FCGR3A Monoclonal Antibody,PE Conjugated

What is FCGR3A and what biological role does it play?

FCGR3A (Fc gamma receptor IIIa, also known as CD16A) is a receptor for the Fc portion of immunoglobulin G that plays crucial roles in immune function. It is primarily involved in the removal of antigen-antibody complexes from circulation and mediates antibody-dependent cellular responses. FCGR3A is expressed on natural killer (NK) cells, T cells, monocytes, and macrophages as an integral membrane glycoprotein anchored through a transmembrane peptide. Unlike its close relative FCGR3B (which is expressed on polymorphonuclear neutrophils), FCGR3A utilizes a transmembrane peptide for anchoring rather than a phosphatidylinositol (PI) linkage .

The receptor serves multiple immunological functions including phagocytosis, secretion of enzymes and inflammatory mediators, antibody-dependent cytotoxicity, and clearance of immune complexes. These functions make FCGR3A a critical component in both innate and adaptive immune responses .

What is the structural difference between FCGR3A and related receptors?

The extracellular domain of FCGR3A shares varying degrees of amino acid identity with paralogs in other species: 63% with mouse Fc gamma RIV, 61% with rat Fc gamma RIIIa, 65% with feline CD16, 59% with bovine CD16, and 58% with porcine Fc gamma RIIIb .

What are the key considerations for handling PE-conjugated FCGR3A antibodies?

When working with PE-conjugated FCGR3A monoclonal antibodies, researchers should follow these methodological guidelines:

• Storage: Store at 4°C and avoid freezing to maintain conjugate stability and fluorescence properties .

• Buffer composition: The antibody is typically provided in PBS buffer at pH 7.4 with 0.09% sodium azide and 0.2% BSA to maintain stability .

• Light sensitivity: Protect from prolonged exposure to light as PE conjugates are photosensitive.

• Working dilution: Determine optimal working dilution empirically for each specific application. Manufacturer recommendations provide starting points, but assay-specific optimization is essential .

What are the primary research applications for PE-conjugated FCGR3A monoclonal antibodies?

PE-conjugated FCGR3A monoclonal antibodies are valuable tools for multiple research applications:

• Flow Cytometry: The primary application for these conjugated antibodies is identifying and quantifying FCGR3A-expressing cells such as NK cells, monocytes, and certain T cell subsets. The PE fluorophore provides excellent signal intensity for detection .

• Immunofluorescence: Used to visualize FCGR3A expression in cell preparations or tissue sections. The bright fluorescence of PE makes it ideal for detecting expression patterns and co-localization studies .

• Immune Cell Phenotyping: Particularly useful in combination with other markers (such as CD56 for NK cells or CD14 for monocytes) to identify specific immune cell populations and subsets .

• Functional Studies: Can be employed to track changes in FCGR3A expression during immune responses, disease progression, or therapeutic interventions .

Methodology for flow cytometry application typically involves:

• Cell preparation (isolation of PBMCs or relevant cell populations)

• Blocking of Fc receptors to prevent non-specific binding

• Staining with optimized concentration of PE-conjugated anti-FCGR3A

• Co-staining with other lineage markers as needed

• Analysis on a flow cytometer with appropriate laser and filter settings for PE detection

How can FCGR3A monoclonal antibodies be used to study genetic polymorphisms?

FCGR3A exhibits important genetic polymorphisms, particularly a single nucleotide polymorphism that creates high binding (176V) and low binding (176F) forms. These variants, when homozygous, may influence susceptibility to autoimmune diseases or response to therapeutic IgG antibodies .

Methodological approach for studying these polymorphisms:

• Genotyping samples for the FCGR3A V176F polymorphism using PCR-based methods.

• Using PE-conjugated anti-FCGR3A antibodies to assess receptor expression levels by flow cytometry.

• Correlating expression levels with genotype data and clinical parameters.

• Functional assays to compare antibody binding capacity, NK cell activity, or ADCC potential between different genotypes.

This approach can be particularly valuable in studying responses to therapeutic antibodies and understanding individual variability in immune-mediated diseases .

What methods are recommended for analyzing FCGR3A expression in tissue samples?

For analyzing FCGR3A expression in tissue samples, immunohistochemistry or immunofluorescence approaches are recommended:

• Tissue Preparation:

  • For paraffin-embedded sections, perform heat-induced epitope retrieval using basic antigen retrieval reagents

  • For frozen sections, fixation with 4% paraformaldehyde is typically sufficient

• Staining Protocol:

  • Block endogenous peroxidase activity and non-specific binding

  • Incubate with primary anti-FCGR3A antibody (typically 3 μg/mL for 1-3 hours at room temperature)

  • Apply appropriate secondary detection system (HRP polymer or fluorescent secondary antibody)

  • Develop with DAB for brightfield visualization or use fluorescent detection systems

  • Counterstain with hematoxylin or DAPI as appropriate

• Analysis:

  • Assess distribution patterns, particularly focusing on lymphocytes and monocyte/macrophage populations

  • Quantify intensity and distribution using digital image analysis when possible

This methodology has been successfully applied to human spleen sections where FCGR3A expression was localized to lymphocytes .

How should researchers optimize staining protocols for PE-conjugated FCGR3A antibodies in flow cytometry?

Optimizing staining protocols for PE-conjugated FCGR3A antibodies requires systematic evaluation of multiple parameters:

• Titration Determination:

  • Perform antibody titration using 2-fold serial dilutions

  • Analyze signal-to-noise ratio at each concentration

  • Select the concentration that provides maximum positive signal with minimal background

  • Typical starting point is testing dilutions around manufacturer's recommendations with PBMCs

• Staining Buffer Optimization:

  • Standard buffers include PBS with 0.5-2% BSA or FBS

  • Addition of 0.1% sodium azide helps preserve samples

  • Test inclusion of 2mM EDTA if cells tend to aggregate

• Incubation Parameters:

  • Compare staining efficiency at different temperatures (4°C, room temperature)

  • Optimize incubation time (typically 15-30 minutes for surface staining)

  • Protect from light during incubation to preserve fluorophore

• Blocking Strategy:

  • Include Fc receptor blocking step (critical for FCGR3A since it binds IgG)

  • Use either commercial Fc block or normal IgG from the same species as the primary antibody

• Controls:

  • Isotype control matched to primary antibody's isotype (IgG1 for COC16 clone)

  • FMO (fluorescence minus one) controls to set proper gating

  • Cell lines known to be positive and negative for FCGR3A expression

What are the critical considerations for multiplexing FCGR3A antibodies with other markers?

When designing multiplex panels including PE-conjugated FCGR3A antibodies, researchers should consider these methodological aspects:

• Spectral Compatibility Planning:

  • Choose fluorochromes with minimal spectral overlap with PE (549/565nm)

  • Consider brightness hierarchy - assign brighter fluorochromes to less abundant markers

  • Validate key marker combinations, like FCGR3A(PE) with CD56(APC) for NK cells or FCGR3A(PE) with CD14(APC) for monocytes

• Compensation Requirements:

  • Prepare single-stained controls for each fluorochrome

  • Include unstained and isotype controls

  • Optimize compensation settings using positive populations with similar brightness to test samples

• Staining Sequence:

  • For complex panels, sequential staining may reduce interference

  • Consider antibody competition if targeting closely located epitopes

• Analysis Strategy:

  • Implement appropriate gating strategies for FCGR3A+ populations

  • Use biaxial plots with key lineage markers (CD56 for NK cells, CD14 for monocytes)

  • Consider examining FCGR3A expression levels (MFI) across different cell populations

A validated approach includes co-staining FCGR3A with NCAM-1/CD56 to identify NK cells or with CD14 to identify monocyte populations, as demonstrated in established protocols .

How can researchers accurately quantify FCGR3A expression levels across different cell populations?

To accurately quantify FCGR3A expression levels across different cell populations, researchers should follow these methodological steps:

• Standardization Approach:

  • Use calibration beads with known amounts of PE molecules

  • Convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Apply quality control standards across experiments

• Population Identification:

  • Implement a gating strategy that clearly defines cell populations

  • For NK cells: CD3-CD56+FCGR3A+

  • For monocytes: CD14+FCGR3A+

  • For macrophages: CD68+FCGR3A+

• Quantification Methods:

  • Report median fluorescence intensity (MFI) ratios relative to isotype controls

  • Calculate percentage of FCGR3A+ cells within each population

  • Consider density of expression (molecules per cell) when possible

• Statistical Analysis:

  • Use Mann-Whitney U tests to evaluate effects of FCGR3A copy numbers on expression levels

  • Implement FDR-adjusted P values for multiple comparisons

  • Consider regression models adjusted for covariates like sex and age

This approach has been successfully employed to demonstrate associations between FCGR3A copy number variations and expression levels in immune cells in the context of autoimmune diseases .

How can FCGR3A monoclonal antibodies be applied to study immune infiltration in cancer?

FCGR3A expression has been identified as a valuable marker for studying immune infiltration in various cancer types, particularly in low-grade gliomas (LGG). A systematic approach includes:

• Tumor Microenvironment Analysis:

  • Use flow cytometry with PE-conjugated FCGR3A antibodies to quantify infiltrating NK cells and macrophages in tumor samples

  • Compare FCGR3A expression between tumor tissue and adjacent normal tissue

  • Correlate expression with clinical parameters and outcomes

• Correlation with Immune Checkpoint Molecules:

  • Analyze relationships between FCGR3A expression and immune checkpoint molecules (PD-1, PD-L1, PD-L2, CTLA4, LAG-3, TIM-3)

  • Evaluate associations with tumor-associated macrophage (TAM) markers

  • Develop multiplexed immunophenotyping panels combining these markers

• Immune Infiltration Score Development:

  • Generate immune infiltration scores based on FCGR3A expression and other immune markers

  • Correlate scores with tumor purity, B cell infiltration, T cell subsets, macrophage levels, neutrophil levels, and dendritic cell levels

  • Apply these scores to predict prognosis and therapeutic responses

Research has demonstrated that FCGR3A expression positively correlates with various immune cell infiltration levels in LGG and 30 other cancer types, suggesting its potential as a prognostic biomarker .

What is the role of FCGR3A copy number variations in autoimmune diseases, and how can it be studied?

FCGR3A copy number variations (CNVs) have been implicated in autoimmune diseases like systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). A comprehensive methodological approach includes:

• CNV Determination:

  • Use quantitative PCR or digital droplet PCR to determine FCGR3A copy number

  • Validate findings with orthogonal methods like MLPA (Multiplex Ligation-dependent Probe Amplification)

  • Categorize samples into low (<2 copies), normal (2 copies), and high (>2 copies) groups

• Association Analysis:

  • Compare CNV distribution between patients and healthy controls

  • Calculate odds ratios for disease risk for each CNV category

  • Implement logistic regression models adjusted for sex and age

  • Apply FDR correction for multiple testing

• Clinical Correlation:

  • Investigate relationships between CNVs and specific disease manifestations

  • Analyze impact on treatment responses and disease progression

  • Evaluate interactions with other genetic risk factors

Research has shown significant associations between FCGR3A CNVs and both SLE (OR 3.26 for <2 copies vs. 2 copies) and RA (OR 2.82 for <2 copies vs. 2 copies). Notably, both low and high copy numbers of FCGR3A are associated with SLE risk, suggesting complex disease mechanisms .

How can researchers investigate the functional differences between FCGR3A polymorphic variants?

FCGR3A exhibits a functionally significant polymorphism (V176F) that affects binding affinity for IgG. To investigate functional differences between these variants:

• Genotype-Phenotype Correlation Study Design:

  • Genotype samples for V176F polymorphism using allele-specific PCR or sequencing

  • Isolate NK cells from individuals with different genotypes

  • Use PE-conjugated FCGR3A antibodies to quantify receptor expression levels

  • Perform functional assays to measure antibody binding capacity

• Antibody-Dependent Cellular Cytotoxicity (ADCC) Assays:

  • Compare ADCC efficiency between NK cells from individuals with different FCGR3A genotypes

  • Measure cytotoxicity against antibody-coated target cells

  • Analyze dose-response relationships with therapeutic antibodies

  • Quantify cytokine production during ADCC responses

• Therapeutic Antibody Response Prediction:

  • Analyze clinical responses to therapeutic IgG antibodies based on FCGR3A genotype

  • Develop predictive models incorporating both genotype and expression level data

  • Test receptor binding affinity using surface plasmon resonance or flow cytometry

This approach can provide insights into why individuals with specific FCGR3A genotypes show different susceptibilities to autoimmune diseases or varying responses to therapeutic antibodies like rituximab .

What are common issues in FCGR3A detection by flow cytometry and how can they be resolved?

When detecting FCGR3A using PE-conjugated antibodies in flow cytometry, researchers may encounter several technical challenges:

For specific resolution of weak or inconsistent FCGR3A staining:

• Ensure proper sample preparation - fresh samples typically provide better results than frozen

• Optimize fixation conditions - excessive fixation can mask FCGR3A epitopes

• Consider enzymatic pre-treatment if working with tissue samples or certain cell types

• Validate results with alternative detection methods or antibody clones

How should researchers approach validation of FCGR3A antibody specificity?

Proper validation of FCGR3A antibody specificity is critical for research reliability and reproducibility:

• Cross-Reactivity Testing Protocol:

  • Test antibody against cell lines with known FCGR3A expression status

  • Include positive controls (NK cells, monocytes) and negative controls (A549 human lung carcinoma cells)

  • Perform parallel staining with multiple anti-FCGR3A antibody clones targeting different epitopes

  • Verify results with genetic knockdown/knockout systems when possible

• Specificity Differentiation from FCGR3B:

  • Use cell-specific markers to distinguish FCGR3A (NK cells, monocytes) from FCGR3B (neutrophils)

  • Employ epitope-specific antibodies that can distinguish between the highly similar FCGR3A and FCGR3B proteins

  • Validate with genetic information when available

• Verification Methods:

  • Western blotting to confirm appropriate molecular weight (50-70 kDa)

  • Competitive binding assays with unlabeled antibody

  • Peptide blocking experiments with the immunizing peptide

  • Correlation of protein detection with mRNA expression data

What methodological approaches should be used to study soluble FCGR3A in clinical samples?

The extracellular domain of FCGR3A can be proteolytically cleaved and retain binding activity in soluble form. To study soluble FCGR3A in clinical samples:

• Sample Collection and Processing:

  • Collect plasma or serum using standardized protocols

  • Process samples within recommended timeframes to prevent artificial elevation of soluble receptors

  • Store aliquots at -80°C to avoid freeze-thaw cycles

• Quantification Methods:

  • Enzyme-linked immunosorbent assay (ELISA) specific for soluble FCGR3A

  • Multiplex bead-based assays for simultaneous measurement of multiple soluble receptors

  • Immunoprecipitation followed by western blotting for molecular characterization

• Clinical Correlation Analysis:

  • Compare levels between healthy controls and disease groups

  • Analyze relationship with disease activity markers

  • Evaluate potential as a biomarker for conditions like rheumatoid arthritis or coronary artery disease

• Functional Assessment:

  • Test the ability of soluble FCGR3A to compete with cell-bound receptors for immune complexes

  • Evaluate potential immunomodulatory effects on NK cell and monocyte functions

  • Analyze correlation between soluble receptor levels and cellular expression of membrane-bound FCGR3A

Research indicates that soluble FCGR3A levels are increased in rheumatoid arthritis and coronary artery diseases, suggesting potential diagnostic or prognostic utility in these conditions .