IZUMO1R Antibody

What is IZUMO1R and why is it significant in reproductive biology?

IZUMO1R (also known as JUNO, FR-delta, or Folbp3) is a pseudo-folate receptor that functions as the egg cell receptor for sperm IZUMO1 protein. This interaction forms an essential adhesion complex during mammalian fertilization . IZUMO1R is structurally unique as it lacks the capacity to bind folates despite its evolutionary relationship to folate receptors.

The IZUMO1:IZUMO1R interaction represents a necessary but not sufficient event for fertilization - while it mediates tight adhesion between gametes, it is not itself a membrane fusogen . Notably, after fertilization, IZUMO1R is shed from the egg membrane to prevent polyspermy, representing a key regulatory mechanism in fertilization.

What are the key experimental applications for IZUMO1R antibodies?

IZUMO1R antibodies are valuable tools in multiple experimental contexts:

• Western Blotting: Detection of IZUMO1R in tissue or cell lysates (typically appearing at ~28-30 kDa)

• Immunocytochemistry/Immunofluorescence: Localization of IZUMO1R on oocyte membranes and in certain T cell populations

• Flow Cytometry: Quantification of IZUMO1R expression in immune cell subsets, particularly Treg cells

• Immunohistochemistry: Detection of IZUMO1R in fixed tissue sections

• ELISA: Quantitative measurement of IZUMO1R in biological samples

• Functional Blocking Studies: Investigation of fertilization processes using antibodies that interfere with IZUMO1-IZUMO1R binding

What validation criteria should be used when selecting an IZUMO1R antibody?

When selecting an IZUMO1R antibody, researchers should consider the following validation criteria:

• Specificity: The antibody should detect IZUMO1R but not closely related folate receptors (FOLR1-3)

• Species Reactivity: Confirm reactivity with target species (human, mouse, etc.) as sequence homology varies

• Application Compatibility: Verify the antibody has been validated for your specific application

• Epitope Information: Select antibodies with defined epitopes that won't interfere with binding studies

• Knockout Validation: Antibodies validated against knockout controls offer superior specificity confirmation

• Citation History: Published usage provides confidence in antibody performance in similar experimental contexts

• Conjugation Options: Consider whether native, tagged, or directly conjugated formats best suit your experimental needs

How should IZUMO1R antibodies be optimized for immunofluorescence studies of oocytes?

Optimizing IZUMO1R antibodies for oocyte immunofluorescence requires special consideration:

Protocol Recommendations:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature; avoid methanol fixation which can disrupt membrane proteins

• Permeabilization: Gentle permeabilization with 0.1% Triton X-100 for 10 minutes

• Blocking: Extended blocking (2+ hours) with 5% normal serum from the species of secondary antibody origin plus 1% BSA

• Primary Antibody: Dilute IZUMO1R antibody typically between 1:100-1:500; incubate overnight at 4°C

• Secondary Detection: Use high-sensitivity fluorophore conjugates; far-red dyes often provide optimal signal-to-noise ratio

• Controls: Include a negative control omitting primary antibody and, if possible, IZUMO1R-knockout samples

Additional Considerations:

• Zone pellucida penetration can be challenging; extended antibody incubation times may be necessary

• IZUMO1R undergoes redistribution and shedding post-fertilization, so careful staging of oocytes is essential

• Co-staining with membrane markers can help contextualize IZUMO1R localization

What methods are effective for studying the IZUMO1-IZUMO1R interaction in vitro?

Several methods have proven effective for investigating the IZUMO1-IZUMO1R interaction:

Biolayer Interferometry (BLI):

• Load IZUMO1-Fc onto protein A sensors and measure binding kinetics with IZUMO1R in solution

• Typical binding affinity (KD) between murine IZUMO1 and IZUMO1R is approximately 11 μM

• Can be used to assess antibody blocking effects on the interaction

Surface Binding Assays:

• Express GPI-anchored IZUMO1R on HEK293F cells and measure binding of IZUMO1-Fc fusion proteins

• Detection using fluorophore-conjugated anti-Fc antibodies and flow cytometry

• IZUMO1-Fc typically shows EC50 values around 10.7 nM to murine JUNO on cell surfaces

Structural Studies:

• X-ray crystallography has been used to determine structures of IZUMO1 alone and in complex with inhibitory antibodies like OBF13

• These studies reveal conformational changes in IZUMO1 upon binding to IZUMO1R/JUNO

Cell-Cell Adhesion Assays:

• Co-culture IZUMO1-expressing cells with IZUMO1R-expressing cells and quantify adhesion events

• Antibodies against either protein can be used to test disruption of binding

How can IZUMO1R antibodies be utilized in immunological research beyond reproductive biology?

Recent research has revealed unexpected immunological functions of IZUMO1R, expanding antibody applications:

T Cell Immunophenotyping:

• IZUMO1R is expressed in CD4+ T cells, particularly Tregs, under Foxp3 control

• Flow cytometry panels can include IZUMO1R antibodies alongside markers like CD44, CD62L, and CD73

• Highest IZUMO1R expression is found in "experienced" Tregs (CD44hiCD62Llo)

Tissue-Specific Treg Analysis:

• IZUMO1R expression varies across tissue compartments, being highest in lymphoid organs and mucosal tissues but lower in skin and visceral adipose tissue

• Immunohistochemistry with IZUMO1R antibodies can help identify specific Treg subpopulations

Functional Studies:

• IZUMO1R on Tregs appears to regulate γδT cell-driven inflammation in skin

• Blocking antibodies against IZUMO1R can be used to investigate this regulatory function

• IZUMO1 (the ligand for IZUMO1R) is expressed by γδT cells in skin, suggesting a novel immune cell interaction system

How can allosteric inhibition of IZUMO1-IZUMO1R interactions be studied using antibodies?

Studying allosteric inhibition of the IZUMO1-IZUMO1R interaction represents an advanced research application:

Structural Framework:
The OBF13 antibody provides a model for allosteric inhibition of IZUMO1. This antibody:

• Binds to the apex of IZUMO1's four-helix domain, distant from the IZUMO1R/JUNO binding site

• Stabilizes IZUMO1 in its apo-conformation, preventing necessary conformational changes

• Decreases binding affinity of IZUMO1 for IZUMO1R by approximately 3-fold in solution and 50-fold on cell surfaces

Experimental Approaches:

• Competitive Binding Assays: Using BLI or SPR to measure how antibody binding affects IZUMO1-IZUMO1R kinetics

• Structural Analysis: X-ray crystallography or cryo-EM of antibody-bound IZUMO1 compared to apo and IZUMO1R-bound states

• Conformational Reporter Assays: Developing FRET-based sensors to detect IZUMO1 conformational changes

• Functional Blocking: Testing antibodies for inhibition of sperm-egg binding without direct competition at the binding interface

IZUMO1 Conformational States:

• Apo-IZUMO1 and OBF13-bound IZUMO1 structures share similar conformations

• IZUMO1R/JUNO binding induces an 8.2 Å shift in the L2 loop of IZUMO1

• This creates a ~9 Å upward shift of the OBF13 epitope region

What strategies can be used to engineer improved affinity IZUMO1R antibodies for research applications?

Engineering higher affinity IZUMO1R antibodies can enhance research applications. The approach used for the OBF13 antibody against IZUMO1 provides a valuable model:

Deep Mutational Scanning Approach:

• Structure-based paratope analysis to identify key contact residues

• Library construction using trinucleotide encoding for all 20 amino acids at each paratope position

• Yeast surface display of antibody variants with fluorescence-based selection

• Sequential rounds of selection with decreasing antigen concentration

• Sequence analysis of enriched variants to identify beneficial mutations

Affinity Maturation Case Study:
Research with OBF13 demonstrated substantial improvements:

• Original OBF13 scFv: KD = 1.0 μM

• Affinity-matured OBF13 (HAC): KD = 7.9 nM

• 126-fold improvement through directed evolution

• Five key substitutions (D31Q, Y104W in heavy chain; T31W, Y49F, S56P in light chain)

Validation Strategies:

• BLI or SPR for precise affinity measurements

• Cell-based binding assays to confirm functional improvement

• Structural analysis to understand the molecular basis of affinity enhancement

• Functional assays to confirm retained specificity and biological activity

What analytical approaches can resolve contradictory findings regarding IZUMO1R interactions with potential binding partners?

Recent literature contains contradictory findings about IZUMO1R interactions, particularly regarding the FCRL3/MAIA protein. Resolving these conflicts requires sophisticated analytical approaches:

Critical Experimental Validation:

• Multiple interaction detection methods:

  • Co-immunoprecipitation with appropriate controls

  • Surface plasmon resonance with reversed orientation immobilization

  • Proximity ligation assays in native cellular contexts

  • FRET/BRET-based interaction studies in live cells

• Cellular context considerations:

  • Membrane composition effects on protein orientation and accessibility

  • Post-translational modifications specific to gametes

  • Temporal dynamics of potential multistep binding events

• Advanced controls:

  • Both knockout/knockin validation approaches

  • Domain-specific mutations to pinpoint interaction surfaces

  • Cross-species comparative analyses to identify conserved interaction mechanisms

Case Study: FCRL3/MAIA as IZUMO1 Receptor:
Initial research suggested FCRL3/MAIA replaced IZUMO1R/JUNO after initial adhesion , but recent findings challenge this:

• More recent research found no direct extracellular interaction between FCRL3/MAIA and IZUMO1

• ELISA, BLI, and cell-based assays all failed to detect binding between purified proteins

• Preincubation of oocytes with anti-JUNO antibodies blocked all IZUMO1 binding, suggesting JUNO is the only IZUMO1 receptor

This highlights the importance of:

• Testing interactions in multiple experimental systems

• Using both binding assays and functional validation

• Carefully controlling for potential artifacts in each system

How does species-specificity in IZUMO1R antibody research impact experimental design and interpretation?

Species-specificity has profound implications for IZUMO1R antibody research:

Cross-Species Binding Variations:

• Murine IZUMO1 binds hamster IZUMO1R/JUNO with ~20-fold higher affinity than murine IZUMO1R/JUNO

• This creates complex interspecies fertilization compatibility patterns

• Antibodies may have species-specific effects on these interactions

Experimental Design Considerations:

• Antibody selection: Choose antibodies validated against IZUMO1R from your species of interest

• Cross-reactivity testing: Validate antibody performance across species when conducting comparative studies

• Control design: Include species-matched positive and negative controls

• Interpretation caveats: Consider that species differences may reflect true biological variation rather than experimental artifacts

Research Applications:
These species differences provide opportunities to study:

• Molecular basis of fertilization barriers between species

• Structural determinants of IZUMO1-IZUMO1R binding specificity

• Evolution of gamete recognition systems

What is the current understanding of IZUMO1R expression in immune cells and implications for antibody-based research?

Recent discoveries about IZUMO1R in the immune system open new research areas:

Expression Patterns:

• IZUMO1R is expressed by CD4+ T cells, with highest levels in Tregs under Foxp3 control

• Expression correlates with CD73 and defines a specific Treg subset (IZUMO1RhiCD73hi)

• This subset increases with age and belongs to antigen-experienced (CD44hiCD62Llo) Treg pool

• Expression varies by tissue: high in lymphoid organs and mucosal tissues, lower in skin and visceral adipose tissue

Functional Significance:

• Treg-specific knockout of IZUMO1R (Iz1rTrKO) shows normal Treg development and homeostasis

• Iz1rTrKO mice uniquely susceptible to imiquimod-induced skin inflammation

• Mechanism involves dysregulation of γδT cells expressing RORγ+

• Intriguingly, dermal γδT cells express IZUMO1, suggesting a direct IZUMO1-IZUMO1R interaction in immune regulation

Research Applications:

• IZUMO1R antibodies can identify specific Treg subsets in flow cytometry

• Blocking antibodies may modulate skin inflammation in experimental models

• Co-staining with IZUMO1 and IZUMO1R can identify potential cell-cell interactions in tissue sections

What potential exists for IZUMO1R antibodies in the development of immunocontraceptives or fertility treatments?

The structural and functional understanding of IZUMO1R offers translational opportunities:

Contraceptive Development Strategy:

• Antibodies targeting IZUMO1R could block sperm-egg binding

• The OBF13 antibody against IZUMO1 provides a model for allosteric inhibition

• Engineering of single-chain antibody fragments (scFvs) could enhance delivery and efficacy

• Structure-based design could lead to small molecule inhibitors targeting critical interaction surfaces

Therapeutic Considerations:

• Species-specificity must be carefully addressed in development

• Allosteric inhibition may offer advantages over direct blocking of binding interfaces

• Small molecule development can utilize structural information from antibody-antigen complexes

• The β-hairpin hinge region of IZUMO1 represents a potential target for inducing conformational changes

Diagnostic Applications:

• Anti-IZUMO1R antibodies could help diagnose specific forms of immunological infertility

• Detecting anti-IZUMO1R or anti-IZUMO1 autoantibodies in patients with unexplained infertility

• Monitoring IZUMO1R expression patterns in eggs during fertility assessment

Ethical and Safety Considerations:

• Target specificity must be carefully evaluated to avoid off-target effects on immune function

• Reversibility of contraceptive approaches is a key consideration

• Long-term immunological consequences require thorough investigation

What are common technical challenges when working with IZUMO1R antibodies and how can they be addressed?

Researchers may encounter several challenges when working with IZUMO1R antibodies:

Challenge: Low Signal in Western Blots

• Solution: Optimize protein extraction with membrane protein-specific lysis buffers

• Solution: Avoid harsh reducing conditions that may disrupt epitopes

• Solution: Extended transfer times for membrane proteins (60-90 minutes)

• Solution: Use PVDF membrane instead of nitrocellulose for better protein retention

Challenge: Background in Immunofluorescence

• Solution: Extend blocking time to 2+ hours with 5% normal serum

• Solution: Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding

• Solution: Use F(ab) fragments or monovalent formats to reduce Fc-mediated background

• Solution: Incubate primary antibody at 4°C overnight rather than shorter room temperature incubations

Challenge: Cross-reactivity with Related Proteins

• Solution: Validate specificity against IZUMO1R knockout controls

• Solution: Pre-absorb antibody with related folate receptor proteins

• Solution: Use epitope-mapped antibodies targeting unique IZUMO1R regions

• Solution: Consider monoclonal alternatives if polyclonal antibodies show cross-reactivity

Challenge: Inconsistent Results Across Tissues

• Solution: Optimize fixation conditions for each tissue type

• Solution: Account for variable expression levels in different tissues

• Solution: Consider tissue-specific antigen retrieval methods

• Solution: Validate antibody performance in each new tissue context

What control strategies are essential when using IZUMO1R antibodies in reproductive biology research?

Robust controls are essential for reliable IZUMO1R antibody research:

Genetic Controls:

• IZUMO1R knockout oocytes as negative controls

• IZUMO1R-overexpressing cell lines as positive controls

• Species-matched samples to account for sequence variations

Technical Controls:

• Antibody omission controls to assess secondary antibody specificity

• Isotype controls matched to antibody class and concentration

• Pre-immune serum controls for polyclonal antibodies

• Peptide competition/blocking to confirm epitope specificity

Biological Validation:

• Temporal controls (pre- and post-fertilization oocytes show dramatic IZUMO1R redistribution)

• Physiological controls (IZUMO1R should co-localize with membrane markers)

• Functional validation (antibody blocking should prevent IZUMO1 binding to oocytes)

Reporting Standards:
Document and report:

• Complete antibody information (supplier, catalog number, lot, dilution)

• Validation method for the specific application

• All control procedures performed

• Any optimization steps required for successful detection