CRA1 Antibody

What is the CRA1 antibody and what is its target antigen?

The CRA1 (AER-37) monoclonal antibody specifically recognizes and binds to the FcεRIα subunit, which is the IgE-binding component of the high-affinity IgE receptor. This receptor subunit lacks signal-transducing ability on its own but is essential for IgE binding. The FcεRIα is primarily expressed on mast cells and basophils and plays a crucial role in mediating allergic responses. The expression of FcεRIα is notably upregulated in the presence of IgE, creating a positive feedback mechanism in allergic conditions .

The antibody has been well-characterized in terms of its binding properties. CRA1 binds to a region on FcεRIα that does not overlap with the IgE binding site, making it valuable for studies where you need to detect the receptor without interfering with its natural IgE-binding function . Specifically, the epitope recognized by CRA1 has been mapped to amino acids 1-84 of the FcεRIα protein .

What is the molecular structure of the FcεRI receptor complex that CRA1 recognizes?

The high-affinity IgE receptor (FcεRI) that contains the CRA1 target exists as a tetrameric complex on the cell surface. This complex consists of:

• One α subunit (the direct target of CRA1 antibody)

• One β subunit

• Two γ subunits

The β and γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs) that are essential for signal transduction following receptor cross-linking. While the α subunit binds IgE with high affinity, it requires association with the other subunits to trigger downstream signaling pathways that lead to degranulation and mediator release in allergic reactions . This structural arrangement makes the FcεRI complex a central player in IgE-mediated allergic responses, as it effectively couples allergen recognition to mast cell and basophil activation.

What are the validated applications for CRA1 antibody in research?

The CRA1 (AER-37) antibody has been validated for several research applications, with flow cytometric analysis being the most thoroughly documented. The antibody has been pre-titrated and extensively tested for flow cytometry on peripheral blood cells . The recommended usage is ≤1 μg per test or specifically 5 μL (0.125 μg) per test when using commercially prepared solutions. A test is defined as the amount of antibody required to stain a cell sample in a final volume of 100 μL .

While flow cytometry represents the primary validated application, researchers have also adapted the CRA1 antibody for Western blotting, ELISA, immunohistochemistry, and immunofluorescence with appropriate optimization . When considering these additional applications, it's important to note that each laboratory should perform validation studies to confirm antibody performance in their specific experimental systems.

How can CRA1 antibody be used in combination with other antibodies for comprehensive analysis of the FcεRI receptor?

A particularly powerful research approach involves using CRA1 in combination with the CRA2 (AER24) monoclonal antibody. While CRA1 binds to a non-IgE-binding region of FcεRIα, CRA2 specifically recognizes the IgE binding site and competes with IgE for receptor binding . This complementary binding profile enables researchers to develop sophisticated analytical methodologies.

By using both antibodies in parallel experiments, researchers can quantitatively measure:

• Total FcεRIα expression (using CRA1, which binds regardless of IgE occupancy)

• Free/unoccupied FcεRIα (using CRA2, which only binds receptors not occupied by IgE)

• IgE-bound FcεRIα (calculated by subtracting CRA2 binding from CRA1 binding)

This dual-antibody approach provides critical insights into receptor occupancy states during allergic responses and can help evaluate the efficacy of therapeutic interventions targeting IgE-mediated pathways .

What are the optimal storage and handling conditions for maintaining CRA1 antibody activity?

To maintain optimal activity of the CRA1 antibody, researchers should adhere to specific storage and handling guidelines. The antibody is typically shipped at either 4°C or -20°C, but for long-term storage, it should be maintained at -20°C . Commercial preparations are often supplied as a purified monoclonal antibody at a concentration of 1mg/ml in PBS (pH 7.4) with 50% glycerol. These preparations are filter-sterilized and azide-free to ensure compatibility with functional assays .

How can researchers validate the specificity of CRA1 antibody in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For CRA1 antibody, multiple approaches can be employed:

• Cross-blocking studies: CRA1 (AER-37) and IE7 antibodies have been shown to bind the same or closely positioned epitopes, as demonstrated by mutual cross-blocking . Researchers can use this property to perform competitive binding assays as a specificity control.

• Flow cytometry on known positive and negative cell populations: CRA1 should show positive staining on mast cells and basophils (which express FcεRIα) but not on lymphocytes or other cell types that lack the receptor.

• Western blot analysis: When performed under reducing conditions using SDS-PAGE, CRA1 should recognize a protein band corresponding to the molecular weight of FcεRIα (~45-60 kDa, depending on glycosylation).

• RNA interference or knockout controls: Cells with FcεRIα knocked down or knocked out should show reduced or absent staining with CRA1 antibody.

• Recombinant protein controls: Since CRA1 was generated against the recombinant extracellular portion of human FcεRIα , this recombinant protein can be used as a positive control or in pre-absorption studies.

How can CRA1 antibody be utilized in studies of allergic response mechanisms?

The CRA1 antibody serves as a valuable tool for investigating the molecular mechanisms underlying allergic responses. Since FcεRI is the primary initiator of allergic reactions, researchers can use CRA1 to:

• Quantify receptor expression levels: Flow cytometric analysis with CRA1 allows precise measurement of FcεRIα expression on mast cells and basophils in different disease states or following experimental treatments .

• Monitor receptor regulation: Since FcεRIα expression is upregulated by IgE, CRA1 can be used to track receptor dynamics during allergic sensitization or desensitization protocols.

• Study receptor internalization: By combining CRA1 with pH-sensitive fluorophores or internalization assays, researchers can monitor receptor trafficking following activation.

• Identify FcεRI-expressing cells in tissues: Using CRA1 for immunohistochemistry or immunofluorescence enables mapping of FcεRI-expressing cells in tissue samples from allergic patients or experimental models.

• Assess therapeutic interventions: CRA1 can be used to evaluate how potential therapeutic compounds affect FcεRI expression, which is a critical parameter in allergic disease management .

What approaches can be used to develop engineered versions of CRA1 for specialized research applications?

Researchers have successfully created modified versions of CRA1 (AER-37) for specialized applications. One documented approach involved generating a chimeric human IgG1 version of AER-37 through molecular engineering:

• Cloning strategy: Synthetic coding DNA fragments containing the variable regions of AER-37 were subcloned into expression vectors (such as pEE12.4) .

• Cell transfection and selection: Transfected cells were seeded at low density to obtain clones derived from single colonies, which were then visually identified .

• Screening approach: Clones were selected based on the amount of AER-37 detected in the culture medium by ELISA .

This chimeric antibody approach offers several advantages, including reduced immunogenicity in humanized systems, potential for effector function modulation through isotype selection, and compatibility with human Fc receptor systems. Similar engineering strategies could be employed to create Fab fragments, single-chain variable fragments (scFv), or bispecific antibodies incorporating the CRA1 binding specificity for specialized research applications.

What is the optimal protocol for using CRA1 in flow cytometric analysis of human basophils?

For flow cytometric analysis of human basophils using CRA1 antibody, the following optimized protocol is recommended:

• Sample preparation:

  • Collect peripheral blood in anticoagulant (EDTA or heparin)

  • Isolate peripheral blood cells through density gradient centrifugation or use whole blood lysis approach

  • Wash cells twice in flow cytometry buffer (PBS with 2% FBS and 0.1% sodium azide)

  • Adjust cell concentration to 1-5 × 10^6 cells/ml

• Staining procedure:

  • Aliquot 100 μl of cell suspension (1-5 × 10^5 cells) per tube

  • Add ≤1 μg (or 5 μL/0.125 μg of commercial preparation) of CRA1 antibody

  • Include appropriate isotype control (mouse IgG2b) in a separate tube

  • For multicolor analysis, include additional markers such as CD123 and HLA-DR (to identify basophils as CD123+ HLA-DR−)

  • Incubate for 30 minutes at 4°C in the dark

  • Wash twice with flow cytometry buffer

  • Resuspend in 300-500 μl of buffer for acquisition

• Data acquisition and analysis:

  • Acquire at least 10,000 events in the basophil gate

  • Analyze FcεRIα expression as median fluorescence intensity

  • Compare to isotype control to determine specific binding

This protocol can be adapted for different fluorophore conjugates of CRA1, including APC (excitation: 633-647 nm; emission: 660 nm) .

How can researchers quantitatively measure IgE receptor occupancy using CRA1 and complementary antibodies?

A sophisticated approach to measure IgE receptor occupancy involves using CRA1 in combination with the CRA2 antibody. This dual-antibody strategy enables researchers to distinguish between total, occupied, and free FcεRI receptors:

• Experimental design:

  • Prepare three parallel samples from the same cell population

  • Sample 1: Stain with CRA1 to measure total FcεRIα (binds regardless of IgE occupancy)

  • Sample 2: Stain with CRA2 to measure unoccupied FcεRIα (CRA2 competes with IgE)

  • Sample 3: Stain with isotype control for background determination

• Quantitative analysis:

  • Total FcεRIα expression = CRA1 signal - isotype control

  • Unoccupied FcεRIα = CRA2 signal - isotype control

  • IgE-occupied FcεRIα = (CRA1 signal - isotype control) - (CRA2 signal - isotype control)

• Data representation:

  • Express receptor occupancy as percentage: (IgE-occupied FcεRIα / Total FcεRIα) × 100%

  • Create comparative analyses across different experimental conditions or patient samples

This methodological approach provides valuable insights into the dynamic relationship between IgE levels and receptor occupancy, which is particularly relevant for understanding allergic response mechanisms and evaluating anti-IgE therapeutic strategies .

What are common challenges when using CRA1 antibody and how can they be addressed?

Researchers may encounter several challenges when working with CRA1 antibody. Here are common issues and their solutions:

• Weak or absent signal in flow cytometry:

  • Ensure proper antibody titration; while ≤1 μg per test is recommended , optimal concentration may vary

  • Verify target expression in your cell population (basophils represent only 0.5-1% of peripheral blood)

  • Check for receptor downregulation due to sample processing (avoid activation)

  • Consider fresh samples, as receptor expression may decrease with extended storage

• High background staining:

  • Implement proper blocking steps (10% serum from the same species as secondary antibody)

  • Include Fc receptor blocking for peripheral blood cells

  • Ensure proper washing between steps

  • Use appropriate isotype controls (mouse IgG2b)

• Cross-reactivity concerns:

  • The CRA1 antibody is specific to human FcεRIα and shows cross-reactivity with human samples but not with other species

  • When working with mixed cell populations, use additional markers to identify specific cell types

• Variable results between experiments:

  • Standardize protocol parameters including incubation times and temperatures

  • Use internal controls for normalization between experiments

  • Consider batch effects of antibody preparations

By addressing these common challenges through methodical troubleshooting, researchers can optimize their experimental protocols for consistent and reliable results with CRA1 antibody.

How can real-time PCR be integrated with CRA1 antibody studies to correlate protein and mRNA expression?

Integrating real-time PCR analysis with CRA1 antibody studies provides a comprehensive view of FcεRIα regulation at both protein and mRNA levels. This combined approach is particularly valuable for understanding receptor modulation in response to experimental treatments or disease states:

• Experimental design for integrated analysis:

  • Split cell samples for parallel protein (flow cytometry with CRA1) and RNA analysis

  • Use standardized protocols for RNA isolation, such as the NucleoSpin RNA II extraction kit

  • Perform cDNA synthesis following established protocols

• Real-time PCR methodology:

  • Use validated probes and primers specific for human FcεRI-α (e.g., Hs00758599_m1) and FcεRI-β (e.g., Hs00175091_m1)

  • Include appropriate housekeeping genes (GAPDH) for normalization

  • Perform reactions on standard equipment (e.g., PRISM 7300 sequence detection system)

  • Quantify relative expression using the standard curve method

• Correlation analysis:

  • Plot CRA1 binding (protein expression by flow cytometry) against mRNA levels

  • Calculate correlation coefficients to determine relationship strength

  • Analyze time-course data to identify temporal relationships between mRNA and protein changes

This integrated approach has been successfully implemented to study FcεRI regulation after IgE stimulation, revealing insights into receptor dynamics. For example, research has shown that monomeric IgE stimulation (1.0 μg/ml for 24h) resulted in a relative FcεRI-β mRNA expression ratio of 1.017 ± 0.109 and a relative FcεRI-α mRNA expression ratio of 0.726 ± 0.027 .

How does CRA1 compare with other anti-FcεRIα antibodies in terms of binding properties and applications?

The CRA1 (AER-37) antibody has distinct properties that differentiate it from other anti-FcεRIα antibodies, particularly when compared to antibodies like IE7 and CRA2 (AER24):

The complementary binding properties of CRA1 and CRA2 make them particularly valuable when used in combination, enabling researchers to distinguish between free and IgE-occupied receptors – an approach that neither antibody alone can accomplish .

What are the considerations for selecting between different fluorophore conjugates of CRA1 for multicolor flow cytometry?

When designing multicolor flow cytometry panels incorporating CRA1 antibody, researchers must carefully consider the selection of fluorophore conjugates based on several technical factors:

• Available conjugates and their properties:

  • APC conjugate: Excitation 633-647 nm; Emission 660 nm; suitable for red laser detection

  • PE conjugate: Excitation 488-561 nm; Emission 578 nm; high brightness suitable for low-abundance targets

  • Other available conjugates may include FITC, PE-Cy7, and others (based on commercial availability)

• Panel design considerations:

  • Target abundance: FcεRIα expression levels vary based on cell type and activation state; brighter fluorophores (PE, APC) are recommended for detecting potentially low-expression states

  • Spectral overlap: Select conjugates to minimize compensation requirements with other markers in your panel

  • Laser configuration: Choose conjugates compatible with your cytometer's lasers and filter sets

• Experimental factors:

  • Autofluorescence: Consider tissue/cell-specific autofluorescence that may interfere with specific channels

  • Stability: Some conjugates may be more photostable or pH-stable than others

  • Signal-to-noise ratio: Compare different conjugates for optimal separation of positive and negative populations

• Titration requirements:

  • Each conjugate may require specific titration to determine optimal staining concentration

  • For APC conjugates, a starting recommendation is 5 μL (0.125 μg) per test

  • For other conjugates, follow manufacturer recommendations initially, then optimize

By carefully considering these factors, researchers can select the most appropriate CRA1 conjugate for their specific experimental design and cytometer configuration, ensuring optimal detection of FcεRIα in complex multicolor panels.

How might CRA1 antibody contribute to developing novel therapeutic approaches for allergic diseases?

The CRA1 antibody's specific binding properties make it a valuable tool for developing and evaluating therapeutic strategies targeting FcεRI-mediated allergic responses:

• Therapeutic target validation:

  • CRA1 can be used to quantify FcεRIα expression levels before and after experimental treatments

  • This allows for direct assessment of therapies designed to modulate receptor expression

  • The non-competing nature of CRA1 enables monitoring of receptor levels even in the presence of therapeutic IgE-targeting antibodies

• Monitoring receptor occupancy during immunotherapy:

  • When used in combination with CRA2, CRA1 enables quantitative measurement of receptor occupancy

  • This approach can track changes in IgE-FcεRI binding during allergen immunotherapy

  • Correlations between clinical improvement and receptor occupancy changes can identify optimal therapeutic protocols

• Development of targeted drug delivery systems:

  • Engineered versions of CRA1 could be developed as carriers for targeted delivery of drugs to FcεRIα-expressing cells

  • This approach could increase therapeutic efficacy while reducing systemic side effects

  • Chimeric versions of CRA1, such as the huIgG1 version already produced , provide platforms for such developments

• Screening platforms for drug discovery:

  • CRA1-based assays can be developed to screen compounds that modulate FcεRIα expression or function

  • High-throughput flow cytometry systems using CRA1 could identify novel anti-allergic compounds from chemical libraries

  • This approach has already contributed to studies on suppression of IgE-mediated anaphylaxis and food allergy

As allergic diseases continue to increase in prevalence globally, these CRA1-facilitated approaches may contribute significantly to the development of more effective and targeted therapeutic strategies.

What emerging technologies might enhance the research applications of CRA1 antibody?

Several cutting-edge technologies show promise for expanding the research applications of CRA1 antibody:

• Single-cell technologies:

  • Integration of CRA1 with single-cell RNA sequencing could reveal heterogeneity in FcεRIα-expressing cell populations

  • Mass cytometry (CyTOF) using metal-labeled CRA1 could enable high-dimensional analysis of receptor expression in relation to dozens of other parameters

  • Single-cell proteomics could correlate FcεRIα expression with broader proteomic signatures at individual cell resolution

• Advanced imaging approaches:

  • Super-resolution microscopy with fluorescently-labeled CRA1 could reveal nanoscale organization of FcεRIα on cell membranes

  • Intravital microscopy using CRA1 derivatives could track mast cell and basophil activation in living tissues

  • Correlative light and electron microscopy could connect FcεRIα distribution with ultrastructural features

• Biosensor development:

  • CRA1-based FRET (Förster Resonance Energy Transfer) sensors could enable real-time monitoring of receptor conformational changes

  • Surface plasmon resonance systems incorporating CRA1 could provide detailed kinetic analyses of receptor-ligand interactions

  • Engineered CRA1 fragments could be integrated into microfluidic devices for point-of-care allergy diagnostics

• Artificial intelligence integration:

  • Machine learning algorithms analyzing CRA1-based flow cytometry data could identify novel cell populations or disease signatures

  • Deep learning approaches could predict FcεRIα expression patterns from genomic or clinical data

  • AI-assisted image analysis could enhance quantification of CRA1 staining in complex tissue samples

These emerging technologies, when combined with the specific binding properties of CRA1 antibody, offer exciting possibilities for advancing our understanding of allergic mechanisms and developing more effective diagnostic and therapeutic approaches.