IL5RA Antibody

What is IL5RA and what is its biological significance in immune signaling?

IL5RA (Interleukin 5 receptor alpha subunit), also known as CD125, is a type I transmembrane protein belonging to the type I cytokine receptor family. It serves as a critical component of the high-affinity IL-5 receptor complex. This receptor complex consists of the alpha chain (IL5RA) that specifically binds to IL-5 and the common beta chain (CD131) that is shared with receptors for IL-3 and GM-CSF .

The IL-5/IL-5RA signaling axis plays crucial roles in:

• Differentiation, proliferation, recruitment, and activation of eosinophils

• Pathological eosinophilia development

• Survival and chemotaxis of eosinophils

Mechanistically, IL5RA acts by forming a heterodimeric receptor with the CSF2RB subunit and subsequently binding to interleukin-5. In unstimulated conditions, IL5RA interacts constitutively with JAK2. When the heterodimeric receptor is activated, it leads to JAK2 stimulation and subsequent activation of the JAK-STAT pathway, triggering downstream signaling cascades .

What cell types express IL5RA and how does this impact experimental design?

IL-5RA expression is predominantly found in:

• Eosinophils (highest expression)

• Basophils

• CD34+ progenitor cells

• T helper 2 (TH2) cells

• Mast cells

• Natural killer T cells (NK T cells)

• Type 2 innate lymphoid cells (ILC2)

When designing experiments involving IL5RA:

• Select appropriate cell models (TF-1/IL-5Rα cells are commonly used as they express the receptor and can demonstrate IL-5–dependent proliferation)

• Consider using primary human eosinophils from patients with eosinophilic conditions for more clinically relevant studies

• Account for variable expression levels across different cell types when interpreting results

• Include appropriate controls to verify receptor expression in your experimental system

What are the different types of IL5RA antibodies available for research?

Several types of IL5RA antibodies are available for research applications:

Examples include polyclonal antibodies like Proteintech's 12655-1-AP and recombinant monoclonal antibodies like Abcam's EPR5450(2) and CAL40 .

How do IL5RA antibodies function in antibody-dependent cell-mediated cytotoxicity (ADCC) assays?

ADCC is a key mechanism by which therapeutic anti-IL5RA antibodies like benralizumab deplete eosinophils. The process occurs through these steps:

• The Fc portion of anti-IL5RA antibodies binds to FcγRIIIa receptors on natural killer (NK) cells

• This binding triggers NK cells to release cytotoxic granules containing perforin and granzymes

• These cytotoxic molecules induce apoptosis in the target cells (eosinophils and basophils expressing IL5RA)

For example, benralizumab induces apoptosis through ADCC, where NK cells target eosinophils and basophils expressing IL5RA and induce their cytotoxic action . Benralizumab has higher affinity to human FcγRIIIa, resulting in enhanced ADCC action compared to other anti-IL-5 monoclonal antibodies .

Engineering approaches can enhance ADCC activity, as demonstrated with the antibody 5R65.7, which showed more potent biological activities than benralizumab analogue in ex vivo assays with peripheral eosinophils from patients with severe eosinophilic asthma (SEA) .

What experimental approaches can determine epitope binding differences between anti-IL5RA antibodies?

Several methodologies can distinguish epitope binding differences:

• Domain-swapped variants: Creating domain-swapped IL-5Rα extracellular-domain variants where human IL-5Rα domains (D1, D2, D3) are replaced with corresponding murine IL-5Rα sequences. For example, research showed benralizumab binds to human D1 (hD1), while antibody 5R65.7 recognizes epitopes within human D3 (hD3) .

• Yeast Surface Display (YSD) system: This facilitates expression of stable functional protein domains and allows mapping of antibody-binding regions to individual variants .

• Competition assays: These determine if different antibodies compete for the same binding site. In one study, a competition ELISA revealed that affinity-matured antibodies blocked the binding of soluble IL-5Rα to human IL-5 with different IC50 values .

• Crystallography/structural analysis: While not explicitly mentioned in the search results, this technique can provide atomic-level insights into antibody-antigen interactions.

How can researchers engineer IL5RA antibodies with improved affinity and specificity?

Engineering improved anti-IL5RA antibodies involves several sophisticated techniques:

• Humanization of murine antibodies:

  • Clone the variable domains of heavy chain (VH) and light chain (VL) genes from hybridoma cells

  • Graft complementarity-determining regions (CDRs) onto human framework regions

  • Example: The humanization of murine antibody m2B7 to create hu2B7

• Affinity maturation using yeast surface display:

  • Introduce random mutations in CDRs using error-prone PCR

  • Display mutant antibody fragments on yeast cell surface

  • Select high-affinity binders through fluorescence-activated cell sorting

  • This approach improved binding from KD ≈ 47.8 nM (hu2B7) to KD ≈ 11.8-24.1 nM in improved variants

• Fc engineering for enhanced ADCC:

  • Modify the Fc region to increase binding to FcγRIIIa receptors on NK cells

  • Optimize glycosylation patterns to enhance ADCC activity

A successful example is antibody 5R65.7, which achieved stronger affinity (KD ≈ 4.64 nM) than a benralizumab analogue (KD ≈ 26.8 nM) and demonstrated improved neutralizing activity toward IL-5–dependent cell proliferation .

What are the critical considerations for validating IL5RA antibody specificity?

Proper validation of IL5RA antibody specificity requires multiple approaches:

• Cross-reactivity testing:

  • Test against structurally related receptors (IL-1 RI, IL-2 R, IL-3 R, IL-4 R, etc.)

  • Example: One IL5RA antibody showed less than 5% cross-reactivity with recombinant human IL-1 RII, IL-2 R beta, IL-2 R gamma, and IL-3 R, and less than 1% cross-reactivity with IL-1 RI, IL-4 R, IL-6 R, IL-7 R, IL-9 R, and IL-10 R

• Multi-antigen non-specificity ELISA:

  • Test binding against structurally unrelated antigens (dsDNA, insulin, hemocyanin, cardiolipin)

  • Example: Engineered antibodies bound specifically to soluble IL-5Rα but showed negligible binding activity for these off-target antigens

• Species cross-reactivity assessment:

  • Test antibodies against IL5RA from different species

  • Many anti-IL5RA antibodies are human-specific with no cross-reactivity to murine IL-5Rα

• Positive and negative cell lines:

  • Use known IL5RA-expressing cells (TF-1/IL-5Rα, eosinophils) and non-expressing controls

  • Validate with techniques such as flow cytometry or Western blot

What are optimal protocols for using IL5RA antibodies in Western blot analysis?

For optimal Western blot results with IL5RA antibodies:

Sample preparation:

• Use appropriate cell lysates known to express IL5RA (human platelets, Jurkat cells)

• Include positive controls (recombinant IL5RA protein)

Protocol recommendations:

• SDS-PAGE: Use 12% Tris-HCl polyacrylamide gels

• Transfer: Use CN membrane and block with 5% skim milk for at least one hour

• Primary antibody: Dilute to manufacturer-recommended concentration (e.g., 1:500-1:1000 for Proteintech 12655-1-AP)

• Secondary antibody: Use appropriate HRP-conjugated antibody (e.g., anti-rabbit or anti-human IgG)

• Detection: Perform chemiluminescent detection

Expected results:

• Expected molecular weights: 48 kDa and 38 kDa (calculated)

• Observed molecular weights: 70-75 kDa and 43 kDa (may vary due to glycosylation)

• When using reduced conditions, expect heavy chain and light chain bands at approximately 50 kDa and 25 kDa, respectively

How can researchers measure IL5RA antibody affinity and binding kinetics?

Several methodologies are available for measuring antibody-antigen interactions:

• Bio-layer interferometry:

  • Using instruments like Octet QKe (ForteBio)

  • Protocol: Dilute purified antibody to 1 μg/ml in kinetics buffer and immobilize on anti-human IgG Fc capture biosensors

  • Monitor binding isotherms by exposing sensors to different concentrations of soluble IL-5Rα

  • Measure association for 300s and dissociation for 600s

  • Use reference sensors without antigen to account for non-specific binding

  • Calculate association and dissociation rate constants by fitting to sensorgrams

• Competitive ELISA:

  • Coat plates with IL-5-mFc protein (100 ng/well)

  • Add various concentrations of anti-IL-5Rα antibodies (0-1 μM) with soluble IL-5Rα (50 nM)

  • Detect residual binding of sIL-5Rα using HRP-conjugated anti-His antibody

  • Present data as percentage of sIL-5Rα bound relative to no antibody competition

  • Calculate IC50 by fitting normalized dose-response data to nonlinear sigmoidal curve

• Surface Plasmon Resonance (SPR):

  • While not explicitly mentioned in the search results, this is another common method for determining binding kinetics

What functional assays can assess the biological activity of IL5RA antibodies?

Several functional assays can evaluate IL5RA antibody activity:

• Cell proliferation inhibition assay:

  • Use cell lines stably expressing IL-5Rα (e.g., TF-1/IL-5Rα cells)

  • Stimulate with recombinant human IL-5 to induce proliferation

  • Add various concentrations of anti-IL5RA antibodies

  • Measure cell proliferation and calculate IC50 values

  • Example: Antibody 5R65 showed comparable antiproliferative activity to benralizumab analogue in this assay

• ADCC assay:

  • Isolate primary human eosinophils as target cells

  • Use NK cells as effector cells

  • Co-culture with anti-IL5RA antibodies

  • Measure target cell depletion or apoptosis

  • Example: 5R65.7 manifested more potent biological activities than benralizumab analogue in ex vivo assays with peripheral eosinophils from patients with SEA

• Receptor binding inhibition:

  • Test the ability of antibodies to block IL-5 binding to IL-5Rα

  • Can be measured by competitive binding assays

  • Important for therapeutic antibodies that aim to block IL-5 signaling

How should researchers approach experimental design when studying IL5RA in eosinophilic diseases?

When designing experiments to study IL5RA in eosinophilic diseases:

• Patient sample selection:

  • Include patients with confirmed eosinophilic conditions (e.g., SEA)

  • Consider disease severity, treatment history, and eosinophil counts

  • Include appropriate healthy controls

• Cell isolation methods:

  • For primary eosinophils: Use density gradient centrifugation and negative selection

  • Culture in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin

• Experimental controls:

  • Include isotype controls for antibodies

  • Use established therapeutic antibodies (e.g., benralizumab) as positive controls

  • Include unstimulated and IL-5 stimulated conditions

• Translational relevance:

  • Correlate in vitro findings with clinical parameters

  • Consider ex vivo assays with patient-derived cells

  • Evaluate potential biomarkers of response

• Ethical considerations:

  • Obtain proper ethical approval for human sample collection

  • Follow institutional guidelines for patient consent and data protection

By following these systematic approaches, researchers can generate robust and clinically relevant data on IL5RA biology in eosinophilic diseases.

What are the common technical challenges when working with IL5RA antibodies and how can they be addressed?

Several technical challenges may arise when working with IL5RA antibodies:

Challenge 1: Variable glycosylation affecting detection

• IL5RA can have multiple observed molecular weights (43 kDa, 70-75 kDa) compared to calculated weights (38 kDa, 48 kDa)

• Solution: Use deglycosylation enzymes before Western blot; include recombinant protein controls; use antibodies targeting different epitopes

Challenge 2: Low expression levels in certain cell types

• Solution: Enrich for IL5RA-expressing cells; use more sensitive detection methods; consider transfection to increase expression

Challenge 3: Cross-reactivity with related receptors

• Solution: Perform thorough validation with known positive and negative controls; use antibodies with demonstrated specificity (<5% cross-reactivity with related receptors)

Challenge 4: Variable results in functional assays

• Solution: Standardize experimental conditions; use multiple donor samples; include positive controls like benralizumab

How can researchers interpret seemingly contradictory results with different IL5RA antibodies?

When faced with contradictory results using different IL5RA antibodies:

• Consider epitope differences:

  • Different antibodies may recognize distinct domains of IL5RA

  • Example: Benralizumab binds to domain 1 (D1), while 5R65.7 recognizes domain 3 (D3)

  • These differences can affect function and detection in different assays

• Evaluate antibody formats and properties:

  • Antibody isotype can affect ADCC activity

  • Affinity differences impact sensitivity (KD ranging from 4.64 nM to 47.8 nM for different antibodies)

  • Polyclonal vs. monoclonal detection capabilities differ

• Assess experimental conditions:

  • Buffer conditions, pH, and temperatures can affect epitope accessibility

  • Fixation methods in IHC may mask certain epitopes

  • Reducing vs. non-reducing conditions in Western blot can reveal different results

• Validate with multiple techniques:

  • Confirm findings using orthogonal methods (e.g., flow cytometry, Western blot, and functional assays)

  • Use genetic approaches (siRNA knockdown) to confirm specificity

What quality control parameters should researchers evaluate when selecting IL5RA antibodies?

Critical quality control parameters to assess include:

Researchers should request validation data and technical specifications from manufacturers to ensure antibodies meet these quality parameters for their specific applications.

How might emerging technologies advance IL5RA antibody development and applications?

Several emerging technologies may transform IL5RA antibody research:

• Single B cell sequencing and antibody discovery:

  • Enables faster identification of novel anti-IL5RA antibodies with unique properties

  • Allows mining of the immune repertoire of patients with eosinophilic conditions

• CRISPR/Cas9 gene editing:

  • Creates precise IL5RA knockout or knock-in cell lines for antibody validation

  • Enables engineering of reporter cell lines with modified IL5RA signaling components

• Structural biology advances:

  • Cryo-EM and X-ray crystallography to determine precise antibody-IL5RA binding interfaces

  • Structure-guided design of antibodies with improved properties

• Bispecific antibody platforms:

  • Development of bispecific antibodies targeting IL5RA and additional targets

  • Potential for enhanced therapeutic effects in eosinophilic diseases

• Advanced functional screening assays:

  • High-throughput systems to screen antibodies for specific functional properties

  • Microfluidic systems for single-cell analysis of antibody effects

What are unresolved questions in IL5RA biology that antibody-based approaches might address?

Several key questions remain in IL5RA biology that could be addressed with antibody tools:

• Receptor complex dynamics:

  • How does IL5RA interact with the common beta chain in different cellular contexts?

  • What are the precise conformational changes upon IL-5 binding?

• Signaling pathway specificity:

  • How does IL5RA specifically activate JAK2 and the JAK-STAT pathway?

  • What determines signaling outcomes in different cell types?

• Role in non-eosinophil cells:

  • What is the functional significance of IL5RA expression on mast cells, NK T cells, and ILC2 cells?

  • How do these non-eosinophil effects contribute to disease pathology?

• Tissue-specific functions:

  • How does IL5RA function differ between circulating and tissue-resident eosinophils?

  • Are there tissue-specific cofactors that modify IL5RA signaling?

• Therapeutic resistance mechanisms:

  • Why do some patients with eosinophilic diseases not respond to anti-IL5RA therapy?

  • Are there compensatory pathways that bypass IL-5/IL5RA signaling?