LILRA5 Antibody

What is LILRA5 and what are its primary biological functions?

LILRA5 (Leukocyte Immunoglobulin-Like Receptor A5) is an activating immune receptor expressed on human phagocytes that plays a role in triggering innate immune responses. The canonical protein has a length of 299 amino acid residues with a molecular mass of approximately 32.8 kDa . LILRA5 co-localizes with FcRγ and functions as an activating receptor that can stimulate reactive oxygen species (ROS) production in phagocytes .

Key characteristics include:

• Subcellular localization: Cell membrane and secreted forms

• Expression profile: Primarily in hematopoietic tissues including bone marrow, spleen, lymph node, and peripheral leukocytes

• Post-translational modifications: Glycosylation has been documented

• Alternative names: CD85 antigen-like family member F, immunoglobulin-like transcript 11 protein (ILT11), leucocyte Ig-like receptor A5, leukocyte Ig-like receptor 9, and CD85f

Recent research has demonstrated that LILRA5 functions to induce ROS production in innate immune cells, suggesting a role in bacterial defense mechanisms .

What are the standard applications for LILRA5 antibodies in experimental research?

LILRA5 antibodies serve multiple experimental purposes based on the current literature:

Most commercially available LILRA5 antibodies show reactivity with human samples, with some products reporting cross-reactivity with mouse and rat orthologs . When selecting an antibody, researchers should consider the specific epitope recognition, as this may affect detection of different LILRA5 isoforms .

How can I confirm the specificity of an anti-LILRA5 antibody?

Confirming antibody specificity is critical for reliable research results. Based on the methods described in recent studies, a comprehensive validation approach includes:

• Recombinant protein binding tests: Test antibody binding to recombinant LILRA5 compared to related proteins (other LILRs and LAIR1) using techniques like ELISA or bead-based assays .

• Cell line validation: Compare antibody binding between:

  • LILRA5-transfected cell lines versus control cell lines

  • Cell lines known to express LILRA5 versus negative controls

• Flow cytometry comparison:

  • Use proper isotype controls to establish threshold for specific staining (typically set at 3% false positive cells)

  • Calculate specific binding as [MFI LILRA5 - MFI Isotype control]

• Western blot validation:

  • Verify molecular weight (31-33 kDa for canonical form)

  • Include positive control samples (K-562 cells have been reported as positive)

• Knockout/knockdown controls: If available, use LILRA5 knockout or knockdown cells as negative controls.

A high-quality validation example from recent research demonstrated specificity through flow cytometry analysis where "anti-LILRA5 P4-11A mAb binds to human rLILRA5, but not to any of the closely related human rLILR that are most likely to be cross-reactive, nor to an immune receptor from a different family called LAIR1" .

What methodologies are most effective for investigating LILRA5's role in ROS production?

Recent research has established robust protocols for studying LILRA5-mediated ROS production in phagocytes . An optimized methodological approach includes:

• Antibody-mediated crosslinking assay:

  • Plate-coating: Coat microplates with 5 μg/ml of anti-LILRA5 antibody in PBS overnight at 4°C

  • Controls: Include isotype-matched control antibodies and positive controls (such as anti-CD3ζ antibodies)

  • Cell preparation: Isolate primary monocytes or neutrophils, or use suitable cell lines

  • ROS detection: Use luminol-enhanced chemiluminescence or fluorescent ROS indicators

• Reporter cell system approach:

  • Generate LILRA5CD3ζ reporter cell lines using the following strategy:

    • Create a DNA construct containing LILRA5 extracellular and transmembrane domains fused to CD3ζ cytoplasmic tail

    • Transduce into NFAT-GFP reporter cells (e.g., 2B4 NFAT-GFP T cells)

    • Confirm expression using flow cytometry

  • Measure activation through GFP induction upon antibody crosslinking

• Ex vivo stimulation protocols:

  • Whole blood or isolated leukocyte stimulation with LPS (50 ng/ml E. coli 026:B6 LPS for 18h has been reported)

  • Compare ROS production capacity in different activation states

  • Correlate with surface LILRA5 expression levels

When interpreting results, it's important to account for the dynamic regulation of surface LILRA5 expression, which may impact ROS production capacity under different conditions or time points.

How does LILRA5 expression change during infection and sepsis, and what are the implications for biomarker development?

LILRA5 shows complex expression patterns during infection and sepsis that are important for potential biomarker applications:

Research findings indicate:

• Transcript vs. protein discrepancy: While LILRA5 transcripts are significantly increased in human bacterial infections and sepsis, surface protein expression remains relatively unchanged .

• Post-transcriptional regulation: Surface LILRA5 expression appears to be dynamically regulated post-transcriptionally, with LPS stimulation affecting expression levels while bacterial infection of whole blood does not .

• Soluble LILRA5 increase: Enhanced levels of soluble LILRA5 in sepsis patients' sera and in supernatants of LPS-stimulated monocytes suggest either shedding from cell surfaces or expression of soluble isoforms .

For biomarker development, these findings suggest:

• Soluble LILRA5 may be more promising than surface expression for diagnostic applications

• A combined approach measuring both transcript and protein levels could provide complementary information

• Time-course studies would be essential to understand the dynamics of LILRA5 expression during disease progression

Researchers should note that "altered surface LILRA5 expression influences LILRA5-induced ROS production capacity" , suggesting functional consequences of expression changes that could affect disease pathophysiology.

What strategies can be used to distinguish between membrane-bound and soluble LILRA5 isoforms in experimental settings?

Distinguishing between membrane-bound and soluble LILRA5 isoforms requires a multi-faceted approach:

• Antibody-based discrimination:

  • Epitope-specific antibodies: Use antibodies targeting regions present in all isoforms versus those specific to certain isoforms

  • Flow cytometry: Detects surface (membrane-bound) LILRA5 on intact cells

  • ELISA/immunoassays: Quantify soluble LILRA5 in serum or culture supernatants

• Molecular characterization:

  • Western blotting with size discrimination: The canonical membrane-bound form is approximately 32.8 kDa, while soluble forms may show different molecular weights

  • PCR-based isoform detection: Design primers to amplify specific isoforms based on known splice variants

    • Up to 4 different isoforms have been reported for LILRA5

    • One 35 kDa soluble form shows a 27 aa substitution for aa 239-299

• Functional separation techniques:

  • Ultracentrifugation: Separate membrane fragments containing LILRA5 from soluble forms

  • Immunoprecipitation: Use antibodies specific to membrane-associated proteins to deplete membrane-bound forms

• Recombinant expression systems:

  • Generate cell lines expressing specific LILRA5 isoforms as reference standards

  • Create chimeric proteins with tags to distinguish isoforms

When conducting experiments, researchers should consider that "shedding of LILRA5 from cell surfaces or expression of sLILRA5 isoforms provides a mechanism to regulate surface LILRA5 expression levels" , which may have functional significance in different physiological contexts.

What are the best experimental designs to investigate LILRA5's potential role in bacterial defense mechanisms?

Based on recent research findings suggesting LILRA5's involvement in bacterial defense , optimal experimental designs should:

• Infection models:

  • Ex vivo whole blood infection:

    • Challenge with different bacterial pathogens (e.g., E. coli, S. aureus)

    • Assess LILRA5 expression at transcript and protein levels

    • Measure functional outputs (ROS production, cytokine release)

  • Primary cell culture systems:

    • Isolate monocytes and neutrophils from healthy donors

    • Challenge with live bacteria or bacterial components (LPS, peptidoglycan)

    • Monitor LILRA5 expression and function over time

• Functional modulation approaches:

  • Antibody-mediated crosslinking: Use agonistic anti-LILRA5 antibodies to trigger signaling

  • Blocking experiments: Use antagonistic antibodies or soluble LILRA5 to inhibit function

  • Genetic manipulation: Knockdown or overexpress LILRA5 in relevant cell types

• ROS production assessment:

  • Chemiluminescence assays for quantitative measurement

  • Fluorescent indicators for single-cell or microscopy-based detection

  • Correlate ROS production with bacterial killing capacity

• Clinical correlation studies:

  • Compare LILRA5 expression patterns in patients with different infectious diseases

  • Correlate expression with disease severity, clinical outcomes, and traditional biomarkers

  • Analyze both surface expression on immune cells and soluble LILRA5 in serum

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and functional assays

  • Identify potential regulatory networks and downstream targets

  • Map LILRA5 signaling pathways in response to bacterial challenges

One research group recently found that "LILRA5 transcripts are significantly increased in human bacterial keratitis and in human sepsis" , supporting its potential role in bacterial defense. The investigators concluded that "this is suggestive that LILRA5 has a role in bacterial defence and could be a useful biomarker for rapid diagnosis of inflammation triggered by bacterial pathogens" .

How can researchers effectively study the post-transcriptional regulation of LILRA5 expression?

Recent research has revealed that surface LILRA5 expression is dynamically regulated post-transcriptionally , making this an important area for investigation. A comprehensive approach includes:

• Transcript-protein correlation analysis:

  • qPCR for transcript quantification alongside flow cytometry for surface protein expression

  • Time-course studies following stimulation with different agents (e.g., LPS, cytokines, bacterial pathogens)

  • Single-cell analysis to identify cell-specific regulation patterns

• mRNA stability and translation studies:

  • Actinomycin D chase experiments to measure mRNA half-life under different conditions

  • Polysome profiling to assess translational efficiency

  • Analysis of 3'UTR regulatory elements that might influence stability or translation

• Protein trafficking and turnover assessment:

  • Pulse-chase experiments to measure protein half-life

  • Surface biotinylation to track internalization and recycling

  • Inhibitor studies targeting different degradation pathways (proteasome, lysosome)

• Soluble isoform characterization:

  • Analysis of alternative splicing patterns through RNA-seq

  • Examination of proteolytic processing using inhibitors of different proteases

  • Quantification of soluble LILRA5 release following different stimuli

• Regulatory factor identification:

  • RNA-binding protein immunoprecipitation to identify factors controlling mRNA stability or translation

  • CRISPR screens targeting potential regulatory factors

  • Analysis of microRNAs that might target LILRA5 mRNA

Previous studies have observed that "Ex vivo bacterial infection of whole blood did not alter surface LILRA5 expression, but LPS stimulation changed expression levels" , highlighting the complexity of LILRA5 regulation. Additionally, researchers found that "soluble (s)LILRA5 was enhanced in sera from sepsis patients and in supernatants of monocytes that were LPS-stimulated" , suggesting multiple mechanisms of regulation.

What are the most common technical challenges when working with LILRA5 antibodies, and how can they be addressed?

Researchers working with LILRA5 antibodies may encounter several technical challenges:

One research team developed a "highly-specific anti-LILRA5 monoclonal antibody that has agonistic properties" , demonstrating the importance of thorough antibody characterization. Their validation approach included:

• Testing binding to recombinant LILRA5 versus related proteins

• Comparing binding between LILRA5-transfected and control cell lines

• Establishing concentration-dependent binding

• Confirming specificity through multiple techniques

For researchers developing new antibodies, hybridoma techniques have been successfully employed, with one group describing: "Anti-LILRA5 P4-11A mAb was purified from a hybridoma derived from fusion of myeloma NS1/0 cells with spleen cells from Balb/c mice immunised with rLILRA5-His" .

How should researchers design experiments to investigate the functional relationship between LILRA5 and FcRγ signaling?

LILRA5 co-localizes with FcRγ and transduces signals through association with immunoreceptor tyrosine-based activation motif (ITAM)-containing FcRγ chains . To effectively study this functional relationship:

• Co-immunoprecipitation approaches:

  • Precipitate LILRA5 and probe for FcRγ association

  • Use cross-linking reagents to stabilize transient interactions

  • Include controls for specificity (other LILR family members)

• Functional reconstitution systems:

  • Express LILRA5 in cells lacking endogenous FcRγ

  • Co-express LILRA5 with wild-type or mutant FcRγ

  • Measure functional outputs (calcium flux, reporter activation)

• Mutational analysis:

  • Generate LILRA5 mutants affecting putative FcRγ interaction sites

  • Create chimeric receptors with other LILR extracellular domains

  • Assess functional consequences of mutations

• Microscopy-based approaches:

  • Fluorescence resonance energy transfer (FRET) to measure direct interaction

  • Co-localization studies using confocal microscopy

  • Live cell imaging to track dynamics of association

• Signaling pathway analysis:

  • Phospho-flow cytometry to assess ITAM-dependent signaling events

  • Inhibitor studies targeting specific components of the signaling cascade

  • Compare signaling patterns between LILRA5 and other FcRγ-associated receptors

• CRISPR/knockdown approaches:

  • Generate FcRγ-deficient cells and assess LILRA5 function

  • Rescue experiments with different FcRγ variants

  • Target downstream signaling components to map pathway dependencies

A technical consideration is that "LILRAs transduce signals through an association with immunoreceptor tyrosine-based activation motif (ITAM)-containing high affinity IgE Fc epsilon receptor type I γ chain (FcεRIγ)" , requiring careful design of experiments to distinguish LILRA5-specific effects from those mediated by other FcRγ-associated receptors.

What controls and validation steps are essential when developing a new assay to measure soluble LILRA5 in clinical samples?

Developing robust assays for soluble LILRA5 in clinical samples requires rigorous controls and validation:

• Analytical validation parameters:

  • Limit of detection and quantification

  • Linearity across the physiological range of concentrations

  • Precision (intra- and inter-assay coefficients of variation)

  • Accuracy (spike-recovery experiments)

  • Specificity (cross-reactivity with related proteins)

• Sample-related controls:

  • Matrix effect assessment (comparing standards in buffer vs. biological matrix)

  • Stability studies (freeze-thaw cycles, temperature, storage time)

  • Interference testing (hemolysis, lipemia, common medications)

• Antibody selection considerations:

  • Epitope mapping to ensure detection of all relevant isoforms

  • For sandwich assays, use antibody pairs recognizing non-overlapping epitopes

  • Validation with recombinant soluble LILRA5 standards

  • Testing with samples from diverse clinical conditions

• Reference standards and calibrators:

  • Develop stable, well-characterized recombinant standards

  • Include international reference materials if available

  • Prepare multi-level calibrators covering the entire measurement range

• Clinical validation approaches:

  • Establish reference ranges in healthy populations

  • Analyze samples from relevant disease states (sepsis, infections)

  • Compare against existing biomarkers or gold standard methods

  • Assess clinical sensitivity and specificity for intended use

• Quality control measures:

  • Include internal QC samples at multiple concentrations

  • Participate in external quality assessment programs if available

  • Implement Westgard rules for run validation

Research has shown that "soluble (s)LILRA5 was enhanced in sera from sepsis patients" , indicating the potential clinical utility of such assays. Additionally, since "shedding of LILRA5 from cell surfaces or expression of sLILRA5 isoforms provides a mechanism to regulate surface LILRA5 expression levels" , measurement of soluble forms could provide insights into the biology of LILRA5 regulation.