C1QBP Antibody

What is C1QBP and why is it significant in immunological research?

C1QBP (complement component 1, q subcomponent binding protein), also known as gC1q-R, p32/p33, or HABP-1 (hyaluronan-binding protein 1), is a multicompartmental and multifunctional protein that undergoes spatiotemporal changes in its distribution in response to various cellular and systemic stimuli. It plays an essential role in mitochondrial biogenesis through mRNA binding activity, while surface-exposed C1QBP can serve as an entry route for numerous pathogens and act as an inflammatory trigger in diseases including vascular disorders and various cancers .
C1QBP is significant in immunological research because it binds to the globular heads of C1q molecules, inhibiting C1 activation and modulating complement activity. This interaction helps regulate the classical pathway of the serum complement system, which is essential for clearance of immune complexes and pathogens . The protein's expression in various cell types, including vascular endothelial cells, B cells, neutrophils, and mast cells, highlights its importance in both complement and coagulation systems .

How do I select the appropriate C1QBP antibody for my specific research application?

Selection of the appropriate C1QBP antibody depends on several factors:

• Target species compatibility: Ensure the antibody recognizes C1QBP in your experimental model. For example, the H-9 antibody detects C1QBP in mouse, rat, and human samples .

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

  • Western blotting (WB)

  • Immunoprecipitation (IP)

  • Immunofluorescence (IF)

  • Immunohistochemistry (IHC)

  • Enzyme-linked immunosorbent assay (ELISA)

• Antibody format: Consider whether you need:

  • Non-conjugated antibody

  • Conjugated variants (agarose, HRP, PE, FITC, or Alexa Fluor conjugates)

• Epitope recognition: Different antibodies recognize distinct epitopes on C1QBP, which may affect function. For instance, mAb-60.11 interferes with C1q recognition, while mAb-74.5.2 disrupts interaction with HMWK .
  For structural studies or investigations of specific domains of C1QBP, consider antibodies with characterized epitopes, such as mAb-1 which recognizes the first acidic loop of C1QBP .

What are the standard protocols for validating C1QBP antibody specificity?

Validation of C1QBP antibody specificity should incorporate multiple complementary approaches:

• Western blotting with wild-type and mutant proteins: Compare reactivity with wild-type C1QBP versus deletion mutants (such as gC1qR-D1, gC1q-D2, and gC1qR-DD) to confirm epitope specificity .

• Surface plasmon resonance (SPR) analysis: Measure binding kinetics of the antibody to immobilized C1QBP. For example, mAb-1 showed differential binding to wild-type gC1qR and gC1qR-D2 but not to gC1qR-D1 or gC1qR-DD, confirming its epitope within the first acidic loop .

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody pulls down C1QBP rather than cross-reactive proteins.

• Competitive binding assays: AlphaScreen bead-based competition binding assays can evaluate interactions between anti-gC1qR mAbs and biotinylated wild-type gC1qR in the presence of potential competitors .

• Knockout/knockdown validation: Demonstrate loss of signal in cells where C1QBP has been knocked out or down through genetic approaches.

How should I design experiments to investigate C1QBP's role in specific cellular compartments?

Designing experiments to investigate C1QBP's compartment-specific roles requires multiple strategic approaches:

• Subcellular fractionation combined with immunoblotting:

  • Separate cellular compartments (mitochondria, cytosol, plasma membrane, nucleus)

  • Probe fractions with C1QBP antibody

  • Validate compartment purity with organelle-specific markers

• Immunofluorescence with co-localization studies:

  • Use C1QBP antibody alongside organelle markers

  • Employ high-resolution confocal or super-resolution microscopy

  • Quantify co-localization coefficients using appropriate software

• Proximity labeling approaches:

  • Express C1QBP fused to BioID or APEX2 in different compartments

  • Identify compartment-specific interacting partners

  • Validate interactions with co-immunoprecipitation using C1QBP antibodies

• Mutation of targeting sequences:

  • Create mutants lacking mitochondrial targeting sequence or other localization signals

  • Compare phenotypes using C1QBP antibodies to track mislocalization

  • Correlate with functional readouts relevant to compartment-specific functions
    This methodological approach accounts for C1QBP's known multicompartmental distribution and can help distinguish between its mitochondrial functions in biogenesis versus its cell surface roles in pathogen binding or inflammatory signaling .

What controls should be included when using C1QBP antibodies for immunoprecipitation experiments?

Robust immunoprecipitation experiments with C1QBP antibodies require careful inclusion of the following controls:

• Input control:

  • Reserve 5-10% of pre-cleared lysate to confirm target protein presence

  • Compare with immunoprecipitated fraction to assess efficiency

• Isotype control antibody:

  • Use matched isotype (e.g., mouse IgG2b kappa for H-9 antibody)

  • Process identical to experimental samples

  • Confirms specificity of C1QBP antibody binding

• Negative control lysates:

  • Cells with C1QBP knockdown/knockout

  • Demonstrates antibody specificity for target protein

• Blocking peptide control:

  • Pre-incubate C1QBP antibody with excess antigen peptide

  • Should abolish specific immunoprecipitation

• Non-denaturing vs. denaturing conditions:

  • Compare results under different lysis conditions

  • Important for C1QBP as it exists as a trimer in native conditions

  • May help distinguish between direct and indirect interactions

• Reverse immunoprecipitation:

  • Immunoprecipitate with antibodies against suspected interacting partners

  • Probe for C1QBP to confirm interaction bidirectionally
    These controls are essential when investigating C1QBP's interactions with complement components, pathogens, or other cellular proteins, ensuring that observations represent genuine biological interactions rather than experimental artifacts.

How can I investigate the structural basis of C1QBP interactions with ligands using antibodies?

Investigating the structural basis of C1QBP-ligand interactions requires sophisticated approaches combining antibodies with structural methods:

• Epitope mapping with domain-specific antibodies:

  • Use characterized antibodies like mAb-1 (first acidic loop-specific), mAb-60.11 (interferes with C1q binding), or mAb-74.5.2 (disrupts HMWK interaction)

  • Perform competition assays between antibodies and ligands

  • Correlate epitope recognition with functional interference

• Co-crystallization studies with Fab fragments:

  • Generate Fab fragments from monoclonal antibodies

  • Co-crystallize with C1QBP and solve structure by X-ray crystallography

  • Similar to the approach used for solving the gC1qR-DD structure at 2.2 Å resolution

• SPR-based binding interference assays:

  • Immobilize biotinylated C1QBP on streptavidin surface

  • Pre-incubate with domain-specific antibodies

  • Measure binding kinetics of ligands

  • Quantify changes in association/dissociation rates

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium uptake patterns of C1QBP alone versus antibody-bound

  • Identify regions with altered solvent accessibility

  • Correlate with potential ligand binding sites

• Single-particle cryo-EM analysis:

  • Visualize C1QBP-antibody complexes

  • Compare with C1QBP-ligand complexes

  • Identify structural changes upon binding
    This methodological framework can help determine how the trimeric core structure of C1QBP and its acidic loops contribute to interactions with various ligands, building on the molecular replacement techniques previously used with PHASER and the PHENIX software suite .

What approaches can reveal the role of C1QBP's acidic loops in ligand binding?

C1QBP contains two acidic loops that play important roles in ligand binding. To investigate their specific contributions:

• Deletion mutant analysis:

  • Utilize established mutants: gC1qR-D1 (first loop deleted), gC1qR-D2 (second loop deleted), and gC1qR-DD (both loops deleted)

  • Compare binding affinities to various ligands using SPR

  • Examine functional outcomes in cellular assays

• Site-directed mutagenesis:

  • Create point mutations of charged residues within acidic loops

  • Systematically test effects on ligand binding

  • Identify critical interaction residues

• Loop-specific antibody blocking:

  • Use mAb-1 which specifically recognizes the first acidic loop

  • Assess inhibition of ligand binding (e.g., FXII-FNII)

  • Compare with effects of antibodies targeting other epitopes

• Peptide competition assays:

  • Synthesize peptides corresponding to acidic loop sequences

  • Test competition with full-length C1QBP for ligand binding

  • Use AlphaScreen bead-based competition assays

• Cross-linking coupled with mass spectrometry:

  • Identify precise contact points between C1QBP acidic loops and ligands

  • Map interaction interfaces at amino acid resolution
    These approaches build on findings that mAb-1 binds to the first acidic loop of C1QBP, which appears to play a role in binding to certain ligands, notably FXII-FNII . The differential binding observed with gC1qR-D2 (enhanced binding to mAb-1 compared to wild-type) suggests that removal of the second acidic loop may increase accessibility of the first loop epitope .

How can C1QBP antibodies be utilized to evaluate potential therapeutic approaches?

C1QBP antibodies offer valuable tools for evaluating therapeutic strategies targeting this protein:

• Target validation in disease models:

  • Use antibodies to confirm C1QBP overexpression/mislocalization in disease tissues

  • Correlate expression levels with clinical outcomes (as observed in breast cancer)

  • Screen patient samples to identify populations likely to respond to C1QBP-targeted therapy

• Therapeutic antibody development:

  • Screen antibodies for functional inhibition of C1QBP-ligand interactions

  • Test candidate antibodies in cellular and animal models

  • Evaluate effects on tumor growth (as demonstrated in breast cancer models) or inflammation

• Companion diagnostic development:

  • Develop immunohistochemistry protocols with validated C1QBP antibodies

  • Create scoring systems for C1QBP expression/localization

  • Correlate with response to targeted therapies

• Mechanism of action studies:

  • Use domain-specific antibodies to understand which C1QBP interactions are essential for disease progression

  • Determine whether blocking complement binding, pathogen entry, or inflammatory signaling is most therapeutic

  • Guide development of small molecule inhibitors targeting specific interaction interfaces
    These approaches are supported by emerging evidence that anti-gC1qR therapy slows tumor growth in animal models of breast cancer and may be beneficial in treating specific types of angioedema . The methodological framework provides a systematic approach to translate basic C1QBP biology into clinically relevant applications.

What methodological considerations are important when studying C1QBP antibodies for inhibiting pathogen entry?

Since surface-exposed C1QBP serves as an entry route for numerous pathogens , C1QBP antibodies have potential as tools to inhibit pathogen entry. Key methodological considerations include:

• Infection model selection:

  • Choose appropriate cell lines expressing surface C1QBP

  • Establish baseline infection rates for each pathogen

  • Develop quantifiable readouts (e.g., fluorescent pathogen tracking, PCR-based quantification)

• Antibody epitope considerations:

  • Test panel of antibodies recognizing different C1QBP domains

  • Determine which epitopes are accessible on surface-exposed C1QBP

  • Identify antibodies that specifically block pathogen binding without affecting host functions

• Dose-response and timing studies:

  • Pre-incubate cells with antibodies before pathogen exposure

  • Test different antibody concentrations to establish IC50 values

  • Determine optimal treatment duration

• Specificity controls:

  • Include isotype control antibodies

  • Perform studies in C1QBP-knockdown/knockout cells

  • Use soluble C1QBP to compete with antibody binding

• Mechanism assessment:

  • Distinguish between blocking initial binding versus preventing internalization

  • Perform live-cell imaging with fluorescently-labeled antibodies and pathogens

  • Analyze co-localization during the infection process
    These methodological approaches address C1QBP's role as a "danger associated" or "damage associated" molecular pattern with enhanced surface expression during infection or inflammatory processes , potentially offering new strategies for preventing pathogen entry.

How can I resolve issues with non-specific binding when using C1QBP antibodies?

Non-specific binding is a common challenge when working with C1QBP antibodies. To address this issue systematically:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • For C1QBP studies, 0.1% (w/v) bovine serum albumin in buffer may be effective

  • Consider longer blocking times (1-2 hours at room temperature)

• Adjust antibody concentration:

  • Perform titration experiments to determine optimal concentration

  • For AlphaScreen assays with anti-C1QBP mAbs, optimize in pair-wise fashion with biotinylated gC1qR

  • Follow manufacturer guidelines for starting dilutions

• Modify washing procedures:

  • Increase number of washes (5-6 washes instead of 3)

  • Add low concentrations of detergent (0.05% Tween-20)

  • Consider higher salt concentrations (140-500 mM NaCl) for more stringent washes

• Include competitors for non-specific interactions:

  • Add non-immune serum from the same species as secondary antibody

  • Include 0.1-1% carrier proteins in antibody diluent

• Validate signal specificity:

  • Perform peptide competition assays

  • Include C1QBP-depleted samples as negative controls

  • Compare multiple antibodies targeting different C1QBP epitopes
    These optimization strategies are based on protocols that have been successful in published C1QBP research, including SPR and AlphaScreen assays , and should help distinguish specific from non-specific signals.

What are the key considerations for preserving C1QBP structure during immunoprecipitation and pull-down assays?

C1QBP exists as a trimer in its native state , which is critical for many of its functions. To preserve its structure during immunoprecipitation and pull-down assays:

• Buffer composition optimization:

  • Use buffers that maintain native structure: 20 mM HEPES (pH 7.4), 140 mM NaCl

  • Include protease inhibitor cocktails to prevent degradation

  • Consider adding low concentrations of stabilizing agents (glycerol 5-10%)

• Lysis condition considerations:

  • Use non-denaturing lysis buffers to maintain trimeric structure

  • Avoid harsh detergents (SDS) that disrupt protein-protein interactions

  • Consider mild detergents (0.05% Tween-20) or digitonin for membrane extraction

• Temperature management:

  • Perform all steps at 4°C to minimize denaturation

  • Avoid freeze-thaw cycles of samples containing C1QBP

  • Pre-chill all buffers and equipment

• Cross-linking strategies:

  • Consider mild cross-linking to stabilize the trimeric structure

  • Use membrane-permeable cross-linkers for intact cell studies

  • Optimize cross-linker concentration to avoid over-cross-linking

• Antibody selection considerations:

  • Choose antibodies that recognize native conformational epitopes

  • Consider using a mixture of antibodies targeting different epitopes

  • Employ gentle elution conditions (competitive peptide elution rather than harsh pH changes)
    These methodological considerations align with published approaches for studying C1QBP interactions, including the SPR methods used to characterize antibody binding to wild-type and mutant forms of C1QBP .

How can surface plasmon resonance be optimized for studying C1QBP antibody interactions with various ligands?

Surface plasmon resonance (SPR) has been successfully used to characterize C1QBP interactions with antibodies and other ligands. For optimal results:

• Surface preparation strategies:

  • Due to C1QBP's asymmetric charge distribution , avoid direct coupling to biosensor surface

  • Use streptavidin surface to capture biotinylated C1QBP

  • Include polyethylene glycol-based spacer arms to minimize steric hindrance

• Experimental design parameters:

  • For antibody kinetic analysis, use concentration ranges from 1.6 to 1000 nM

  • Consider flow rates of 30 μl/min for optimal mass transport

  • Design cycles with 2-minute association phases followed by appropriate dissociation phases (up to 60 minutes for high-affinity interactions)

• Regeneration optimization:

  • Develop regeneration protocols that maintain ligand activity

  • For C1QBP studies, three consecutive injections of 0.1 M glycine (pH 2.2), 2 M NaCl for 30 seconds has proven effective

  • Validate consistent binding capacity after regeneration

• Data analysis approaches:

  • Use appropriate binding models (Langmuir, bivalent analyte kinetic model)

  • Perform reference subtraction to account for non-specific binding

  • Utilize Biacore T-200 Evaluation software v3.2 or similar tools for fitting kinetic parameters

• Validation experiments:

  • Compare SPR results with orthogonal methods (ELISA, AlphaScreen)

  • Confirm specificity with competition assays

  • Verify data reproducibility across multiple independent experiments
    These SPR optimization strategies have been successfully applied to characterize the binding of monoclonal antibodies like mAb-1 to various forms of C1QBP, revealing important insights about epitope accessibility and binding kinetics .

What approaches can be used to detect and quantify cell surface-exposed C1QBP versus intracellular forms?

Distinguishing between cell surface-exposed and intracellular C1QBP is crucial given its different functions in these locations. Key methodological approaches include:

• Flow cytometry-based detection:

  • Non-permeabilized cells: Detect only surface-exposed C1QBP

  • Permeabilized cells: Detect total cellular C1QBP

  • Calculate ratio to determine surface exposure levels

  • Use domain-specific antibodies to assess orientation on cell surface

• Surface biotinylation techniques:

  • Label cell surface proteins with membrane-impermeable biotinylation reagents

  • Immunoprecipitate with C1QBP antibodies

  • Detect biotinylated fraction with streptavidin

  • Compare with total C1QBP levels

• Protease protection assays:

  • Treat intact cells with proteases that cannot penetrate membrane

  • Analyze C1QBP degradation by immunoblotting

  • Protected fraction represents intracellular pool

• Super-resolution microscopy techniques:

  • Perform immunofluorescence without permeabilization

  • Use membrane markers for co-localization

  • Quantify signal intensity at membrane versus intracellular compartments

  • Apply 3D reconstruction to visualize distribution patterns

• Electron microscopy immunogold labeling:

  • Apply primary C1QBP antibodies to non-permeabilized or permeabilized samples

  • Detect with gold-conjugated secondary antibodies

  • Provides high-resolution localization at plasma membrane versus mitochondria
    These approaches are particularly relevant given that gC1qR becomes localized at the cellular surface in response to various stimuli, where it serves as a route for pathogens and acts as an inflammatory trigger , despite its normal intracellular functions in mitochondrial biogenesis.

How should researchers interpret differences in binding affinity between various anti-C1QBP monoclonal antibodies?

Interpreting differences in binding affinity between anti-C1QBP monoclonal antibodies requires careful analysis:

• Kinetic parameter comparison:

  Antibody	Association Rate (kon)	Dissociation Rate (koff)	Apparent Affinity (KD)	Epitope Region
  mAb-12/13	Moderate	Very slow	<10 nM	Non-loop region
  mAb-3/5/18	Moderate	Faster	<10 nM	Non-loop region
  mAb-1	Very fast	Moderate	<10 nM	First acidic loop
  These differences reflect:

  • Epitope accessibility (fast kon for exposed epitopes like mAb-1)

  • Structural stability of antibody-antigen complex (slow koff for stable complexes)

  • Whether binding follows Langmuir kinetics or more complex models

• Structural interpretation:

  • Antibodies like mAb-12/13 that follow Langmuir binding models likely bind to a single epitope on the C1QBP trimer

  • Higher response levels for mAb-1, mAb-3, mAb-5, and mAb-18 suggest they may bind multiple sites on the C1QBP trimer

  • Removal of the second acidic loop (gC1qR-D2) enhances mAb-1 binding, indicating interdomain influences on epitope accessibility

• Functional correlation:

  • Compare binding parameters with functional inhibition of C1QBP-ligand interactions

  • Determine whether high affinity correlates with better inhibition of pathogen binding

  • Assess whether certain epitopes are more effective targets for therapeutic development
    This analytical framework provides insights beyond simple affinity measurements, revealing important structural and functional characteristics of C1QBP and its interactions with antibodies.

What criteria should be used to evaluate the potential of C1QBP antibodies for therapeutic applications?

Evaluating C1QBP antibodies for therapeutic potential requires a comprehensive assessment framework:

• Target specificity metrics:

  • Cross-reactivity with related proteins (minimal desired)

  • Specificity for disease-relevant C1QBP conformations/locations

  • Ability to distinguish surface-exposed versus intracellular C1QBP

• Functional inhibition parameters:

  • IC50 values for blocking pathogen binding or inflammatory signaling

  • Comparison with existing inhibitors or competing antibodies

  • Preservation of essential C1QBP functions in normal cells

• Pharmacological properties:

  • Binding kinetics (ideally kon >1×10^5 M^-1s^-1, koff <1×10^-4 s^-1)

  • Stability under physiological conditions

  • Tissue penetration capabilities

• In vivo efficacy criteria:

  • Dose-dependent response in disease models

  • Comparison with proof-of-concept studies showing anti-gC1qR therapy slowing tumor growth in breast cancer models

  • Potential application to other diseases like specific types of angioedema

• Safety assessment metrics:

  • On-target, off-tissue effects

  • Immunogenicity profile

  • Complement or antibody-dependent cellular cytotoxicity potential
    This evaluation framework builds on emerging evidence that therapeutic targeting of cell surface-exposed gC1qR may be beneficial in certain cancers and potentially in other diseases , providing a systematic approach to advance the most promising antibody candidates.

How can researchers leverage new structural insights about C1QBP's acidic loops to develop more specific therapeutic agents?

Recent structural insights into C1QBP's acidic loops provide opportunities for developing targeted therapeutics:

• Structure-guided antibody engineering:

  • Utilize the 2.2 Å resolution structure of gC1qR-DD

  • Design antibodies specifically targeting functional regions of acidic loops

  • Engineer antibody complementarity-determining regions (CDRs) to enhance specificity for disease-relevant conformations

• Peptide mimetic development:

  • Design peptides based on acidic loop sequences

  • Test competitive inhibition of C1QBP-ligand interactions

  • Optimize for stability, specificity, and pharmacokinetic properties

• Small molecule screening approaches:

  • Use the defined structure of acidic loops to create in silico screening models

  • Design focused libraries targeting the charge distribution and structural features

  • Validate hits with competition assays against characterized antibodies like mAb-1

• Bifunctional molecule development:

  • Create molecules linking acidic loop-binding domains with effector functions

  • Explore targeted protein degradation approaches (PROTACs) specific for surface-exposed C1QBP

  • Develop antibody-drug conjugates targeting acidic loop epitopes

• Computational design strategies:

  • Perform molecular dynamics simulations of acidic loop flexibility

  • Identify "druggable" pockets that appear transiently

  • Design conformation-specific inhibitors
    These approaches leverage the structural understanding gained from techniques like molecular replacement using PHASER and the PHENIX software suite , as well as the characterization of antibodies like mAb-1 that specifically recognize the first acidic loop of C1QBP .

What methodological advances are needed to better understand the regulation of C1QBP surface expression during disease states?

Understanding the regulation of C1QBP surface expression during disease states requires several methodological advances:

• Live-cell imaging technologies:

  • Develop non-perturbing labeling strategies for C1QBP in living cells

  • Implement time-lapse imaging to track C1QBP translocation to the surface

  • Correlate with disease-relevant stimuli (infection, inflammation, oncogenic signaling)

• Single-cell analysis approaches:

  • Apply mass cytometry (CyTOF) with C1QBP antibodies

  • Correlate surface C1QBP with other markers of cell state

  • Identify heterogeneity within diseased tissues

• Pathway analysis tools:

  • Develop reporter systems for monitoring C1QBP trafficking

  • Screen for regulators of surface translocation

  • Identify druggable nodes in pathways controlling surface expression

• Improved animal models:

  • Generate knockin mice with tagged C1QBP for in vivo tracking

  • Develop tissue-specific and inducible C1QBP expression systems

  • Create humanized models for testing therapeutic antibodies

• Biomarker development approaches:

  • Establish protocols for detecting soluble versus membrane-bound C1QBP

  • Correlate with disease progression

  • Develop companion diagnostics for anti-C1QBP therapies
    These methodological advances would address the current knowledge gap regarding how C1QBP undergoes spatiotemporal changes in its distribution in response to various cellular and systemic stimuli , particularly focusing on its enhanced surface expression during infection or inflammatory processes when it acts as a "danger associated" or "damage associated" molecular pattern .