LILRB5 Antibody, Biotin conjugated

What is LILRB5 and what role does it play in immune regulation?

LILRB5 (Leukocyte immunoglobulin-like receptor subfamily B member 5), also known as CD85c, LIR-8, or CD antigen CD85c, is an inhibitory receptor that plays an important role in innate immunity. It belongs to the inhibitory leukocyte immunoglobulin-like receptor family, which regulates immune responses . The receptor contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that bind to Src homology 2-containing tyrosine phosphatase 2 (SHP-2) . Research indicates that LILRB5 specifically binds to MHC class I heavy chains and β2-microglobulin, suggesting its role in MHC class I-dependent immune responses . Furthermore, LILRB5 has been shown to upregulate major histocompatibility complex (MHC) class I and β2-microglobulin gene expression, as well as transporter associated with antigen processing 1-2 (TAP1-2) expression, indicating its function as a transcriptional regulator of MHC class I pathway components .

What are the structural features of LILRB5 antibodies and how does biotin conjugation affect their utility?

LILRB5 antibodies are typically generated against specific regions of the human LILRB5 protein. These antibodies can be:

Biotin conjugation enhances the utility of these antibodies by enabling secondary detection systems that use streptavidin conjugates. This provides greater flexibility in detection methodologies and can significantly amplify signals due to the high-affinity interaction between biotin and streptavidin. The biotin-conjugated LILRB5 antibodies maintain their specificity while gaining versatility in experimental applications such as ELISA, flow cytometry, and immunohistochemistry . The typical preservation buffer for biotin-conjugated LILRB5 antibodies contains 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain antibody stability and function .

What are the major applications of LILRB5 antibody in immunological research?

LILRB5 antibodies have several key applications in immunological research:

The biotin-conjugated format enhances signal detection across these applications through streptavidin-based amplification systems, particularly beneficial in flow cytometry and immunohistochemistry applications where signal strength is critical .

What are the optimal conditions for using biotin-conjugated LILRB5 antibodies in flow cytometry?

For optimal flow cytometry results with biotin-conjugated LILRB5 antibodies, researchers should follow these methodological guidelines:

• Cell Preparation: Prepare cells (1.0 × 10^6) by suspending them in staining buffer containing 10% FBS, 15 mM HEPES, and 2 mM EDTA in PBS .

• Antibody Dilution: The optimal dilution should be determined experimentally, but typically ranges from 1:100 to 1:500 for biotin-conjugated LILRB5 antibodies .

• Staining Protocol:

  • Incubate cells with the primary biotin-conjugated LILRB5 antibody for 30 minutes on ice

  • Wash once with staining buffer

  • Add fluorophore-conjugated streptavidin (e.g., PE-streptavidin) and incubate for an additional 30 minutes on ice

  • Wash again with staining buffer before analysis

• Controls:

  • Include an isotype control (e.g., rabbit IgG-biotin) treated identically to the experimental samples

  • Include unstained controls and single-color controls for compensation settings

  • For validation studies, consider using a 293T cell line transfected with a human LILRB5 expression vector as a positive control

• Analysis Parameters:

  • Analyze using standard flow cytometry equipment (e.g., BD FACSAria II)

  • Gate appropriately on monocyte populations when analyzing peripheral blood samples

  • Data should be acquired using software such as BD FACSDiva and analyzed with programs like FlowJo

This methodology has been validated in studies examining LILRB5 expression on peripheral blood monocytes, showing successful detection with properly optimized protocols .

How should storage and handling of biotin-conjugated LILRB5 antibodies be optimized for long-term experiments?

Proper storage and handling are crucial for maintaining the activity of biotin-conjugated LILRB5 antibodies:

• Storage Temperature:

  • Upon receipt, store antibodies at -20°C or -80°C as recommended by manufacturers

  • Avoid repeated freeze-thaw cycles, which can cause protein denaturation and biotin-conjugate degradation

• Aliquoting Strategy:

  • For long-term experiments, create small single-use aliquots immediately after receiving the antibody

  • Use sterile microcentrifuge tubes and aseptic technique when preparing aliquots

  • Document preparation date and freeze-thaw cycle count for each aliquot

• Working Solution Preparation:

  • When preparing diluted working solutions, use high-quality, filtered buffer solutions

  • Typical dilution buffers should contain 0.01M PBS (pH 7.4) with a stabilizer such as 1% BSA

  • Fresh working solutions should be prepared for each experiment rather than stored

• Stability Considerations:

  • The typical shelf-life is 12 months when stored properly at -20°C or -80°C

  • Biotin conjugates may gradually lose activity even under optimal storage conditions

  • Consider including positive controls from previous batches to monitor potential decline in antibody activity

• Handling During Experiments:

  • Keep antibodies on ice or at 4°C during experiments

  • Minimize exposure to light, particularly for dual-labeled antibodies

  • Return to -20°C storage promptly after use

These guidelines help ensure consistent antibody performance across extended experimental timeframes and reduce variability in results due to reagent degradation .

What protocol modifications are necessary when using biotin-conjugated LILRB5 antibodies for co-immunoprecipitation studies?

When using biotin-conjugated LILRB5 antibodies for co-immunoprecipitation studies, especially to investigate interactions with MHC class I and β2-microglobulin, the following protocol modifications are necessary:

• Pre-clearing Step:

  • Pre-clear cell lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding

  • Use lysis buffers containing protease inhibitors to preserve protein interactions

• Antibody-Bead Coupling:

  • Instead of directly adding biotin-conjugated antibodies to the sample, first couple them to streptavidin-coated beads

  • Incubate 2-5 μg of biotin-conjugated LILRB5 antibody with streptavidin beads for 30-60 minutes at room temperature

  • Wash beads thoroughly to remove unbound antibody

• Immunoprecipitation Procedure:

  • Add antibody-coupled beads to pre-cleared lysates

  • Incubate overnight at 4°C with gentle rotation

  • Wash beads 3-5 times with cold washing buffer containing 0.01M PBS and 0.1% Tween-20

• Detection Considerations:

  • For Western blot detection of co-immunoprecipitated proteins, avoid using biotin-conjugated secondary antibodies as they may cross-react with the biotin on the primary antibody

  • Instead, use HRP-conjugated or fluorescently-labeled antibodies that recognize the Fc region of the detecting antibody

  • When probing for MHC class I, expect bands at approximately 42 kDa

  • For β2-microglobulin detection, expect bands at approximately 14 kDa

• Controls:

  • Include appropriate negative controls such as isotype-matched biotin-conjugated IgG

  • Include a non-transfected cell line as a negative control when using transfected cells

  • When possible, perform reverse co-IP using antibodies against suspected binding partners (MHC class I or β2-microglobulin)

This approach has been validated in studies investigating LILRB5's interaction with MHC class I molecules, showing that LILRB5 immunoprecipitates contain both MHC class I proteins and β2-microglobulin .

How can researchers validate the specificity of biotin-conjugated LILRB5 antibodies in their experimental systems?

Validating antibody specificity is crucial for generating reliable data. For biotin-conjugated LILRB5 antibodies, a multi-step validation approach is recommended:

• Positive and Negative Cell Controls:

  • Use cells known to express LILRB5 (e.g., human peripheral blood monocytes) as positive controls

  • Use cell lines that lack LILRB5 expression as negative controls

  • Compare staining patterns between these controls to evaluate specificity

• Transfection Experiments:

  • Generate cell lines transfected with LILRB5 expression vectors (e.g., LILRB5-eGFP constructs)

  • Compare antibody binding between transfected and non-transfected cells

  • Quantify transfection efficiency using flow cytometry and verify protein expression by Western blotting

• Cross-Reactivity Assessment:

  • Test antibody against a panel of closely related proteins (other LILR family members)

  • Verify that the antibody recognizes the targeted epitope but not homologous regions in related proteins

  • If possible, test against LILRB5 from different species to confirm species reactivity claims

• Blocking/Competition Experiments:

  • Pre-incubate the antibody with recombinant LILRB5 protein before adding to samples

  • A true specific antibody will show diminished or absent staining after this blocking step

  • Use this approach to confirm that binding is to the intended target rather than non-specific interactions

• Knockout/Knockdown Validation:

  • When available, use LILRB5 knockout or knockdown systems as the gold standard for antibody validation

  • Compare staining patterns in wild-type versus knockout/knockdown samples

  • Absence of staining in knockout/knockdown samples strongly supports antibody specificity

These validation steps have been successfully used in studies of LILRB5, particularly when examining its interactions with MHC class I molecules and its expression on monocytes .

What are common pitfalls when analyzing LILRB5 expression data from flow cytometry experiments?

When analyzing LILRB5 expression using biotin-conjugated antibodies in flow cytometry, researchers should be aware of these common pitfalls:

• Streptavidin-Biotin Background Issues:

  • Endogenous biotin in samples can lead to false-positive signals

  • Solution: Block endogenous biotin using avidin/biotin blocking kits before adding biotin-conjugated antibodies

• Compensation Challenges:

  • Improper compensation when using streptavidin-fluorophore conjugates can lead to spillover artifacts

  • Solution: Run single-color controls with each fluorophore and set compensation matrices correctly

• Monocyte Autofluorescence:

  • Monocytes (primary LILRB5-expressing cells) exhibit significant autofluorescence

  • Solution: Include unstained controls and use fluorophores with emission spectra distant from autofluorescence peaks

• Inconsistent Gating Strategies:

  • Variations in gating between experiments can lead to apparent differences in LILRB5 expression

  • Solution: Develop standardized gating protocols based on cell size, granularity, and lineage markers

• Cell Activation During Processing:

  • Cell processing can alter receptor expression through activation or shedding

  • Solution: Minimize processing time and maintain samples at 4°C to reduce activation-induced changes

• Expression Level Inconsistencies:

  • LILRB5 expression levels can vary between donors and with inflammatory status

  • Solution: Include appropriate reference samples and report relative rather than absolute expression when comparing between experiments

• Antibody Titration Errors:

  • Insufficient titration can lead to suboptimal signal-to-noise ratios

  • Solution: Perform antibody titration experiments to determine optimal concentration for each specific application

During flow cytometry analysis of transfected cells expressing LILRB5, studies have shown that proper gating and antibody concentration are critical for accurately distinguishing positive from negative populations, with successful detection requiring careful optimization of these parameters .

How should researchers interpret apparent discrepancies in LILRB5 binding studies between different experimental systems?

When encountering discrepancies in LILRB5 binding studies across different experimental systems, researchers should consider these key interpretive factors:

In comparative studies examining LILRB5 binding to MHC class I molecules, researchers found that proteins from different genetic lines (6.3 vs. 7.2) showed dramatically different binding properties, demonstrating the importance of considering genetic variants when interpreting seemingly discrepant results .

How can biotin-conjugated LILRB5 antibodies be used to investigate the receptor's role in immune regulation and disease pathogenesis?

Biotin-conjugated LILRB5 antibodies offer powerful tools for investigating this receptor's immunoregulatory functions:

• Multi-parameter Flow Cytometry:

  • Combine biotin-conjugated LILRB5 antibodies with other immune markers to characterize expression patterns

  • Use fluorescent streptavidin conjugates for detection in multi-color panels

  • This approach allows correlation of LILRB5 expression with activation state, disease progression, or response to therapy

• Immunomodulatory Studies:

  • Utilize antibodies to block or activate LILRB5 signaling in functional assays

  • Assess effects on T cell proliferation, cytokine production, and antigen presentation

  • Studies with related inhibitory receptors (LILRB3) demonstrated that ligation by agonistic antibodies can significantly inhibit T cell proliferation by up to 50%

• MHC Class I Interaction Analysis:

  • Employ biotin-conjugated antibodies in co-localization studies with MHC class I molecules

  • Use techniques like proximity ligation assay (PLA) with streptavidin-conjugated detection systems

  • Research has shown that LILRB5 binds to MHC class I and β2-microglobulin, with this interaction regulating innate immune responses

• JAK/STAT Signaling Pathway Investigations:

  • Monitor how LILRB5 activation influences JAK/STAT pathway components

  • Combine LILRB5 detection with phospho-specific antibodies against STAT proteins

  • Studies indicate LILRB5 can activate the JAK/STAT signaling pathway in macrophages and induce cytokine expression

• Cytokine Response Profiling:

  • Assess how LILRB5 engagement alters cytokine production profiles

  • Research shows LILRB5 activation significantly upregulates expression of IFN-γ (up to 44-fold), IL-17A, and IL-12p40

  • Protein levels of these cytokines also increase substantially (e.g., IFN-γ levels of 1312-1461 ng/mL compared to 55 ng/mL in controls)

These approaches have revealed that LILRB5 functions as a transcriptional regulator of MHC class I pathway components and plays a key role in innate immune regulation through its interactions with SHP-2, MHC class I, and β2-microglobulin .

What methodological approaches can be used to study LILRB5's interactions with MHC class I molecules using biotin-conjugated antibodies?

To investigate LILRB5's interactions with MHC class I molecules, researchers can employ these methodological approaches using biotin-conjugated antibodies:

• Flow-based Binding Assays:

  • Generate LILRB5-expressing cell lines and assess binding of fluorescently-labeled MHC class I tetramers

  • Use biotin-conjugated LILRB5 antibodies to block binding sites and determine specificity

  • Flow cytometry analysis can quantify the percentage of binding inhibition

  • Research shows LILRB5 binds specifically to HLA-B27 free heavy chain dimers, with binding blocked by anti-LILRB5 antisera

• FRET/BRET Interaction Studies:

  • Couple biotin-conjugated LILRB5 antibodies with streptavidin-conjugated donor fluorophores

  • Use acceptor-labeled MHC class I molecules to measure energy transfer upon binding

  • This approach provides real-time interaction kinetics in live cells

• Surface Plasmon Resonance (SPR):

  • Immobilize biotinylated LILRB5 antibodies on streptavidin-coated sensor chips

  • Capture recombinant LILRB5 in a functionally active orientation

  • Measure binding kinetics with various MHC class I heavy chains and β2-microglobulin

  • This allows precise determination of association/dissociation constants

• Co-immunoprecipitation with Sequential Elution:

  • Use biotin-conjugated LILRB5 antibodies coupled to streptavidin beads for immunoprecipitation

  • Perform sequential elution steps to identify differential binding affinities

  • Western blot analysis of precipitates can detect MHC class I (42 kDa) and β2-microglobulin (14 kDa)

  • Research has demonstrated that LILRB5 immunoprecipitates contain both MHC class I and β2-microglobulin proteins

• Gene Expression Analysis Following Receptor Ligation:

  • Use biotin-conjugated LILRB5 antibodies to ligate the receptor on target cells

  • Measure changes in expression of MHC class I pathway genes using qRT-PCR

  • Studies show LILRB5 upregulates MHC class I genes (BF-I, BF-IV, HLA-A) by 42.7-174.8 fold and related genes (TAP-1, TAP-2) by up to 86.9-fold

These approaches have been successfully employed to demonstrate that LILRB5 preferentially binds to MHC class I heavy chains compared to β2-microglobulin, with binding to MHC class I being significantly stronger .

What techniques can be employed to investigate LILRB5's role in modulating innate immune signaling pathways?

Advanced techniques for investigating LILRB5's role in innate immune signaling include:

• Phospho-specific Flow Cytometry:

  • Use biotin-conjugated LILRB5 antibodies for receptor identification alongside phospho-specific antibodies

  • Determine activation status of key signaling molecules (SHP-2, JAK/STAT) following LILRB5 engagement

  • Quantify changes in phosphorylation state at single-cell resolution

  • Research has shown LILRB5 binds to SHP-2, affecting downstream signaling cascades

• CRISPR/Cas9 Receptor Editing:

  • Generate LILRB5 knockout or domain-specific mutant cell lines

  • Use biotin-conjugated antibodies to verify knockout efficiency

  • Compare signaling responses between wild-type and edited cells

  • Assess functional consequences on cytokine production and MHC class I expression

• Proximity Ligation Assay (PLA):

  • Combine biotin-conjugated LILRB5 antibodies with antibodies against signaling molecules

  • Visualize protein-protein interactions at <40nm resolution in situ

  • Quantify interaction frequency under different stimulation conditions

• Reporter Gene Assays:

  • Construct LILRB5-responsive reporter systems (e.g., using NFAT-GFP)

  • Evaluate signaling activation upon receptor cross-linking

  • Studies show that LILRB5 cross-linking in chimeric receptor systems induces NFAT activation and GFP expression

• Multi-omics Integration:

  • Combine transcriptomic, phosphoproteomic, and epigenetic analyses following LILRB5 engagement

  • Identify integrated signaling networks and feedback mechanisms

  • Data shows LILRB5 activation significantly alters expression of:

    • Th1 cytokines (IFN-γ increased 44.5-fold at mRNA level, 1312 ng/mL at protein level)

    • Th17 cytokines (IL-17A and IL-12p40)

    • MHC class I pathway genes (up to 174.8-fold increase)

• Live Cell Imaging:

  • Use biotin-streptavidin systems to visualize LILRB5 clustering and signaling complex formation

  • Track receptor internalization and trafficking following ligand binding

  • Correlate receptor dynamics with activation of downstream signaling events

These methodologies have revealed that LILRB5 plays a crucial role in activating the JAK/STAT signaling pathway and controlling cytokine expression in macrophages, suggesting potential therapeutic applications in immune regulation .

How can researchers utilize biotin-conjugated LILRB5 antibodies to explore the functional differences between LILRB5 genetic variants?

Research has identified functionally distinct LILRB5 genetic variants, including LILRB5R and LILRB5S. To explore these differences using biotin-conjugated antibodies:

• Variant-Specific Expression Analysis:

  • Develop genotyping assays to identify LILRB5 variants in research samples

  • Use biotin-conjugated LILRB5 antibodies in flow cytometry to quantify expression levels of different variants

  • Compare surface density between variants using calibration beads

  • Research has demonstrated that variant expression affects binding capacity to MHC molecules

• Differential Binding Assays:

  • Express different LILRB5 variants in cell lines and compare binding to potential ligands

  • Use competitive binding assays with biotin-conjugated antibodies to assess binding site differences

  • Studies show LILRB5R exhibits higher binding to MHC class I and β2m than LILRB5S

• Signaling Consequence Evaluation:

  • Compare signaling outcomes between variants using phospho-flow cytometry

  • Measure activation of downstream pathways following antibody-mediated receptor cross-linking

  • Correlate with functional readouts such as cytokine production

  • Research demonstrates LILRB5R induces significantly higher upregulation of:

    • IFN-γ (44.5-fold vs. 15.3-fold for LILRB5S)

    • Protein levels (1312 ng/mL for LILRB5R vs. 537 ng/mL for LILRB5S)

• Variant-Specific Immunoprecipitation:

  • Perform co-immunoprecipitation studies to compare protein interaction networks

  • Use biotin-conjugated antibodies coupled to streptavidin beads

  • Identify differential binding partners through mass spectrometry

  • Research shows variants differ in their association strength with MHC class I (stronger for LILRB5R)

• Transcriptional Regulation Analysis:

  • Compare gene expression profiles induced by different LILRB5 variants

  • Measure upregulation of MHC class I pathway genes and cytokines

  • Studies indicate LILRB5R induces stronger expression of MHC-related genes:

    • BF-I (42.7-fold), BF-IV (174.8-fold), and HLA-A (78.0-fold)

    • TAP-1 and TAP-2 (up to 86.9-fold)

• Functional Consequences in Primary Cells:

  • Isolate primary cells from donors with different LILRB5 genotypes

  • Compare functional responses to stimulation

  • Assess effect on T cell proliferation and cytokine production

  • Flow cytometry analysis shows variant-dependent differences in MHC class I surface expression:

    • LILRB5R increases MHC class I expression by 51.8% vs. 41.2% for LILRB5S

    • β2m expression increases by 29.5% for LILRB5R vs. 24.2% for LILRB5S

These approaches have revealed significant functional differences between LILRB5 variants, with proteins from line 6.3 showing dramatically higher binding to MHC class I than those from line 7.2, suggesting important implications for immune regulation .

What emerging technologies might enhance the utility of biotin-conjugated LILRB5 antibodies in immunotherapy research?

Several emerging technologies show promise for expanding the applications of biotin-conjugated LILRB5 antibodies in immunotherapy research:

• Bispecific Antibody Engineering:

  • Develop bispecific constructs combining LILRB5 targeting with T cell engagement

  • Engineer biotin-tagged fragments for modular assembly with streptavidin scaffolds

  • This approach could allow selective modulation of myeloid cell function in the tumor microenvironment

  • Studies of related receptors (LILRB3) demonstrate that antibody-mediated targeting can reprogram myeloid cells

• CAR-Macrophage Development:

  • Utilize LILRB5 antibody-derived binding domains in chimeric antigen receptors

  • Engineer macrophages with these constructs for targeted phagocytosis

  • The selective expression pattern of LILRB5 on myeloid cells makes this receptor particularly suitable for myeloid cell engineering approaches

• Antibody-Drug Conjugates:

  • Leverage biotin-streptavidin chemistry for modular assembly of LILRB5-targeted drug conjugates

  • Develop conjugates delivering immunomodulatory compounds to LILRB5-expressing cells

  • This strategy could enable selective drug delivery to myeloid populations

• Single-Cell Multiomics Integration:

  • Combine biotin-conjugated antibody detection with single-cell RNA/ATAC-seq

  • Correlate LILRB5 surface expression with transcriptional and epigenetic states

  • This could identify novel regulatory networks influenced by LILRB5 signaling

• In Vivo Imaging Applications:

  • Utilize biotin-conjugated antibodies with streptavidin-coupled imaging agents

  • Monitor LILRB5-expressing cell trafficking and localization in disease models

  • This approach could provide insights into myeloid cell dynamics during immune responses

• Nanobody and Alternative Scaffold Development:

  • Engineer smaller binding domains derived from LILRB5 antibodies

  • Improve tissue penetration while maintaining targeting specificity

  • Biotin-conjugation of these smaller formats enables versatile detection systems

These technologies could help address the currently unexplored immunotherapeutic potential of LILRB5, building on findings that LILRB5 activation significantly modulates cytokine production and MHC class I expression . Given the observed inhibitory effect of related receptors on T cell proliferation, LILRB5-targeted approaches might offer novel strategies for treating inflammatory and autoimmune conditions.

What are the key unresolved questions regarding LILRB5's biological functions that could be addressed using biotin-conjugated antibodies?

Despite advances in understanding LILRB5, several key questions remain unresolved that could be addressed using biotin-conjugated antibodies:

• Ligand Specificity Beyond MHC Class I:

  • While studies confirm LILRB5 binding to MHC class I heavy chains , other potential ligands remain unexplored

  • Research question: Does LILRB5 recognize non-classical MHC molecules or stress-induced ligands?

  • Methodology: Use biotin-conjugated antibodies in binding inhibition assays with candidate ligands

• Tissue-Specific Expression Patterns:

  • Current knowledge of LILRB5 expression is largely limited to peripheral blood monocytes

  • Research question: How does LILRB5 expression vary across tissue-resident myeloid populations?

  • Methodology: Apply biotin-conjugated antibodies in multi-parameter immunohistochemistry of tissue sections

• Developmental Regulation:

  • Little is known about when LILRB5 expression is initiated during myeloid differentiation

  • Research question: How is LILRB5 expression regulated during monocyte/macrophage development?

  • Methodology: Use biotin-conjugated antibodies to track expression during in vitro differentiation models

• Regulatory T Cell Interactions:

  • LILRB5's effects on T cell responses may involve regulatory T cells

  • Research question: Does LILRB5 signaling modulate Treg generation or function?

  • Methodology: Co-culture LILRB5-activated monocytes with T cells and analyze Treg induction

• Cross-talk with Other Inhibitory Receptors:

  • Potential synergistic effects with other inhibitory receptors remain unexplored

  • Research question: How does LILRB5 functionally interact with other inhibitory receptors like PD-1/PD-L1?

  • Methodology: Use combinatorial antibody targeting approaches with biotin-conjugated LILRB5 antibodies

• Epigenetic Regulation of Target Genes:

  • Mechanisms by which LILRB5 upregulates MHC class I genes (by up to 174.8-fold) remain unclear

  • Research question: Does LILRB5 signaling induce epigenetic modifications at MHC loci?

  • Methodology: Combine biotin-conjugated antibody-mediated cell sorting with ChIP-seq analysis

• Role in Disease Pathogenesis:

  • LILRB5's contribution to infectious, autoimmune, or malignant diseases is poorly understood

  • Research question: How does LILRB5 expression or function change in disease states?

  • Methodology: Compare LILRB5 expression and signaling in samples from healthy and disease cohorts