LILRA2 extracellular domains

  • Monitor GFP expression as an indicator of LILRA2 activation

This approach has revealed that LILRA2 binds specifically to N-truncated Igs with relatively weak affinity but fast kinetics, and that cell surface-bound LILRA2 exhibits enhanced interactions with N-truncated Igs due to avidity effects .

What strategies can resolve contradictory data when studying LILRA2-mediated responses in different inflammatory contexts?

Resolving contradictory findings regarding LILRA2 function requires careful experimental design and consideration of contextual factors:

Known contradictions in LILRA2 biology:

• LILRA2 stimulation promotes antimicrobial activity against some pathogens while inhibiting TLR-mediated antimicrobial responses against others .

• LILRA2 increases inflammatory cytokine production in some contexts but inhibits LPS-mediated TNF-α secretion in others .

Resolution strategies:

• Consider activation context:

  • Examine the effect of pre-existing TLR engagement before LILRA2 stimulation

  • Test multiple timepoints to capture temporal dynamics of responses

  • Compare outcomes with different LILRA2 ligands (N-truncated Igs vs. fibrinogen)

• Cell-type specific analysis:

  • Use LILRA2 antibodies for precise immunophenotyping of responding cells

  • Apply single-cell techniques to resolve heterogeneity in LILRA2+ populations

  • Compare neutrophil vs. monocyte vs. macrophage responses

• Blocking antibody approach:

  • Use validated anti-LILRA2 antibodies (e.g., clone 600007) at optimal concentrations (10 μg/mL)

  • Include appropriate isotype controls

  • Compare multiple LILRA2 antibody clones (e.g., clone 600007 vs. clone 135)

• Comprehensive readout panel:

  • Measure diverse cytokines beyond IL-8 and TNF-α

  • Assess both antimicrobial functions (ROS, phagocytosis) and inflammatory mediators

  • Incorporate transcriptomic analysis (e.g., RNA-seq)

Studies show that LILRA2 engagement can have context-dependent effects on immune cell function, with the nature of the ligand (microbial vs. endogenous) and concurrent receptor engagement influencing the outcome .

What criteria should guide selection of the appropriate LILRA2 antibody clone for specific applications?

Selecting the optimal LILRA2 antibody requires consideration of several technical parameters:

Selection criteria by application:

Epitope considerations:
The hydrophobic region of LILRA2 domain 2 has been identified as the N-truncated Ig-binding site , making antibodies targeting this region particularly useful for functional studies. When selecting antibodies for mechanistic studies of LILRA2-ligand interactions, prioritize clones validated for blocking this domain.

Clone-specific performance:
Research has demonstrated that clone 600007 provides superior blocking of LILRA2-fibrinogen interaction compared to clone 135, while both effectively inhibit LILRA2 function to varying degrees .

How do post-translational modifications of LILRA2 impact antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) of LILRA2 can significantly influence antibody recognition and experimental interpretation:

Key LILRA2 modifications:

• N-linked glycosylation: The LILRA2 extracellular domain contains seven potential N-linked glycosylation sites that may affect antibody binding.

• Phosphorylation: LILRA2 signaling involves phosphorylation events that may alter conformation.

• Proteolytic processing: Potential cleavage during isolation or sample preparation.

Experimental approaches to address PTM variation:

• Treatment with glycosidases: Compare antibody binding to native and deglycosylated LILRA2 to assess glycan dependence.

• Multiple epitope targeting: Use antibodies recognizing different LILRA2 domains to ensure robust detection regardless of PTM status.

• Recombinant standards: Include purified recombinant LILRA2 (glycosylated and non-glycosylated) as controls in quantitative assays.

• Sample preparation considerations:

  • Inclusion of protease inhibitors during cell lysis

  • Standardized protocols for fixation in IHC/ICC to preserve epitope accessibility

  • Fresh vs. frozen sample comparison when establishing protocols

• Cell activation state awareness: Surface expression of LILRA2 can change upon cellular activation , potentially exposing different epitopes.

Understanding these considerations will improve experimental reproducibility and interpretation of LILRA2 antibody-based assays across different research contexts.

How can LILRA2 antibodies contribute to investigating the role of LILRA2 in specific infectious diseases?

LILRA2 antibodies serve as crucial tools for elucidating this receptor's role in infectious disease pathogenesis:

Methodological approaches for infectious disease research:

• Mycobacterial infections (e.g., M. tuberculosis, M. leprae):

  • Use anti-LILRA2 antibodies to assess receptor expression in patient samples

  • Apply blocking antibodies (10 μg/mL) in ex vivo infection models to evaluate effects on bacterial control

  • Compare with LILRB2 blockade, which enhances killing of intracellular M. tuberculosis

• Bacterial pneumonia (e.g., S. pneumoniae):

  • Analyze LILRA2 expression in bronchoalveolar lavage samples

  • Examine interaction with N-truncated Igs in patient pus fluid samples

  • Block LILRA2 to determine effects on neutrophil antimicrobial functions

• Research workflow for infectious disease studies:

  • Compare LILRA2 expression between healthy controls and infected patients by flow cytometry

  • Correlate expression with disease severity and bacterial burden

  • Use blocking antibodies in functional assays measuring ROS production, phagocytosis, and cytokine production

  • Examine the ratio of cleaved to intact Igs in patient samples

Studies have shown that LILRA2 is upregulated in lepromatous leprosy lesions compared to tuberculoid forms, and blocking LILRA2 reduces TLR-mediated antimicrobial activity in some contexts . Conversely, LILRA2 activation inhibits L. pneumophila growth in monocytes , highlighting context-dependent roles.

What are the methodological considerations when using LILRA2 antibodies to study inflammatory conditions involving fibrinogen recognition?

When investigating LILRA2's role in inflammatory conditions through fibrinogen recognition, several methodological considerations are critical:

Experimental design considerations:

• Fibrinogen preparation and presentation:

  • LILRA2 specifically recognizes solid-phase fibrinogen, not soluble fibrinogen

  • Pre-immobilize fibrinogen on surfaces for functional assays

  • Block plate surfaces with BSA to prevent non-specific fibrinogen immobilization when testing soluble fibrinogen

• Blocking strategy optimization:

  • Use anti-LILRA2 antibody (clone 600007) at 10 μg/mL for effective blocking

  • Include appropriate isotype controls

  • Compare fibrinogen-induced responses with and without LILRA2 blockade

• Readout selection:

  • Measure IL-8 production as a validated LILRA2-dependent readout

  • Consider comprehensive analysis using RNA-seq to capture the full response profile

  • Compare responses to other LILRA2 ligands (N-truncated Igs)

• Control for other fibrinogen receptors:

  • LILRB2 and LILRA3 also bind fibrinogen/fibrin

  • Include specific blocking antibodies against these receptors as controls

  • Consider the differential recognition of fibrinogen vs. fibrin by different LILRs

Research has demonstrated that LILRA2 activation by solid-phase fibrinogen promotes expression of various inflammation-related genes in primary monocytes, and this response can be effectively blocked with anti-LILRA2 antibodies . This suggests that LILRA2 functions as a critical sensor for immobilized fibrinogen in inflammatory settings.

How might emerging antibody engineering technologies enhance LILRA2 research applications?

Advanced antibody engineering approaches offer promising opportunities to develop next-generation tools for LILRA2 research:

Emerging technologies with potential applications:

• Bispecific antibodies:

  • Designing antibodies that simultaneously target LILRA2 and potential ligands

  • Creating tools that co-engage LILRA2 with other receptors (TLRs, FcRs) to study receptor crosstalk

• Intrabodies and nanobodies:

  • Developing smaller antibody formats capable of tracking LILRA2 intracellular trafficking

  • Creating tools for real-time visualization of LILRA2 signaling dynamics

• Optogenetic antibody systems:

  • Engineering light-controlled anti-LILRA2 antibodies for precise temporal control of receptor blockade

  • Enabling reversible manipulation of LILRA2 signaling in living cells

• Site-specific conjugation strategies:

  • Producing homogeneously labeled LILRA2 antibodies with defined fluorophore/payload locations

  • Optimizing antibody orientation for surface immobilization in biosensor applications

• Antibody fragments with enhanced tissue penetration:

  • Developing Fab or scFv formats for improved tissue distribution in animal models

  • Creating tools for in vivo imaging of LILRA2 expression patterns

These approaches could significantly advance understanding of LILRA2's role in recognizing both microbially cleaved antibodies and solid-phase fibrinogen , potentially revealing new therapeutic targets for infectious and inflammatory diseases.

What strategies can address challenges in reproducing LILRA2 antibody-based experimental findings across different research contexts?

Reproducibility challenges in LILRA2 research can be addressed through systematic methodological approaches:

Standardization strategies:

• Detailed reporting of antibody characteristics:

  • Document complete antibody information: clone, supplier, catalog number, lot, concentration

  • Specify conjugates used and validation performed for specific applications

  • Report titration data demonstrating optimal antibody concentration (e.g., 10 μg/mL for blocking)

• Experimental context documentation:

  • Standardize cell isolation procedures for primary monocytes and neutrophils

  • Document donor characteristics and activation states of cells

  • Report precise details of ligand preparation (N-truncated Igs, fibrinogen immobilization)

• Multi-parameter validation:

  • Verify LILRA2 antibody specificity using multiple approaches:

    • Flow cytometry on known positive/negative cell populations

    • Binding assays with recombinant LILRA2 vs. other LILR family members

    • Functional validation in reporter systems

• Cross-laboratory validation initiatives:

  • Establish shared protocol repositories with detailed methods

  • Implement round-robin testing of key LILRA2 antibody clones across laboratories

  • Create biobanks of standardized control samples

Research has shown that different anti-LILRA2 antibody clones (600007 vs. 135) have varying efficacy in blocking LILRA2-ligand interactions , highlighting the importance of clone-specific validation and standardization in experimental approaches.

What are common technical pitfalls when working with LILRA2 antibodies and how can they be resolved?

Researchers frequently encounter specific technical challenges when working with LILRA2 antibodies:

Common challenges and solutions:

• Low detection signal in flow cytometry:

  • Problem: Weak staining despite known LILRA2 expression

  • Solutions:

    • Optimize fixation protocols (avoid harsh fixatives that may destroy epitopes)

    • Test multiple antibody clones targeting different LILRA2 domains

    • Use signal amplification systems or brighter fluorophores

    • Ensure cells are analyzed fresh, as LILRA2 expression may change with prolonged storage

• Non-specific binding in Western blots:

  • Problem: Multiple bands observed beyond expected 51 kDa (unglycosylated) size

  • Solutions:

    • Implement stringent blocking conditions (5% BSA often superior to milk for glycoproteins)

    • Increase washing stringency

    • Consider the contribution of glycosylation to apparent molecular weight

    • Use recombinant LILRA2 as positive control to identify specific band

• Inconsistent blocking efficiency:

  • Problem: Variable outcomes in LILRA2 blocking experiments

  • Solutions:

    • Use validated blocking antibody (clone 600007) at optimal concentration (10 μg/mL)

    • Pre-incubate cells with antibody before exposure to ligands

    • Ensure antibody remains present throughout the experiment

    • Verify antibody function with appropriate positive controls

• Conflicting results between different LILRA2 ligands:

  • Problem: Discrepancies between N-truncated Ig and fibrinogen experiments

  • Solutions:

    • Recognize that truncated Ig cannot block LILRA2-fibrinogen interaction

    • Understand that solid-phase vs. soluble presentation dramatically affects recognition

    • Control for potential contribution of other LILR family members (LILRB2, LILRA3)

These approach modifications have been validated in published studies and can significantly improve LILRA2 antibody application outcomes .

How can researchers verify LILRA2 antibody specificity when studying novel disease contexts or tissue types?

When extending LILRA2 antibody applications to new disease models or tissues, rigorous validation is essential:

Comprehensive validation workflow:

• Cross-reactivity assessment:

  • Test antibody binding to recombinant LILRA2 versus other LILR family proteins

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Validate in cells with genetic manipulation (LILRA2 knockout or overexpression)

• Tissue-specific validation strategies:

  • Use multiple antibody clones targeting different epitopes

  • Implement RNA-protein correlation:

    • Compare antibody staining with LILRA2 mRNA expression (RNA-seq, qPCR, ISH)

    • Assess concordance between protein and transcript levels

  • Include appropriate positive controls (monocytes, neutrophils) alongside tissue samples

• Disease-context considerations:

  • Validate antibodies in both healthy and disease samples

  • Consider potential post-translational modifications in disease states

  • Test for interfering factors in disease samples (rheumatoid factor, autoantibodies)

• Advanced validation approaches:

  • Epitope mapping to confirm antibody binding site

  • Competitive binding assays with known LILRA2 ligands

  • Functional validation using reporter systems expressing LILRA2

• Negative control strategies:

  • Use lymphocytes as natural negative controls for LILRA2 expression

  • Include appropriate isotype controls matched to primary antibody

  • Perform antibody adsorption controls with recombinant LILRA2