LILRB5 Antibody, HRP conjugated

What is LILRB5 and what is its function in the immune system?

LILRB5 (Leukocyte immunoglobulin-like receptor subfamily B member 5) is an inhibitory receptor belonging to the LILR family. It is also known as CD85c, LIR8, or LIR-8. LILRB5 may act as a receptor for class I MHC antigens . Unlike other LILR family members that bind to β2m-associated HLA-class I, LILRB5 demonstrates unique binding specificity for HLA-class I heavy chains, particularly HLA-B27 free heavy chain dimers . This distinctive binding profile results from differences in the D1 and D2 immunoglobulin-like binding domains. LILRB5 likely functions as an inhibitory regulator in immune responses through its interaction with MHC class I molecules, potentially contributing to immune tolerance mechanisms.

What cell types express LILRB5?

LILRB5 expression has been primarily detected on cells of the myeloid lineage. Flow cytometric analysis has demonstrated LILRB5 expression on human peripheral blood monocytes . Western blot analysis has also confirmed LILRB5 presence in human bone marrow lysates . Expression studies using immunohistochemistry have detected LILRB5 in various tissue types, though at varying levels. The expression pattern suggests LILRB5 plays a role in myeloid cell function, particularly in monocytes and potentially other bone marrow-derived cells.

What are the key differences between unconjugated LILRB5 antibodies and HRP-conjugated versions?

HRP-conjugated LILRB5 antibodies have the enzyme directly linked to the antibody molecule, enabling direct detection through enzymatic reaction with a substrate such as TMB or luminol . This conjugation eliminates the need for a secondary antibody step, potentially reducing background and cross-reactivity issues.

What are the optimal conditions for Western blot using LILRB5 antibodies?

For optimal Western blot detection of LILRB5, the following protocol parameters have proven effective:

• Sample preparation: Prepare cell lysates from appropriate sources (human monocytes, bone marrow, or transfected cells expressing LILRB5) using standard lysis buffers containing protease inhibitors.

• Gel conditions: Use reducing SDS-PAGE conditions as demonstrated in published protocols .

• Transfer conditions: Standard wet or semi-dry transfer to PVDF membrane.

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: For unconjugated antibodies, dilutions between 1:500 to 1:2000 have been successful. R&D Systems' anti-LILRB5 was effective at 0.5 μg/mL . For HRP-conjugated antibodies, follow manufacturer's recommendations, typically 1:1000 to 1:5000.

• Incubation: Overnight at 4°C or 2 hours at room temperature.

• Detection: Anti-LILRB5 antibodies typically detect a band at approximately 64 kDa in human samples under reducing conditions .

• Expected results: When using appropriate positive controls such as human bone marrow lysates, a specific band at approximately 64 kDa should be visible .

How should I design flow cytometry experiments with LILRB5 antibodies?

Successful flow cytometry experiments with LILRB5 antibodies require careful attention to controls and protocol optimization:

• Cell preparation: For peripheral blood samples, isolate PBMCs using density gradient centrifugation. Fix cells with 4% paraformaldehyde and permeabilize with 90% methanol for intracellular staining .

• Antibody dilution: A dilution of 1/500 (0.1 μg) has been successfully used for flow cytometry . Titrate new antibody lots to determine optimal concentration.

• Essential controls:

  • Isotype control (matching the primary antibody class, e.g., Rabbit IgG for rabbit anti-LILRB5)

  • Unstained cells (to establish autofluorescence baseline)

  • FMO (fluorescence minus one) controls when using multiple markers

  • Positive control (cells known to express LILRB5, such as monocytes)

• Staining protocol:

  • For direct conjugates: Incubate cells with antibody for 30-45 minutes at 4°C in the dark

  • For indirect detection: Incubate with primary antibody, wash, then incubate with appropriate secondary (e.g., Goat Anti-Rabbit IgG H&L Alexa Fluor 488 at 1/5000 dilution)

• Gating strategy: First gate on monocytes based on FSC/SSC characteristics, then analyze LILRB5 expression within this population.

• Validation: Specificity can be confirmed using blocking experiments with anti-LILRB5 antisera or comparing staining patterns between wild-type and LILRB5-transfected cells .

What is the recommended protocol for immunoprecipitation using LILRB5 antibodies?

Based on published research, the following protocol has been effective for immunoprecipitation of LILRB5 and its binding partners:

• Cell preparation: Culture appropriate cells (e.g., transfected cell lines expressing FLAG-tagged LILRB5) or isolate primary cells expressing endogenous LILRB5.

• Lysis: Use a non-denaturing lysis buffer containing protease inhibitors to preserve protein-protein interactions.

• Pre-clearing: Incubate lysates with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation approaches:

  • For FLAG-tagged LILRB5: Use anti-FLAG antibodies for immunoprecipitation

  • For endogenous LILRB5: Use specific anti-LILRB5 antibodies

  • For interacting partners: Use antibodies against potential binding partners (e.g., HC10 for HLA-class I heavy chains)

• Detection: Western blot using appropriate antibodies. LILRB5 can be detected using anti-FLAG antibodies for tagged constructs or specific anti-LILRB5 antibodies for endogenous protein.

• Expected results: In successful co-immunoprecipitation experiments, LILRB5 has been detected as a band of approximately 120 kDa when tagged with eGFP and FLAG , or at its native molecular weight of approximately 64 kDa .

How does LILRB5 binding to MHC class I heavy chains differ from other LILR family members?

LILRB5 exhibits distinctive binding characteristics compared to other LILR family members:

This unique binding specificity stems from structural differences in the D1 and D2 immunoglobulin-like binding domains. Sequence alignment analysis reveals that although LILRB5 shares 67% sequence similarity with LILRB2, only 5 of the amino acids that have been shown to bind to classical β2m-associated HLA class I in LILRB2 are conserved in LILRB5 .

Co-immunoprecipitation experiments have confirmed that HLA-B7 and B27 heavy chains interact with LILRB5 . This selective binding profile may have significant implications for LILRB5's biological function in immune regulation, particularly in contexts where free MHC heavy chains are present.

What mechanisms regulate LILRB5 signaling pathways?

LILRB5 signaling involves several critical regulatory components:

• Tyrosine phosphorylation: Research demonstrates that LILRB5 is constitutively tyrosine-phosphorylated in transfected cells .

• SHP-2 association: LILRB5 associates with the tyrosine phosphatase SHP-2, with tyrosine phosphorylation appearing essential for this interaction . This association was demonstrated by both immunoprecipitation and flow cytometry analysis.

• Downstream gene regulation: LILRB5 expression significantly upregulates MHC class I-related genes, including:

  • β2m (12.1 to 32.1-fold increase)

  • BF-I, BF-IV, and HLA-A (up to 174.8-fold increase)

  • TAP-1 and TAP-2 (up to 72.6-fold increase)

• Nitric oxide production: Cells transfected with LILRB5 show significantly higher NO concentration compared to control groups , suggesting LILRB5 may influence reactive oxygen species production.

The signaling properties vary between LILRB5 variants, with some forms showing stronger associations with SHP-2 and greater effects on downstream gene expression . These findings suggest LILRB5 plays a role in regulating antigen presentation pathways through its signaling activities.

What are the implications of LILRB5 expression in cancer immunology research?

Research into LILRB5's role in cancer has revealed several important findings:

Given these findings, LILRB5 represents a potential prognostic biomarker and therapeutic target in cancer immunotherapy research, though more studies are needed to fully elucidate its role across different cancer types.

How can I address weak or absent signal when using LILRB5 antibodies?

When facing weak or absent signals with LILRB5 antibodies, consider the following troubleshooting approaches:

• Verify expression:

  • Check LILRB5 mRNA expression by qPCR before protein detection

  • Use positive control samples known to express LILRB5 (human monocytes or bone marrow )

  • Enrich for monocytes if working with mixed cell populations

• Antibody selection:

  • Try antibodies targeting different epitopes of LILRB5

  • Consider antibodies validated for your specific application

  • Verify species reactivity matches your samples

• Protocol optimization:

  • Increase antibody concentration (reducing dilution)

  • Extend incubation time (overnight at 4°C instead of 1-2 hours)

  • Try more sensitive detection methods (chemiluminescent vs. colorimetric)

  • For Western blots, increase protein loading (50-100 μg total protein)

  • For flow cytometry, optimize fixation and permeabilization conditions

• Sample preparation:

  • For Western blot, try different lysis buffers (RIPA vs. NP-40)

  • Consider non-reducing conditions if the epitope is sensitive to reduction

  • For immunohistochemistry, optimize antigen retrieval methods

• Detection system:

  • Use amplification systems (biotin-streptavidin or tyramide signal amplification)

  • For HRP-conjugated antibodies, verify enzyme activity with a test substrate

  • Consider fresher antibody aliquots if degradation is suspected

What strategies can reduce non-specific binding and background with LILRB5 antibodies?

To minimize non-specific binding and background issues:

• Blocking optimization:

  • Extend blocking time (2 hours to overnight)

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • For Western blots, include 0.1-0.5% Tween-20 in blocking and antibody solutions

• Antibody dilution:

  • Increase dilution to reduce non-specific binding (start with manufacturer's recommendation, then increase if background persists)

  • For HRP-conjugated antibodies, typical effective dilutions range from 1:1000 to 1:5000

• Washing protocol:

  • Increase wash frequency (5-6 washes instead of 3)

  • Extend wash duration (10 minutes per wash)

  • Use gentle agitation during washes

• Controls:

  • Include isotype controls to identify non-specific binding

  • Use blocking peptides to confirm antibody specificity

  • Include secondary-only controls (omitting primary antibody)

• For flow cytometry:

  • Use Fc receptor blocking reagents before antibody incubation

  • Include viability dyes to exclude dead cells (high non-specific binders)

  • Optimize compensation when using multiple fluorophores

• For immunohistochemistry:

  • Quench endogenous peroxidase activity (3% H₂O₂ treatment)

  • Block endogenous biotin if using biotin-based detection systems

  • Use more specific detection systems (polymer-based instead of ABC method)

How should I properly store and handle HRP-conjugated LILRB5 antibodies to maintain activity?

Proper storage and handling of HRP-conjugated antibodies is crucial for maintaining enzymatic activity:

• Storage temperature:

  • Store at 2-8°C (refrigerated) as recommended for most antibody products

  • Do not freeze HRP-conjugated antibodies, as this can reduce enzyme activity

  • Avoid room temperature storage for extended periods

• Aliquoting:

  • Upon receipt, prepare small single-use aliquots to avoid repeated freeze-thaw cycles

  • Use sterile tubes and pipette tips when preparing aliquots

  • Include the recommended storage buffer in all aliquots

• Buffer considerations:

  • Ensure preservatives like Proclin 300 are maintained at appropriate concentrations (0.03%)

  • Maintain glycerol content (typically 50%) for stability

  • Avoid sodium azide in working solutions, as it inhibits HRP activity

• Working solutions:

  • Prepare fresh working dilutions on the day of the experiment

  • Return stock solutions to proper storage immediately after use

  • Discard remaining diluted antibody rather than storing

• Protection measures:

  • Minimize light exposure during handling

  • Avoid contamination with microorganisms

  • Keep caps tightly sealed to prevent evaporation

• Quality control:

  • Test activity periodically on positive control samples

  • Note lot number and track performance between experiments

  • Observe expiration dates provided by the manufacturer

By following these guidelines, researchers can maintain optimal HRP activity and ensure consistent experimental results when using HRP-conjugated LILRB5 antibodies.