ASGR1 Antibody Pair

What is ASGR1 and why is it an important research target?

ASGR1 (asialoglycoprotein receptor 1) is a type II integral membrane protein predominantly expressed in hepatocytes. It functions as a major subunit of the asialoglycoprotein receptor (ASGPR), which recognizes terminal β-linked galactose or N-acetylglucosamine (GlcNAc) on circulating glycoproteins. With approximately one million copies per hepatocyte, ASGR1 plays a critical role in serum glycoprotein homeostasis through endocytosis and lysosomal degradation of glycoproteins with terminal galactose residues . Recent genetic studies have identified a loss-of-function variant of ASGR1 associated with decreased non-HDL cholesterol levels and reduced risk of coronary heart disease, making it a promising therapeutic target .

What constitutes an ASGR1 antibody pair and what are their primary applications?

An ASGR1 antibody pair consists of two antibodies that recognize different epitopes on ASGR1: a capture antibody that immobilizes the target antigen, and a detection antibody that enables visualization or quantification. Commercial examples include the MAB27551 (capture) and MAB27552 (detection) pair for mouse ASGR1/ASGPR1 . These pairs are primarily used in sandwich ELISA, cytometric bead arrays, and multiplex immunoassays for quantitative detection of ASGR1 in research samples . The paired approach significantly enhances specificity and sensitivity compared to single-antibody detection methods, enabling precise measurement of ASGR1 in complex biological samples.

How do I select the appropriate ASGR1 antibody pair for cross-species applications?

When selecting antibody pairs for cross-species applications, evaluate the protein sequence homology between species and review the antibodies' validated reactivity profiles. For example, certain antibody pairs like Proteintech's recombinant monoclonal 83691 series demonstrate cross-reactivity with human, mouse, and rat ASGR1 . Importantly, verify the epitope recognition regions, as conserved domains across species enhance cross-reactivity potential. Preliminary testing should include titration experiments with positive controls from each species of interest. Western blot analysis can confirm specific recognition of the expected molecular weight (42-46 kDa for ASGR1) . For definitive validation, perform parallel sandwich ELISA experiments with recombinant ASGR1 proteins from each species to establish limits of detection and quantification.

What are the optimal conditions for developing a sandwich ELISA using ASGR1 antibody pairs?

For optimal ASGR1 sandwich ELISA development, start with antibody pair validation using recombinant ASGR1 protein. Coat a clear polystyrene microplate with the capture antibody (e.g., MAB27551 for mouse ASGR1) at 1-10 μg/mL in coating buffer (typically carbonate-bicarbonate buffer, pH 9.6) overnight at 4°C . After blocking with 1-5% BSA, add samples or standards in a suitable diluent containing calcium (2 mM CaCl₂), as calcium is essential for ASGR1's conformation and function . For detection, use a biotinylated detection antibody (e.g., MAB27552) followed by streptavidin-HRP . Develop with a substrate solution (e.g., TMB) and stop with acid solution. Critical parameters include:

Sensitivity can be enhanced by implementing signal amplification systems and extending incubation times at critical steps .

How should I design experiments to assess pH/Ca²⁺-dependent binding properties of anti-ASGR1 antibodies?

Design experiments to evaluate pH/Ca²⁺-dependent binding by comparing antibody-ASGR1 interactions under physiological versus endosomal conditions. Use surface plasmon resonance (SPR) to measure binding kinetics at neutral pH (7.4) with high calcium (2 mM CaCl₂) and at acidic pH (5.5-6.0) with low calcium (2 μM CaCl₂), mimicking endosomal conditions . Immobilize recombinant ASGR1 on a sensor chip and flow antibodies at various concentrations across the surface under both conditions.

A robust experimental design should include:

• Multiple antibody concentrations (0.1-100 nM) to determine kinetic parameters (k​on​, k​off​, K​D​)

• Control antibodies with known pH/Ca²⁺-independent binding

• "Catch-and-release" (CAR) antibodies for comparison, which show strong binding at neutral pH/high calcium but minimal binding at acidic pH/low calcium

• Validation of SPR results through complementary methods such as ELISA or biolayer interferometry

Calculate fold-changes in binding affinity between conditions to classify antibodies as pH/Ca²⁺-dependent (>10-fold change) or pH/Ca²⁺-independent (<3-fold change) . This characterization is critical for developing antibodies with improved pharmacokinetic properties for therapeutic applications.

What methodological approaches should be used to validate ASGR1 antibody specificity in liver tissue samples?

To validate ASGR1 antibody specificity in liver tissue samples, implement a multi-method approach combining immunohistochemistry (IHC), western blotting, and appropriate controls. For western blotting, prepare liver lysates from multiple species alongside negative control tissues (where ASGR1 is not expressed) and positive control recombinant ASGR1 protein. Probe with ASGR1 antibodies (e.g., MAB27552) at 0.5-1 μg/mL to confirm detection of the expected 42 kDa band in liver samples only .

For IHC validation:

• Test paraffin-embedded liver sections with the ASGR1 antibody at 5-10 μg/mL alongside isotype control antibodies

• Include ASGR1-knockout or siRNA-treated tissue sections as negative controls

• Verify hepatocyte-specific cytoplasmic and membrane staining patterns consistent with ASGR1 biology

• Perform peptide competition assays to confirm binding specificity

• Use dual-immunofluorescence with established hepatocyte markers to confirm cell-type specificity

Additional validation techniques include RNA-protein correlation through parallel qRT-PCR and immunodetection in the same samples, and comparison of staining patterns across multiple validated ASGR1 antibodies targeting different epitopes . This comprehensive approach ensures confident interpretation of ASGR1 detection in complex liver tissue samples.

How can ASGR1 antibody pairs be utilized to study target-mediated drug disposition (TMDD) of therapeutic antibodies?

To study TMDD of therapeutic antibodies targeting ASGR1, design pharmacokinetic (PK) studies employing dose escalation with labeled antibodies. Administer intravenous doses of anti-ASGR1 antibodies (ranging from 0.3-30 mg/kg) to transgenic mice expressing human FcRn, as demonstrated in previous research . Collect serum samples at predetermined timepoints (0.25, 1, 4, 24, 48, 96, 168, 240, and 336 hours post-dosing) and quantify antibody concentrations using an ASGR1 antibody pair-based sandwich ELISA.

A robust experimental design should compare:

• "Catch-and-release" (CAR) antibodies with pH/Ca²⁺-dependent binding

• Conventional antibodies with pH/Ca²⁺-independent binding

• Antibody variants with modified FcRn binding (e.g., YTE and IHH mutations)

Analyze the resulting data using non-compartmental analysis to determine clearance rates, volume of distribution, and area under the curve (AUC). Plot antibody concentration versus time on semi-log scales to visualize biphasic elimination patterns. Non-dose-proportional increases in exposure indicate TMDD, while target saturation is suggested when exposure becomes dose-proportional at higher doses . This approach provides critical insights into ASGR1-mediated clearance mechanisms and informs optimal dosing strategies for therapeutic antibodies.

What parameters should be analyzed when evaluating catch-and-release (CAR) versus conventional anti-ASGR1 antibodies in vivo?

When evaluating CAR versus conventional anti-ASGR1 antibodies in vivo, analyze multiple parameters across pharmacokinetic, pharmacodynamic, and molecular dimensions. For pharmacokinetic assessment, determine clearance rates, terminal half-life (t½), and AUC values at multiple dose levels (e.g., 0.3, 3, 10, and 30 mg/kg). Research shows that CAR antibodies demonstrate significantly higher serum exposure (85-fold higher AUC) compared to conventional antibodies at lower doses (0.3 mg/kg), but this advantage diminishes at higher, receptor-saturating doses .

For comparative analysis, measure:

Research indicates that the pH/Ca²⁺-dependent binding of CAR antibodies enables endosomal dissociation from ASGR1, leading to antibody recycling and improved pharmacokinetics, but this advantage is context-dependent and influenced by FcRn binding affinity and target expression levels .

How can researchers analyze ASGR1 antibody competitive binding to distinguish epitope-specific effects?

To analyze ASGR1 antibody competitive binding and distinguish epitope-specific effects, implement a comprehensive competition binding assay system. Begin by expressing mouse or human ASGR1 in a suitable cell line (e.g., CHO-S cells) and pre-incubate with varying concentrations of the test antibodies (1-1000 nM). Subsequently challenge with natural ligands (e.g., GalNAc and asialofetuin) at their predetermined EC₅₀ concentrations . Measure residual ligand binding using flow cytometry or fluorescence-based detection to generate competition curves and calculate IC₅₀ values.

To map distinct epitopes:

• Perform pairwise competition experiments with a panel of anti-ASGR1 antibodies

• Generate competition matrices based on percent inhibition values

• Construct epitope bins using hierarchical clustering analysis

• Compare IC₅₀ values for ligand displacement across antibody classes

How should ASGR1 antibody pairs be employed to evaluate potential biomarkers in clinical samples?

To employ ASGR1 antibody pairs for biomarker evaluation in clinical samples, develop a standardized sandwich ELISA or multiplex immunoassay protocol with rigorous quality control measures. Select antibody pairs validated for human ASGR1 detection with minimal cross-reactivity to similar proteins. For clinical sample analysis:

• Establish a reference range using at least 100 healthy control samples stratified by age and sex

• Develop calibration curves using recombinant human ASGR1 protein spanning physiological concentrations

• Include quality control samples at low, medium, and high concentrations in each assay

• Validate the assay according to Clinical and Laboratory Standards Institute (CLSI) guidelines for precision, accuracy, analytical sensitivity, and specificity

When analyzing samples from patients with potential liver pathologies:

• Measure alkaline phosphatase (ALP) levels in parallel, as research shows elevated ALP is associated with ASGR1 inhibition (up to 251% increase observed in clinical trials)

• Correlate ASGR1 levels with lipid profiles and cardiovascular risk markers

• Consider the confounding effects of medications that might affect ASGR1 expression or function

• Compare results with established liver function tests to determine the added diagnostic value

For longitudinal studies, ensure sample collection, processing, and storage conditions are standardized to minimize pre-analytical variability, as this is critical for reliable biomarker assessment .

How does ASGR1 deficiency impact experimental models of liver injury, and how can antibody pairs help quantify these effects?

ASGR1 deficiency significantly exacerbates both acute and chronic liver injury in experimental models. In ASGR1-knockout (Asgr1-/-) mice, acetaminophen-induced acute liver injury and CCl₄-induced chronic liver injury are markedly intensified compared to wild-type controls . These ASGR1-deficient mice exhibit significantly elevated serum levels of liver enzymes (ALT, AST, ALP, and GGT), increased hepatic ballooning degeneration, enhanced inflammatory cell infiltration, and elevated expression of proinflammatory cytokines (TNF-α, IL-6, MCP1, and IL-1β) .

To quantify these effects using antibody pairs:

• Develop sandwich ELISAs using validated ASGR1 antibody pairs to measure circulating ASGR1 levels in different experimental groups

• Establish multiplex assays combining ASGR1 detection with inflammatory biomarkers

• Implement tissue immunostaining protocols to assess hepatic ASGR1 distribution and expression levels

• Correlate ASGR1 levels with apoptotic markers detected by TUNEL staining and expression of pro-apoptotic genes (Bax, Caspase3, Caspase9) and anti-apoptotic genes (Bcl-2)

A comprehensive experimental approach should compare multiple models:

• ASGR1 genetic knockout models

• ASGR1 antibody-mediated blockade (using validated antibodies at 10-30 mg/kg)

• ASGR1 overexpression systems

• Hepatocyte-specific conditional knockout models

This multi-faceted approach provides mechanistic insights into how ASGR1 modulates liver injury responses and offers potential therapeutic strategies for liver diseases .

What methodological considerations are important when designing phase 1 clinical trials for anti-ASGR1 antibody therapeutics?

When designing phase 1 clinical trials for anti-ASGR1 antibody therapeutics, several critical methodological considerations must be addressed based on existing clinical experience. The trial design should employ a randomized, placebo-controlled, double-blind, ascending single-dose approach to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics . Key design elements include:

• Dose selection strategy:

  • Start with doses substantially below receptor saturation (e.g., 21 mg SC)

  • Include multiple ascending dose cohorts (e.g., 21, 70, 210, 420, and 700 mg SC)

  • Consider both subcutaneous and intravenous administration routes

  • Use 3:1 randomization ratio (active:placebo) for efficient safety assessment

• Subject selection criteria:

  • Healthy adults aged 18-55 years

  • Exclusion of liver diseases or conditions affecting lipid metabolism

  • Balanced representation of sex and ethnicity

• Endpoint selection:

  • Primary: Adverse events, safety laboratory analytes, vital signs, ECGs

  • Secondary: Antibody pharmacokinetics, alkaline phosphatase levels, lipid profiles

• Biomarker strategy:

  • Monitor total alkaline phosphatase (ALP) as a pharmacodynamic marker, as dose-related increases (up to 251% at 700 mg SC) have been observed

  • Implement detailed lipid panel measurements to detect potential cardioprotective effects

  • Develop assays to measure target engagement and receptor occupancy

Based on previous clinical experience, researchers should anticipate potential dichotomous exposure patterns at intermediate doses due to target-mediated clearance, with more consistent exposures at higher doses suggesting target saturation . Include intensive PK sampling during the first 24-48 hours to capture potential rapid target-mediated elimination.

How can researchers address ASGR1 antibody target-mediated drug disposition challenges in pharmacokinetic studies?

To address ASGR1 antibody target-mediated drug disposition (TMDD) challenges in pharmacokinetic studies, implement a multi-faceted experimental strategy. TMDD presents a significant hurdle due to ASGR1's high expression level (approximately one million receptors per hepatocyte) and rapid recycling . To overcome these challenges:

• Design antibody engineering approaches:

  • Develop "catch-and-release" (CAR) antibodies with pH/Ca²⁺-dependent binding properties that enable dissociation in the acidic endosomal environment

  • Incorporate FcRn-binding enhancing mutations (e.g., YTE) to improve antibody recycling

  • Consider bispecific formats that combine ASGR1 targeting with extended half-life domains

• Implement advanced sampling strategies:

  • Use frequent early sampling timepoints (0.25, 1, 4, 8, 12, 24 hours) to capture rapid initial clearance phase

  • Extend sampling duration (up to 14 days) to fully characterize terminal elimination

  • Consider microsampling techniques to enable more frequent sampling without excessive blood volume collection

• Apply specialized PK modeling approaches:

  • Implement target-mediated drug disposition models that incorporate receptor dynamics

  • Use quasi-steady-state approximation models for high-turnover targets like ASGR1

  • Incorporate receptor occupancy data to inform PK model parameters

What are the key technical challenges in developing highly specific ELISA assays using ASGR1 antibody pairs?

Developing highly specific ELISA assays using ASGR1 antibody pairs presents several technical challenges that require systematic troubleshooting. Based on research experience, the following challenges and solutions are critical:

• Cross-reactivity with ASGR2 (homologous subunit):

  • Challenge: ASGR1 shares structural homology with ASGR2, potentially leading to cross-reactivity

  • Solution: Pre-screen antibody pairs against recombinant ASGR1 and ASGR2 proteins to ensure specificity; select pairs with >100-fold selectivity for ASGR1

• Calcium dependency of detection:

  • Challenge: ASGR1 conformation and epitope accessibility are calcium-dependent

  • Solution: Include 2mM CaCl₂ in all assay buffers to maintain proper receptor conformation ; verify antibody binding in calcium-free conditions to identify calcium-sensitive epitopes

• Hook effect at high ASGR1 concentrations:

  • Challenge: Excess antigen can bridge capture and detection antibodies, reducing signal at high concentrations

  • Solution: Implement sample dilution protocols; incorporate multiple dilutions for each sample; use four-parameter logistic curve fitting to account for hook effect

• Matrix effects from liver-enriched samples:

  • Challenge: Liver tissue lysates and hepatocyte culture supernatants contain interfering substances

  • Solution: Optimize blocking buffers with additional detergents (0.05% Tween-20) and protein blockers (2-5% BSA); perform spike-recovery experiments to quantify and correct for matrix effects

• Isotype-specific interference:

  • Challenge: Endogenous antibodies or rheumatoid factors can bridge detection antibodies

  • Solution: Include nonspecific IgG from the detection antibody species; use F(ab')₂ fragments instead of whole IgG for detection; evaluate heterophilic antibody blockers

Based on the technical data from R&D Systems' ASGR1 ELISA, sample dilution optimization and calcium supplementation are particularly critical for obtaining reproducible results . Additionally, implementing stringent quality control measures including inter-assay calibrators and controls is essential for maintaining assay performance across multiple runs.

How can researchers resolve discrepancies between in vitro binding data and in vivo efficacy of anti-ASGR1 antibodies?

Resolving discrepancies between in vitro binding data and in vivo efficacy of anti-ASGR1 antibodies requires systematic investigation of multiple biological and methodological factors. These discrepancies are common and have been observed in studies comparing "catch-and-release" (CAR) and conventional anti-ASGR1 antibodies . To address these inconsistencies:

• Investigate target accessibility differences:

  • In vitro: Utilize primary hepatocytes rather than recombinant proteins to better simulate the native membrane environment of ASGR1

  • In vivo: Implement intravital microscopy to visualize antibody distribution in liver sinusoids and quantify hepatocyte exposure

• Assess competitive displacement by endogenous ligands:

  • Conduct competition binding studies with physiologically relevant concentrations of natural ASGR1 ligands (asialofetuin, GalNAc)

  • Research shows anti-ASGR1 antibodies exhibit IC₅₀ values of 24-44 nM against GalNAc and 3.7-4 nM against asialofetuin

  • Evaluate if plasma concentrations achieved in vivo are sufficient to compete with these ligands

• Analyze pharmacokinetic-pharmacodynamic relationships:

  • Develop integrated PK-PD models incorporating receptor occupancy, target turnover, and biological response

  • Determine the minimum receptor occupancy required for biological effects

  • Consider implementing receptor occupancy assays in liver biopsies from animal models

• Examine target-mediated drug disposition effects:

  • Recent research demonstrates that despite superior in vitro properties, CAR antibodies show limited benefits at low doses due to ASGR1's high expression and rapid turnover

  • At higher doses (≥10 mg/kg), both CAR and conventional antibodies show similar exposure profiles when target saturation occurs

• Evaluate the influence of FcRn expression:

  • Investigate the relationship between hepatocyte FcRn expression and antibody recycling capacity

  • Consider species differences in FcRn expression and binding affinity when translating between animal models and humans