Havcr1 Antibody Pair

Basic Research Questions

• What is HAVCR1 and what are its main functional domains for antibody targeting?

HAVCR1 (Hepatitis A Virus Cellular Receptor 1) is a class I integral membrane glycoprotein also known as TIM-1, KIM-1, or CD365. Its protein structure consists of distinct functional domains that researchers can target with antibodies:

• N-terminal cysteine-rich region (Ig-like domain) that displays homology to members of the immunoglobulin superfamily

• Threonine-, serine-, and proline-rich (TSP-rich) region with mucin-like characteristics

• Transmembrane domain

• Cytoplasmic domain

The Cys-rich region and its first N-glycosylation site are required for HAV binding and interaction with protective monoclonal antibody 190/4. Studies show that huhavcr-1 (human homolog) is approximately 79% identical to the African green monkey kidney (AGMK) havcr-1, but contains fewer hexameric repeats in the mucin-like region (13 vs. 27) and a 12 amino acid deletion in the cytoplasmic domain .

• What are the primary applications for HAVCR1 antibody pairs in biomedical research?

HAVCR1 antibody pairs serve multiple critical research applications:

These antibody pairs are particularly useful for:

• Studying HAV-receptor interactions

• Investigating kidney injury biomarkers

• Exploring HAVCR1's role in viral pathogenesis

• Examining HAVCR1 expression in various tissues

Researchers should select antibody pairs based on their experimental needs, ensuring they recognize different, non-overlapping epitopes on the HAVCR1 protein .

• How should researchers select the appropriate HAVCR1 antibody pair for their specific experimental design?

Selection of appropriate HAVCR1 antibody pairs requires consideration of several critical factors:

• Target species compatibility: Verify whether you need human, mouse, or rat-reactive antibodies. For instance, commercially available pairs include human KIM-1 (mouse/mouse host combination), mouse KIM-1 (goat/goat combination), and rat KIM-1 (mouse/goat combination) .

• Epitope specificity: Ensure paired antibodies recognize distinct, non-overlapping epitopes. The N-terminal cysteine-rich region and the mucin-like domain offer distinct binding sites for complementary antibodies .

• Clonality considerations: Monoclonal antibodies provide consistency across experiments but may have limited epitope recognition. Polyclonal antibodies offer broader epitope recognition but may introduce batch-to-batch variability.

• Application compatibility: Different experimental techniques require specific antibody characteristics:

  • ELISA: Low background, high signal-to-noise ratio

  • Western blot: Denaturation-resistant epitope recognition

  • IHC: Ability to work in fixed tissues

Cross-validate antibody pairs using positive and negative controls before conducting large-scale experiments to ensure specificity and sensitivity for your experimental system .

Intermediate Research Questions

• How do researchers validate the specificity of HAVCR1 antibody pairs in experimental systems?

Rigorous validation of HAVCR1 antibody pairs is essential to ensure experimental reproducibility. A comprehensive validation approach includes:

• Cell and tissue expression profiling: Compare antibody detection patterns with known HAVCR1 expression profiles. Northern blot analysis has shown HAVCR1 expression in various human organs including liver, small intestine, colon, and spleen, with notably higher expression in kidney and testis .

• Knockout/knockdown controls: Test antibodies on:

  • HAVCR1 knockout tissues/cells

  • siRNA-treated cells with reduced HAVCR1 expression

  • Dog cells transfected with HAVCR1 cDNA vs. vector-only controls

• Cross-reactivity assessment: Verify minimal cross-reactivity with related proteins:

  • Other TIM family members

  • Related membrane glycoproteins

  For example, certain commercial antibody pairs show less than 0.1% cross-reactivity with recombinant mouse TIM-1 when used in sandwich immunoassays .

• Epitope mapping: Confirm antibody binding to predicted epitopes using:

  • Peptide competition assays

  • Deletion mutants (as demonstrated in studies showing the Cys-rich region is required for specific antibody binding)

• Multiple detection methods: Verify concordant results across different techniques:

  • Western blot (typically detecting ~39 kDa protein, though the observed molecular weight may be higher due to glycosylation)

  • Immunohistochemistry

  • ELISA

Proper validation should include documentation of antibody specificity, sensitivity, and reproducibility across different experimental conditions.

• What are the key methodological considerations when designing sandwich ELISA assays using HAVCR1 antibody pairs?

Designing effective sandwich ELISA assays for HAVCR1 requires careful methodological planning:

Critical Parameters for HAVCR1 Sandwich ELISA Development:

Technical considerations specific to HAVCR1:

• Account for HAVCR1's heavy glycosylation, which can affect antibody binding

• Consider potential matrix effects when analyzing urine or serum samples

• Validate assays across physiological and pathological HAVCR1 concentration ranges

• Implement appropriate quality controls, including spike-recovery tests

Thorough optimization is essential, as evidenced by commercially available HAVCR1 antibody pairs that specifically bind distinct epitopes to enable sensitive detection across different species (human, mouse, rat) .

• How can researchers distinguish between membrane-bound and shed forms of HAVCR1 using antibody pairs?

Distinguishing between membrane-bound and shed forms of HAVCR1 is methodologically challenging yet crucial for understanding its biology. Effective approaches include:

Epitope-specific antibody selection:

• Use antibodies targeting the extracellular domain (common to both forms)

• Pair with antibodies specific to the transmembrane or cytoplasmic domains (present only in membrane-bound form)

Isolation strategies:

• Membrane fraction isolation via ultracentrifugation

• Immunoprecipitation from different cellular compartments

• Analysis of cell culture supernatants vs. cell lysates

Technical approaches:

• Western blot analysis with domain-specific antibodies:

  • Full-length HAVCR1: ~39 kDa theoretical size (often observed at 50-72 kDa due to glycosylation)

  • Shed ectodomain: Lower molecular weight band

• Flow cytometry for surface vs. intracellular staining:

  • Surface staining: Membrane-bound form

  • Permeabilized cells: Total HAVCR1

• Sandwich ELISA configurations:

  • Capture with anti-ectodomain, detect with anti-cytoplasmic domain: Membrane-bound only

  • Capture and detect with anti-ectodomain antibodies: Total HAVCR1

Research has shown that HAVCR1 is shed into urine after acute kidney damage, making it a valuable biomarker for renal tubular injury . Researchers must carefully consider antibody epitope specificity when designing assays to discriminate between these forms.

Advanced Research Questions

• How do glycosylation patterns affect HAVCR1 detection with antibody pairs, and what strategies can overcome these challenges?

HAVCR1's extensive glycosylation presents significant challenges for antibody-based detection systems. The protein contains both N-linked and O-linked glycosylation sites that influence its structure and epitope accessibility:

Impact of glycosylation on HAVCR1 detection:

• The first N-glycosylation site in the Cys-rich region is critical for HAV binding and antibody recognition

• The mucin-like TSP-rich region contains numerous O-glycosylation sites

• Glycosylation causes significant discrepancy between calculated (~39 kDa) and observed (50-72 kDa) molecular weight

• Glycosylation patterns vary between tissues and disease states

Methodological strategies to address glycosylation variability:

Experimental validation approach:

• Compare detection before and after enzymatic deglycosylation

• Analyze recombinant HAVCR1 expressed in systems with different glycosylation machinery

• Verify antibody binding to synthetic peptides corresponding to non-glycosylated regions

Understanding glycosylation's impact is particularly important when developing quantitative assays, as glycosylation changes can affect antibody binding affinity and assay sensitivity.

• What are the most effective experimental designs for investigating HAVCR1's role as a receptor for multiple viruses using antibody pairs?

Investigating HAVCR1's multi-viral receptor functionality requires sophisticated experimental designs utilizing antibody pairs:

Competition-based binding assays:

• Establish baseline virus binding using labeled virions or viral proteins

• Pre-treat cells with competitive anti-HAVCR1 antibodies targeting different domains

• Quantify inhibition to map critical binding interfaces for each virus

Domain-specific blockade studies:

• Use antibodies targeting specific HAVCR1 domains:

  • Cys-rich region (critical for HAV binding)

  • Mucin-like region

  • Site-specific glycosylation motifs

• Compare effects on binding of HAV, Ebola, Marburg, Dengue, and Zika viruses

Co-immunoprecipitation strategies:

• Capture HAVCR1 with one antibody

• Probe for co-precipitated viral proteins with virus-specific antibodies

• Map binding interfaces through mutational analysis

Cell-based infection models:

• Use antibody pairs in blocking experiments:

  • Pre-treatment with domain-specific antibodies

  • Post-infection detection with virus-specific antibodies

• Quantify infection rates via flow cytometry or imaging

Experimental controls and validation:

• HAVCR1-transfected vs. vector control cells (as demonstrated with dog cells transfected with huHAVcr-1 cDNA that gained limited susceptibility to HAV infection)

• HAVCR1 knockout/knockdown controls

• Domain deletion mutants (e.g., Cys-rich region deletion mutants)

This methodological framework can reveal both shared and virus-specific binding determinants, informing development of targeted antiviral strategies.

• How should researchers address contradictory results when using different HAVCR1 antibody pairs in complex biological systems?

Addressing contradictory results from different HAVCR1 antibody pairs requires systematic troubleshooting and experimental design adjustments:

Root cause analysis of discrepancies:

Comprehensive validation approach:

• Multi-technique verification:

  • Compare results across Western blot, immunoprecipitation, IHC, and ELISA

  • Validate with functional assays (e.g., virus binding studies)

• Biological context consideration:

  • Tissue-specific expression patterns (higher in kidney and testis)

  • Cell activation state effects

  • Species-specific differences (human vs. simian HAVCR1)

• Quantitative reconciliation:

  • Titrate antibody concentrations

  • Develop standard curves with recombinant proteins

  • Perform spike-recovery experiments

• Independent methodology:

  • Validate with non-antibody methods (mass spectrometry, RNA-seq)

  • Use genetic approaches (CRISPR, siRNA)

Research has shown that even the protective epitope 190/4 shows antigenic variability among primates, highlighting the importance of epitope-specific considerations when interpreting results across species .

• What are the advanced methodological approaches for developing HAVCR1 antibody pairs for high-sensitivity detection in biofluid samples?

Developing high-sensitivity HAVCR1 detection systems for biofluids requires advanced methodological approaches:

Signal amplification strategies:

• Enzyme-mediated amplification systems

• Tyramide signal amplification

• Poly-HRP conjugation

• Nanomaterial-enhanced detection (quantum dots, gold nanoparticles)

Sample preparation optimization:

• Urine samples:

  • Normalization to creatinine

  • Concentration via ultrafiltration

  • Stabilization with protease inhibitors

  • Removal of interfering components

• Serum/plasma samples:

  • Pre-clearing with protein A/G

  • Albumin/IgG depletion

  • Selective enrichment via affinity methods

Advanced detection platforms:

• Single molecule array (Simoa) technology

• Proximity ligation assay (PLA)

• Time-resolved fluorescence resonance energy transfer (TR-FRET)

• Electrochemiluminescence (ECL) platforms

Antibody engineering approaches:

• Recombinant antibody production for batch consistency

• Affinity maturation via directed evolution

• Fragment-based approaches (Fab, scFv)

• Bispecific antibody formats

Experimental validation metrics:

• Establish analytical sensitivity (limit of detection, limit of quantification)

• Determine analytical specificity (cross-reactivity, interference testing)

• Assess precision (intra-assay and inter-assay CV%)

• Verify linearity and recovery in relevant matrices

These advanced approaches are particularly important for detecting shed HAVCR1 in urine following acute kidney injury, where it serves as a sensitive biomarker for renal tubular damage , requiring quantification across a wide dynamic range of concentrations.