C5AR1 Antibody

What is C5AR1 and why is it significant in immunological research?

C5AR1 (Complement C5a Receptor 1), also known as CD88, is a G protein-coupled receptor that binds to complement anaphylatoxin C5a. The protein is approximately 39.3 kilodaltons in mass and functions as a critical mediator in inflammatory responses . The significance of C5AR1 in research stems from its role as a potent pro-inflammatory inducer that regulates numerous cellular functions through complement system activation . Studies targeting C5AR1 have implications for understanding inflammatory disorders, innate immunity, and potential therapeutic interventions for complement-mediated diseases. This receptor is expressed on myeloid cells, endothelial cells, epithelial cells, and smooth muscle cells, making it an important research target across multiple physiological systems .

What are the most reliable methods to verify C5AR1 antibody specificity?

Verification of C5AR1 antibody specificity requires multiple validation approaches:

• Cell Line Validation: Test antibodies on both positive cell lines (e.g., U937 human histiocytic lymphoma cells) and negative control lines (e.g., SH-SY5Y neuroblastoma cells) that differentially express C5AR1 .

• Transfection Studies: Evaluate antibody binding in HEK293 cells transfected with human C5AR1 versus cells transfected with irrelevant proteins and reporter genes like eGFP .

• Direct ELISA Testing: Perform direct ELISA against the immunogen and related peptides to confirm epitope specificity .

• Tissue Cross-Reactivity: Test antibody staining patterns in tissues known to express C5AR1 (e.g., human spleen) using appropriate controls .

• Peptide Blocking: Pre-incubate the antibody with immunizing peptides to confirm binding specificity through signal reduction .

Researchers should document all validation steps in their experimental protocols and select antibodies with comprehensive validation data for their specific applications.

How do monoclonal and polyclonal C5AR1 antibodies differ in research applications?

When selecting between these antibody types, researchers should consider whether epitope-specific recognition (monoclonal) or broader protein detection (polyclonal) better serves their experimental goals.

What are the optimal protocols for using C5AR1 antibodies in flow cytometry?

Optimal flow cytometry protocols for C5AR1 antibody staining should follow these methodological steps:

• Sample Preparation:

  • For whole blood: Use 100 μL of fresh human whole blood per test

  • For isolated cells: Prepare 1×10^6 cells (neutrophils, monocytes, or transfected cell lines) per sample

• Staining Protocol:

  • Block Fc receptors with 2% normal serum for 15 minutes at 4°C

  • Incubate with primary anti-C5AR1 antibody (5-10 μg/mL, e.g., clone 347234) for 30 minutes at 4°C

  • Wash twice with PBS containing 0.5% BSA and 0.1% sodium azide

  • For unconjugated antibodies: Incubate with fluorochrome-conjugated secondary antibody (e.g., Allophycocyanin-conjugated Anti-Mouse IgG) for 30 minutes at 4°C

  • Wash twice and resuspend in appropriate buffer for analysis

• Controls:

  • Include isotype controls (e.g., Mouse IgG1κ isotype)

  • Use known C5AR1-positive (e.g., U937) and C5AR1-negative cell populations

  • For transfected cells, include non-transfected controls and reporter gene (e.g., eGFP) markers for gating

• Gating Strategy:

  • Set quadrant markers based on control antibody staining

  • For whole blood analysis, use forward/side scatter to identify neutrophil and monocyte populations before assessing C5AR1 expression

This methodology allows for reliable quantification of C5AR1 surface expression across different cell populations.

How can researchers optimize C5AR1 antibody staining for immunohistochemistry?

For optimal immunohistochemistry staining of C5AR1, researchers should implement this methodological approach:

• Tissue Preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness)

  • For human samples, tissues with known C5AR1 expression (e.g., spleen) are recommended

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval using basic buffer (pH 9.0)

  • Recommended protocol: 20 minutes at 95-98°C in appropriate retrieval solution

• Staining Protocol:

  • Block endogenous peroxidase with 0.3% H₂O₂

  • Apply protein blocking solution (5-10% normal serum)

  • Incubate with primary C5AR1 antibody (5-8 μg/mL) for 1 hour at room temperature or overnight at 4°C

  • Use detection system appropriate for the primary antibody species (e.g., Anti-Mouse IgG HRP Polymer for mouse monoclonal antibodies)

  • Develop with DAB substrate and counterstain with hematoxylin

• Controls and Validation:

  • Include negative controls (isotype-matched antibodies)

  • Use positive control tissues (human spleen sections show consistent staining in splenocytes)

  • Expected staining pattern: Cytoplasmic localization in C5AR1-positive cells

• Troubleshooting Guidance:

  • For weak staining: Increase antibody concentration or extend incubation time

  • For high background: Optimize blocking or reduce primary antibody concentration

  • For inconsistent results: Ensure proper antigen retrieval and consistent fixation times

This protocol maximizes sensitivity while maintaining specificity for C5AR1 detection in tissue specimens.

What functional assays can effectively evaluate C5AR1 antibody blocking efficiency?

The following functional assays provide robust methodologies to evaluate C5AR1 antibody blocking efficiency:

• C5a-Induced Calcium Flux Assay:

  • Isolate human neutrophils from peripheral blood

  • Load cells with calcium-sensitive fluorescent dye (e.g., Fluo-4 AM)

  • Pre-incubate with C5AR1 blocking antibody (10-50 μg/mL) for 15-30 minutes

  • Stimulate with recombinant human C5a (10-100 nM)

  • Measure calcium flux by fluorescence microscopy or plate reader

  • Effective blocking antibodies will significantly reduce the calcium flux response

• Neutrophil Activation Marker Analysis:

  • Treat whole blood or isolated neutrophils with C5AR1 antibody

  • Stimulate with C5a or Escherichia coli

  • Assess upregulation of activation markers CD11b and CD66b by flow cytometry

  • Quantify the reduction in marker expression compared to non-blocked controls

• iLite® C5a Assay:

  • Utilize the standardized reporter cell line system

  • Pre-incubate cells with serial dilutions of C5AR1 antibody

  • Add C5a stimulant according to manufacturer's protocol

  • Measure luciferase activity as indicator of C5aR1 signaling

  • Calculate IC50 values to compare different antibody clones

• Chemotaxis Assay:

  • Use Transwell migration chambers

  • Place neutrophils in upper chamber pre-treated with C5AR1 antibody

  • Add C5a to lower chamber as chemoattractant

  • Quantify migrated cells after 1-2 hours

  • Express results as percentage inhibition of migration

These methodological approaches provide complementary data on the functional blocking capacity of C5AR1 antibodies in different physiological contexts.

How can researchers distinguish between C5AR1 and C5AR2 (C5L2) in experimental systems?

Distinguishing between C5AR1 and C5AR2 (C5L2) requires careful methodological approaches:

• Antibody Selection and Validation:

  • Use antibodies specifically validated for distinguishing between these receptors

  • Perform specificity testing on C5aR1his- and C5aR2his-expressing Flp-InTM-CHO cells

  • Confirm non-cross-reactivity through ELISA against receptor-specific peptides

• Functional Discrimination:

  • C5AR1 mediates calcium mobilization while C5AR2 does not

  • Perform calcium flux assays to identify C5AR1-specific responses

  • C5AR1 couples to G-proteins while C5AR2 preferentially recruits β-arrestin

  • Use G-protein versus β-arrestin recruitment assays to differentiate receptor activity

• Molecular Approaches:

  • Design PCR primers specific to unique regions of each receptor

  • Use receptor-specific siRNA knockdown to confirm antibody specificity

  • Employ receptor-selective antagonists (e.g., PMX-53 or W-54011 for C5AR1)

• Expression Pattern Analysis:

  • Map cell type-specific expression patterns (C5AR1 is more broadly expressed)

  • Compare subcellular localization (C5AR1 predominantly on cell surface, C5AR2 more intracellular)

This systematic approach ensures accurate discrimination between these related but functionally distinct complement receptors in experimental systems.

What are the critical considerations when generating new C5AR1 monoclonal antibodies?

The generation of new C5AR1 monoclonal antibodies requires careful attention to several critical factors:

• Immunogen Design:

  • Target specific extracellular domains of C5AR1 to ensure accessibility

  • Consider using synthetic peptides corresponding to key regions:

    • N-terminal region (aa 1-36): MNSFNYTTPDYGHYDDFDTLDLNTPVDKTSNTLRVP

    • First extracellular loop (aa 90-109): PILFTSIVQHHHWPFGGAA

    • Second extracellular loop (aa 175-188): RVVREEYFPPKVL

    • Third extracellular loop (aa 266-282): SFLEPSSPTFLLLNKLD

  • Conjugate peptides to carrier proteins (e.g., diphtheria toxoid) to enhance immunogenicity

• Hybridoma Selection Strategy:

  • Implement multi-step screening process:

    • Initial ELISA screening against immunizing peptide

    • Secondary screening for whole blood cell binding

    • Functional screening using calcium flux or reporter assays

  • Select hybridomas producing antibodies that bind native receptor conformation

• Antibody Characterization Requirements:

  • Determine antibody isotype and subclass

  • Map precise epitope recognition

  • Assess cross-reactivity with related receptors

  • Evaluate species cross-reactivity potential

  • Characterize binding affinity through kinetic analyses

• Functional Validation:

  • Test for agonistic versus antagonistic properties

  • Evaluate capacity to block C5a-induced cellular responses

  • Compare performance against established antibodies (e.g., S5/1, 347214, avdoralimab)

  • Generate F(ab')₂ fragments to assess Fc-independent functions

These methodological considerations are essential for developing C5AR1 antibodies with desired specificity and functional properties for research applications.

How can researchers investigate the role of C5AR1 in disease models using antibody-based approaches?

Investigating C5AR1 in disease models using antibody-based approaches requires systematic experimental design:

• Model Selection and Characterization:

  • Choose appropriate disease models with known complement involvement

  • Establish baseline C5AR1 expression in relevant tissues using immunohistochemistry

  • Characterize temporal changes in C5AR1 expression during disease progression

• In Vivo Blocking Studies:

  • Select antibodies with demonstrated in vitro blocking efficiency

  • Consider using F(ab')₂ fragments to eliminate Fc-mediated effects

  • Establish dose-response relationships and therapeutic windows

  • Determine optimal administration routes and schedules

  • Include isotype-matched control antibodies in parallel groups

• Cellular Mechanism Investigation:

  • Use flow cytometry to identify cell populations affected by antibody treatment

  • Assess changes in activation markers (CD11b, CD66b) on neutrophils and monocytes

  • Perform ex vivo functional assays with cells isolated from treated animals

  • Combine with cell-specific depletion studies to determine cellular targets

• Outcome Assessment:

  • Measure disease-specific endpoints (e.g., inflammatory markers, tissue damage)

  • Assess systemic complement activation (C5a levels, terminal complement complex)

  • Evaluate potential compensatory mechanisms through transcriptional analysis

  • Monitor for unexpected consequences of complement modulation

This comprehensive approach allows researchers to establish causal relationships between C5AR1 inhibition and disease outcomes while elucidating underlying cellular mechanisms.

How should researchers address inconsistent C5AR1 antibody staining patterns across different experimental systems?

When encountering inconsistent C5AR1 antibody staining, researchers should systematically troubleshoot using this methodological framework:

• Sample Preparation Variables:

  • Fixation method: Overfixation can mask C5AR1 epitopes; compare methanol vs. paraformaldehyde fixation results

  • Membrane permeabilization: Optimize detergent concentration and incubation time

  • Antigen retrieval: Test different pH buffers and heating protocols

  • Fresh vs. frozen vs. FFPE samples: Determine optimal sample preparation for your antibody

• Antibody Selection Considerations:

  • Epitope accessibility: Antibodies targeting different domains may perform differently across applications

  • Clone-specific characteristics: Compare multiple validated clones (e.g., 347234, 18-41-6)

  • Format suitability: Some conjugated antibodies may lose activity while unconjugated versions perform well

• Biological Variation Factors:

  • C5AR1 expression levels: Verify baseline expression in your model system

  • Receptor internalization: C5a stimulation can cause rapid internalization, altering staining patterns

  • Cell activation status: Inflammatory stimuli can significantly upregulate C5AR1

  • Species differences: Human-specific antibodies may not recognize orthologous epitopes

• Technical Validation Approach:

  • Implement positive and negative controls in every experiment

  • Use multiple detection methods to cross-validate findings

  • Perform blocking experiments with immunizing peptides

  • Correlate results with functional readouts of C5AR1 activity

This systematic approach helps identify the source of inconsistency and establish reliable protocols for C5AR1 detection across different experimental systems.

What are the most common pitfalls in interpreting C5AR1 antibody-based experimental data?

Researchers should be aware of these common pitfalls when interpreting C5AR1 antibody-based data:

• Receptor Modulation Misinterpretation:

  • C5AR1 rapidly internalizes upon C5a binding, potentially leading to false-negative results

  • Surface vs. total cellular expression may yield contradictory findings

  • Recommendation: Include time-course studies and assess both surface and intracellular staining

• Cross-Reactivity Concerns:

  • Antibodies may detect related G-protein coupled receptors

  • C5AR1 and C5AR2 share structural similarities leading to potential cross-reactivity

  • Recommendation: Validate specificity using C5AR1-deficient cells or C5AR2-expressing controls

• Functional Blocking Variability:

  • Antibody concentration affects blocking efficiency

  • F(ab')₂ vs. whole IgG may yield different results due to Fc receptor interactions

  • Recommendation: Establish dose-response curves and include appropriate controls

• Cell Type-Specific Differences:

  • C5AR1 glycosylation patterns vary across cell types, affecting antibody binding

  • Expression density influences detectable signal and functional outcomes

  • Recommendation: Validate findings across multiple cell types relevant to your research question

• Technical Artifacts Table:

Awareness of these pitfalls enables more accurate interpretation of C5AR1-related experimental findings.

How can researchers integrate C5AR1 antibody data with other methodologies to build comprehensive mechanistic models?

Integrating C5AR1 antibody data with complementary methodologies creates robust mechanistic models through this systematic approach:

• Multi-omics Integration Strategy:

  • Correlate antibody-detected C5AR1 protein levels with transcriptomic data

  • Combine with phosphoproteomic analysis of downstream signaling pathways

  • Integrate with metabolomic profiling to identify functional consequences

  • Use systems biology approaches to model C5AR1 signaling networks

• Functional Validation Framework:

  • Pair antibody blocking studies with genetic approaches (siRNA, CRISPR-Cas9)

  • Compare antibody antagonism with small molecule inhibitors (e.g., W-54011, PMX-53, avacopan)

  • Correlate in vitro findings with in vivo phenotypes in relevant disease models

  • Use calcium flux and activation marker assessments to confirm functional outcomes

• Spatial-Temporal Analysis Methods:

  • Combine flow cytometry quantification with imaging to determine cellular localization

  • Implement time-course studies to capture dynamic receptor expression changes

  • Use tissue-specific approaches to map C5AR1 expression across organ systems

  • Develop in vivo imaging using labeled antibodies to track C5AR1-expressing cells

• Translational Research Approaches:

  • Compare findings in experimental models with human patient samples

  • Assess how C5AR1 blocking antibodies affect patient-derived cells

  • Correlate C5AR1 expression patterns with clinical outcomes

  • Evaluate potential biomarkers associated with C5AR1 activity

How are C5AR1 antibodies being utilized to understand the crosstalk between complement and other inflammatory pathways?

C5AR1 antibodies are enabling sophisticated investigations into complement-inflammation crosstalk through these methodological approaches:

• Neutrophil Extracellular Trap (NET) Formation Studies:

  • Use C5AR1 blocking antibodies to assess NET formation in response to various stimuli

  • Combine with inhibitors of other pathways (TLR, NLRP3) to identify synergistic interactions

  • Quantify NETs using immunofluorescence and DNA quantification assays

  • Determine how C5AR1 signaling integrates with other neutrophil activation pathways

• Macrophage Polarization Analysis:

  • Apply C5AR1 antibodies to examine how complement affects M1/M2 polarization

  • Assess changes in polarization markers by flow cytometry and qPCR

  • Combine with cytokine blockade to delineate hierarchical relationships

  • Map temporal relationships between C5AR1 activation and polarization changes

• Adaptive Immunity Modulation:

  • Investigate how C5AR1 blockade affects T cell activation in co-culture systems

  • Examine dendritic cell antigen presentation following C5AR1 targeting

  • Assess B cell responses in the presence of C5AR1 blocking antibodies

  • Determine how complement-immune cell crosstalk shapes adaptive responses

• Cell-Specific Signaling Pathway Integration:

  • Use phospho-specific flow cytometry to map signaling pathway activation

  • Identify common signaling nodes between C5AR1 and other inflammatory receptors

  • Apply small molecule inhibitors with C5AR1 antibodies to dissect pathway interactions

  • Develop computational models predicting pathway crosstalk based on experimental data

These approaches reveal how C5AR1 functions as a critical node connecting complement activation with broader inflammatory networks in various disease contexts.

What novel applications of C5AR1 antibodies are emerging in the field of precision medicine?

Emerging applications of C5AR1 antibodies in precision medicine span multiple domains:

• Patient Stratification Methodologies:

  • Develop flow cytometry panels to quantify C5AR1 expression on circulating leukocytes

  • Correlate expression levels with disease severity and treatment response

  • Identify C5AR1 expression patterns that predict complement-targeted therapy efficacy

  • Create standardized protocols for clinical laboratory implementation

• Companion Diagnostic Development:

  • Use C5AR1 antibodies to assess target engagement of complement inhibitors

  • Design immunohistochemistry protocols to evaluate tissue-specific C5AR1 expression

  • Develop blood-based assays measuring C5AR1 activation status

  • Correlate C5AR1 blockade with clinical response metrics

• Targeted Therapy Approaches:

  • Engineer bispecific antibodies linking C5AR1 targeting with cytotoxic functions

  • Develop antibody-drug conjugates targeting C5AR1-expressing cells

  • Create C5AR1 antibody fragments with enhanced tissue penetration

  • Combine with emerging gene editing technologies for precision targeting

• Personalized Monitoring Systems:

  • Establish longitudinal monitoring protocols using standardized C5AR1 assessment

  • Develop algorithms incorporating C5AR1 status with other biomarkers

  • Create point-of-care testing using simplified C5AR1 detection methods

  • Implement machine learning approaches to predict disease flares based on C5AR1 data

These methodological approaches represent significant advances in translating fundamental C5AR1 biology into clinically actionable information for precision medicine applications.