ITIH1 Antibody, Biotin conjugated

What is ITIH1 and what are its key biological functions?

ITIH1 (Inter-alpha-trypsin inhibitor heavy chain H1) is a heavy chain member of the ITIH family that is primarily synthesized in the liver and circulates in the plasma. The protein contains a signal sequence, propeptide, and a conserved von-Willebrand type A domain characteristic of other heavy chains in this family . ITIH1 notably includes a C-terminal extension multicopper oxidase domain (also present in ITIH3) that undergoes trimming to reveal a C-terminal aspartic acid residue, enabling crosslinking to chondroitin sulfate .

Functionally, ITIH1 serves as the HC1 component of Inter-alpha-inhibitor, a protease inhibitor complex composed of three subunits (HC1, HC2, and bikunin) linked via a chondroitin sulfate moiety . ITIH1 can alternatively associate with hyaluronan (HA) to form the Serum-derived hyaluronan associated protein (SHAP)-hyaluronan complex, which stabilizes HA-rich extracellular matrices during inflammatory processes and ovulation . Research has demonstrated that ITIH1 potentiates CD-44-mediated leukocyte adhesion to hyaluronan substratum and helps recruit immune cells to inflammation sites . Additionally, ITIH1 has shown antitumoral properties by increasing cell attachment and reducing metastases .

What applications are most suitable for biotin-conjugated ITIH1 antibodies?

Biotin-conjugated ITIH1 antibodies are particularly valuable in several experimental applications:

Biotin-conjugated ITIH1 antibodies are particularly effective in sandwich enzyme immunoassays, where the biotin conjugation allows for high-affinity binding to streptavidin-HRP complexes, enhancing detection sensitivity in complex biological samples . When working with tissue samples, these antibodies facilitate visualization of ITIH1 localization patterns and expression levels across different tissue compartments .

How should researchers select between different epitope regions for ITIH1 antibody targets?

The choice of epitope region targeted by an ITIH1 antibody significantly impacts experimental outcomes and should be guided by specific research questions:

When studying ITIH1 processing or cleavage events, antibodies targeting different regions can provide complementary information. For investigation of ITIH1's role in forming complexes with chondroitin sulfate or hyaluronan, antibodies recognizing the C-terminal region may be more informative since this region contains the critical aspartic acid residue involved in crosslinking . Conversely, when examining protein-protein interactions involving the von Willebrand domain, antibodies targeting the middle region may yield more relevant results .

What are the optimal validation protocols for confirming ITIH1 antibody specificity?

Rigorous validation of ITIH1 antibody specificity is crucial before proceeding with experimental applications. A comprehensive validation approach should include:

• Western blot analysis: Verify that the antibody detects a band of the expected molecular weight (approximately 68kDa for ITIH1) . Multiple tissue/cell types should be tested, including those known to express ITIH1 (primarily liver) and those with minimal expression.

• Knockout/knockdown controls: Compare antibody reactivity in samples with normal ITIH1 expression versus those where ITIH1 has been silenced through genetic manipulation or RNA interference.

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish signal in subsequent detection assays. For instance, with antibodies raised against synthetic peptides from the C-terminal region, confirming specificity by blocking with the immunogen peptide "QPSPTHSSSNTQRLPDRVTGVDTDPHFIIHVPQKEDTLCFNINEEPGVIL" is recommended .

• Cross-reactivity assessment: Test reactivity against closely related family members (ITIH2, ITIH3, ITIH4) to ensure specificity within the ITIH family.

• Correlation with orthogonal methods: Results should be confirmed using alternative detection methods or antibodies targeting different epitopes of ITIH1.

For biotin-conjugated antibodies specifically, additional validation should confirm that the conjugation process has not altered antibody specificity or binding efficiency through comparative analysis with the unconjugated version.

How can researchers optimize protocols for detecting ITIH1 in complex biological samples?

Detecting ITIH1 in complex biological samples requires careful optimization of several parameters:

• Sample preparation optimization:

  • For plasma/serum: Dilute samples (typically 1:100 to 1:500) in appropriate buffer to minimize matrix effects

  • For tissue samples: Optimize homogenization methods to maintain protein integrity while maximizing extraction efficiency

  • Consider pre-clearing samples with protein A/G if background issues persist

• Antibody dilution optimization: Titrate both primary and detection antibodies to determine optimal concentrations that maximize specific signal while minimizing background.

• Signal enhancement strategies: For low-abundance samples, consider:

  • Extended incubation times (2 hours at 37°C as standard for many ITIH1 assays)

  • Signal amplification systems compatible with biotin (e.g., tyramide signal amplification)

  • Concentration of samples prior to analysis

• Detection system selection: When using biotin-conjugated antibodies, ensure compatible detection reagents are employed and that endogenous biotin is blocked in biotin-rich samples (especially liver tissues where ITIH1 is abundant).

What strategies help address the challenge of endogenous biotin interference when using biotin-conjugated ITIH1 antibodies?

Endogenous biotin can significantly interfere with detection systems utilizing biotin-conjugated antibodies, especially in biotin-rich tissues like liver, which is also the primary site of ITIH1 expression. Researchers should implement these strategies:

• Biotin blocking steps:

  • Pretreat samples with avidin followed by biotin before adding the biotin-conjugated antibody

  • Use commercial biotin blocking kits that employ sequential avidin and biotin treatments

• Sample preparation modifications:

  • For fixed tissues, extend fixation time moderately to reduce accessible endogenous biotin

  • Consider non-biotin-based detection alternatives for highly biotin-rich samples

• Control implementation:

  • Include negative controls treated with non-specific biotin-conjugated antibodies of the same isotype

  • Run parallel detection with unconjugated primary antibody followed by biotinylated secondary antibody to assess contribution of endogenous biotin

• Alternative conjugation consideration:

  • For samples with persistent biotin interference, consider alternative conjugation methods (fluorophores, enzymes) or detection systems

• Quantitative adjustment:

  • Implement computational correction based on signal from biotin-only controls when analyzing results

When working with liver samples specifically, where both ITIH1 expression and endogenous biotin are abundant, comprehensive blocking protocols are essential for accurate results.

What controls are essential when using biotin-conjugated ITIH1 antibodies?

A robust experimental design with appropriate controls is critical when working with biotin-conjugated ITIH1 antibodies:

For sandwich immunoassays specifically, include the following additional controls:

• Standard curve using recombinant ITIH1 protein (e.g., Recombinant Human ITIH1 His-tag Protein)

• Sample diluent blank wells (100 μL Standard/sample Diluent (R1))

• Serial dilution of positive samples to verify linearity of detection

These controls help distinguish true ITIH1 signal from technical artifacts and enable proper quantitative analysis of results.

How do storage conditions affect the performance of biotin-conjugated ITIH1 antibodies?

Proper storage is crucial for maintaining the integrity and performance of biotin-conjugated ITIH1 antibodies:

• Temperature considerations:

  • Store conjugated antibodies at 4°C for short-term use (up to 12 months)

  • For longer storage (up to 24 months), dilute with up to 50% glycerol and store at -2°C to -8°C

  • Avoid repeated freeze-thaw cycles which can compromise both enzyme activity and antibody binding

• Light protection:

  • Store all biotin-conjugated antibodies in light-protected vials or cover with light-protecting material (e.g., aluminum foil)

  • Minimize exposure to light during experimental procedures to prevent photobleaching

• Buffer and additives:

  • Most commercial biotin-conjugated ITIH1 antibodies are provided in 1x PBS buffer

  • Some formulations may include preservatives or stabilizers

  • When diluting, use buffers recommended by the manufacturer

• Aliquoting recommendations:

  • Upon receipt, consider creating small working aliquots to minimize repeated freeze-thaw cycles

  • Document the date of first use and number of freeze-thaw cycles for each aliquot

• Stability indicators:

  • Monitor for signs of degradation (precipitation, color change)

  • Include positive controls with each experiment to confirm antibody performance over time

Following these storage guidelines will help ensure consistent experimental results and maximize the usable lifespan of biotin-conjugated ITIH1 antibodies.

What are common causes of high background when using biotin-conjugated ITIH1 antibodies and how can they be addressed?

High background is a frequent challenge when working with biotin-conjugated antibodies. The following table outlines common causes and solutions specific to ITIH1 detection:

For particularly challenging samples, consider implementing a step-wise optimization approach:

• Begin with manufacturer's recommended protocol

• Test increased washing stringency (more wash steps, higher salt concentration)

• Evaluate different blocking reagents (BSA vs. casein vs. normal serum)

• Adjust antibody incubation conditions (temperature, time, concentration)

• Implement specific blocking for endogenous biotin if necessary

How should researchers interpret differences in ITIH1 detection patterns across different biological samples?

Interpretation of ITIH1 detection patterns requires consideration of its biological characteristics and experimental variables:

• Tissue-specific expression patterns:

  • ITIH1 is primarily synthesized in the liver

  • Lower expression levels in other tissues should be carefully validated

  • Unexpected detection in non-liver tissues may represent either novel expression sites or technical artifacts

• Molecular weight variations:

  • Full-length ITIH1 appears at approximately 68kDa

  • Higher molecular weight bands may represent:

    • ITIH1 in complex with other proteins

    • Post-translationally modified forms

    • Incompletely denatured protein

  • Lower molecular weight bands may indicate:

    • Proteolytic processing

    • Degradation

    • Alternative splicing variants

• Post-translational modifications:

  • ITIH1 can be linked to chondroitin sulfate or hyaluronan

  • These modifications significantly alter apparent molecular weight

  • Different epitope-targeting antibodies may have variable recognition of modified forms

• Complex formation interpretation:

  • ITIH1 functions as part of larger complexes (Inter-alpha-inhibitor)

  • Signal patterns may reflect intact complexes versus free ITIH1

  • Consider using denaturing versus non-denaturing conditions to distinguish forms

• Quantitative considerations:

  • Normalize ITIH1 levels to appropriate housekeeping proteins or total protein

  • Consider liver-specific markers when comparing samples with varying hepatocyte content

  • Establish physiological reference ranges from healthy control samples

How can researchers effectively use ITIH1 antibodies to study its role in inflammatory processes?

ITIH1's involvement in inflammatory processes, particularly through its association with hyaluronan, presents unique research opportunities:

• Co-localization studies:

  • Use biotin-conjugated ITIH1 antibodies in combination with markers for:

    • Hyaluronan (using biotinylated hyaluronan binding protein)

    • TSG-6 (tumor necrosis factor-stimulated gene-6), which mediates ITIH1 transfer to hyaluronan

    • CD44 (hyaluronan receptor involved in leukocyte adhesion)

  • Implement multi-color fluorescence microscopy with spectrally distinct fluorophores

• Functional assays:

  • Leukocyte adhesion assays with ITIH1-depleted versus normal matrices

  • Migration assays in the presence of ITIH1 antibodies that may disrupt ITIH1-hyaluronan interactions

  • Matrix stability assessments under inflammatory conditions

• Inflammation models:

  • Track ITIH1-hyaluronan complex formation (SHAP-HA) during progression of inflammatory conditions

  • Compare complex formation in acute versus chronic inflammation

  • Correlate ITIH1 complex levels with inflammatory markers

• Clinical correlations:

  • Analyze SHAP-HA complex levels in patient samples from inflammatory conditions

  • The complex has been shown to be upregulated in patients with chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma

  • Compare biomarker potential across different inflammatory etiologies

• Intervention studies:

  • Test treatments that modulate ITIH1-hyaluronan interactions

  • Evaluate effects on inflammatory processes

  • Monitor changes in ITIH1 complex formation as an outcome measure

By leveraging biotin-conjugated ITIH1 antibodies in these approaches, researchers can gain deeper insights into the protein's functional roles in inflammatory regulation and potential therapeutic targets.

How can ITIH1 antibodies contribute to cancer research based on its reported antitumoral properties?

ITIH1 has demonstrated antitumoral properties by increasing cell attachment and reducing metastases . Researchers can leverage ITIH1 antibodies to explore these effects through:

• Expression profiling:

  • Compare ITIH1 levels across tumor versus normal tissues

  • Correlate expression with tumor invasiveness and metastatic potential

  • Analyze changes during cancer progression

• Mechanistic studies:

  • Investigate ITIH1's interaction with extracellular matrix components in tumor microenvironments

  • Examine effects on tumor cell adhesion, migration, and invasion

  • Study relationship between ITIH1 and cancer-relevant signaling pathways

• Prognostic biomarker evaluation:

  • Assess correlation between ITIH1 expression and patient outcomes

  • Develop standardized ITIH1 detection protocols for clinical samples

  • Validate cutoff values for prognostic significance

• Therapeutic targeting approaches:

  • Explore ITIH1 supplementation as a metastasis-inhibiting strategy

  • Develop methods to enhance endogenous ITIH1 activity

  • Test combination approaches with existing cancer therapies

• Model systems development:

  • Generate ITIH1 reporter cell lines for real-time monitoring

  • Create animal models with altered ITIH1 expression

  • Develop in vitro systems that recapitulate ITIH1's extracellular matrix stabilizing functions

These approaches can help elucidate ITIH1's role in tumor biology and potentially identify new therapeutic strategies based on its antitumoral properties.

What emerging techniques might enhance the utility of biotin-conjugated ITIH1 antibodies in research?

Several cutting-edge approaches can extend the capabilities of biotin-conjugated ITIH1 antibodies:

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions involving ITIH1 and potential binding partners

  • Visualize ITIH1 interactions with hyaluronan, chondroitin sulfate, or TSG-6 in situ

  • Quantify interaction dynamics under different physiological conditions

• Mass cytometry (CyTOF):

  • Integrate ITIH1 detection into high-parameter immune profiling

  • Correlate ITIH1 levels with multiple cell surface and intracellular markers

  • Profile ITIH1 in rare cell populations

• Spatial transcriptomics integration:

  • Combine ITIH1 protein detection with spatial mRNA analysis

  • Map ITIH1 production versus localization in tissues

  • Correlate with expression of functionally related genes

• Organoid and 3D culture systems:

  • Monitor ITIH1 distribution and function in three-dimensional tissue models

  • Study dynamics of ITIH1-matrix interactions in controlled microenvironments

  • Test effects of ITIH1 modulation on tissue organization

• Click chemistry modifications:

  • Use biotin-conjugated antibodies as scaffolds for additional functionalization

  • Attach stimulus-responsive moieties for controlled detection or perturbation

  • Develop multi-functional probes for simultaneous detection and manipulation

• Intravital microscopy applications:

  • Track ITIH1 dynamics in living organisms

  • Monitor real-time changes during inflammatory processes

  • Observe interactions with immune cells in native tissue environments

These emerging approaches can provide unprecedented insights into ITIH1 biology while maximizing the utility of biotin-conjugated antibodies.

How might ITIH1 antibodies be integrated into multiplexed detection systems for comprehensive proteomic analysis?

Integrating ITIH1 detection into multiplexed systems enables more comprehensive analysis of biological pathways:

• Multiplex immunoassay platforms:

  • Include ITIH1 in custom antibody panels targeting liver-secreted proteins

  • Develop bead-based assays incorporating ITIH1 alongside related biomarkers

  • Validate antibody performance in multiplex versus singleplex formats

• Sequential detection strategies:

  • Optimize antibody stripping and reprobing protocols for biotin-conjugated antibodies

  • Develop order-specific protocols that maximize epitope preservation

  • Implement computational approaches to correct for incomplete stripping

• Spectral unmixing approaches:

  • Use biotin-conjugated ITIH1 antibodies with spectrally distinct streptavidin conjugates

  • Combine with antibodies using orthogonal detection systems

  • Apply advanced image analysis for accurate signal separation

• Microfluidic integration:

  • Incorporate ITIH1 detection into microfluidic-based proteomics

  • Develop protocols for extremely low sample volume requirements

  • Enable automated, high-throughput profiling of ITIH1 alongside other markers

• Single-cell proteomics applications:

  • Adapt ITIH1 detection for single-cell resolution techniques

  • Correlate with other protein targets at individual cell level

  • Characterize cellular heterogeneity in ITIH1 production and binding

By incorporating these approaches, researchers can position ITIH1 analysis within broader biological contexts while maintaining specific and sensitive detection capabilities.