ITIH1 Antibody

What is ITIH1 and why is it significant in research?

ITIH1 (Inter-alpha-trypsin inhibitor heavy chain 1) is a member of the inter-alpha-trypsin inhibitor heavy chain family that plays crucial roles in multiple physiological and pathological processes. It has significant research importance due to its involvement in metabolic disorders, cancer progression, and potential as a biomarker. In metabolic disease models, ITIH1 has been identified as a protein highly secreted by the liver in association with insulin resistance . In cancer research, ITIH1 expression is frequently altered, with studies showing significant downregulation in liver hepatocellular carcinoma (LIHC) compared to normal tissue . ITIH1's diverse biological functions and disease associations make it an important target for antibody-based research applications.

What detection methods are available for ITIH1 in biological samples?

Several methods can be employed to detect ITIH1 in biological samples:

• Enzyme-Linked Immunosorbent Assay (ELISA): Sandwich ELISA techniques using antibodies specific for human ITIH1 allow quantitative measurement in serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids . This method employs capture antibodies pre-coated onto microplates that bind ITIH1 in samples, followed by detection antibodies and enzyme conjugates for visualization.

• Western Blotting: This technique enables detection and semi-quantification of ITIH1 protein expression, as demonstrated in studies examining ITIH1 levels in renal cell carcinoma (RCC) cells compared to normal kidney cells (HK-2) .

• Immunohistochemistry (IHC): While not explicitly detailed in the provided sources, IHC is recommended for validating ITIH1 expression at the protein level in tissue samples, supplementing mRNA-based findings .

What are the critical factors for successful ITIH1 antibody experiments?

When designing experiments using ITIH1 antibodies, researchers should consider:

• Antibody specificity: High specificity is crucial, as cross-reactivity with analogues can compromise results. ELISA kits should have demonstrated high sensitivity and excellent specificity for ITIH1 detection without significant cross-reactivity .

• Sample preparation: Different biological samples require appropriate processing. For cell cultures, standard protocols involve cell lysis followed by protein extraction. For tissue samples, proper homogenization techniques are essential.

• Assay validation: Positive and negative controls should be included to validate antibody performance, especially when working with new antibody lots or experimental systems.

• Quantification approach: For precise measurement, standard curves with recombinant ITIH1 protein should be established when using quantitative techniques like ELISA.

How can ITIH1 antibodies be utilized in metabolic disease research?

ITIH1 antibodies serve as valuable tools in metabolic disease research based on recent findings indicating ITIH1's role in insulin resistance. Research has demonstrated that:

• Neutralization experiments: ITIH1 neutralizing antibodies can overcome systemic glucose intolerance and insulin resistance in mice . Researchers can design similar neutralization experiments to investigate ITIH1's causal role in metabolic conditions.

• Mechanistic studies: Antibodies can help elucidate how ITIH1 contributes to insulin resistance. For example, glycosyl-modified ITIH1 has been shown to deposit on hyaluronan surrounding adipose tissue and skeletal muscle, creating a physical barrier between insulin and its receptor . Antibodies can be used to visualize this deposition pattern.

• Biomarker evaluation: ITIH1 expression in liver positively correlates with surrogate markers for diabetes in patients with impaired glucose tolerance or overt diabetes . Researchers can use antibodies to develop assays measuring ITIH1 levels as potential biomarkers.

What are the most effective experimental designs to study ITIH1's role in cancer progression?

Based on current research, the following experimental approaches are recommended:

• Gene expression manipulation combined with antibody detection:

  • ITIH1 knockdown using siRNAs (as demonstrated in RCC cell lines A498, ACHN, and 786-O), followed by antibody-based detection of ITIH1 and related pathway proteins .

  • ITIH1 overexpression experiments, with subsequent analysis of cancer cell properties including proliferation, migration, and invasion .

• Pathway analysis designs:

  • Combined treatment approaches using ITIH1 antibodies with pathway inhibitors (such as JSH-23 for NF-κB pathway inhibition) to elucidate molecular mechanisms .

  • Western blotting for multiple pathway components (e.g., phosphorylated NF-κB, IκB, IKK, Cyclin D1) following ITIH1 manipulation .

• Correlation studies:

  • Analysis of ITIH1 expression in relation to tumor mutational burden (TMB), microsatellite instability (MSI), and DNA methylation patterns across cancer types .

  • Validation across multiple independent datasets to confirm findings, as exemplified by studies using TCGA and GEO datasets .

How should researchers interpret conflicting data regarding ITIH1 expression in different cancer types?

Researchers encountering conflicting ITIH1 expression data should consider:

• Tissue context specificity: ITIH1 expression and function can vary dramatically between tissue types. For example, ITIH1 appears highly expressed in normal liver but significantly downregulated in liver hepatocellular carcinoma (LIHC) , while showing different patterns in other cancers.

• Methodological considerations: Discrepancies may result from differences in detection methods (mRNA vs. protein), sample collection, or analysis techniques. This is exemplified by the observation that ITIH1 was downregulated in LIHC tissue samples from TCGA, which was validated in five independent GEO datasets .

• Basal expression levels: Meaningful changes should be interpreted in the context of basal expression. Some cancer types and corresponding normal tissues showed extremely low ITIH1 expression, making differences potentially less biologically significant .

• Environmental factors: In vitro vs. in vivo differences may occur due to microenvironmental factors, as noted in the discrepancy between ITIH1 expression in RCC tissues versus cell lines .

What are the optimal sample preparation protocols for ITIH1 antibody-based detection?

For optimal ITIH1 detection, researchers should follow these sample preparation guidelines:

• Cell culture samples:

  • Harvest cells during logarithmic growth phase

  • Wash cells with PBS to remove media components

  • Lyse cells using appropriate buffers containing protease inhibitors

  • Centrifuge lysates at high speed (≥10,000 g) to remove cellular debris

  • Quantify total protein concentration before antibody-based analysis

• Tissue samples:

  • Flash-freeze tissues immediately after collection

  • Homogenize tissues in proper buffer with protease inhibitors

  • Filter homogenates and centrifuge to remove debris

  • For ELISA applications, ensure samples are within the linear range of detection

• Serum/plasma samples:

  • Process blood samples consistently (timing between collection and processing)

  • Use appropriate anticoagulants as specified by the detection method

  • Centrifuge at recommended speeds to separate cellular components

  • Store aliquots at -80°C to avoid repeated freeze-thaw cycles

How can researchers validate the specificity of ITIH1 antibodies in their experimental systems?

To ensure antibody specificity for ITIH1:

• Positive and negative controls:

  • Use cell lines or tissues with known ITIH1 expression levels

  • Include ITIH1 knockdown samples (using siRNA as demonstrated in studies)

  • Use recombinant ITIH1 protein as a positive control

• Cross-reactivity testing:

  • Test antibody against related ITIH family members (ITIH2, ITIH3, ITIH4)

  • Validate specificity across species if working with non-human models

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of ITIH1

  • Confirm findings using alternative detection methods (e.g., mass spectrometry)

• Preabsorption controls:

  • Pre-incubate antibody with purified ITIH1 antigen before use in experiments

  • Signal should be significantly reduced if antibody is specific

What are the key considerations for quantifying ITIH1 in different experimental contexts?

Accurate ITIH1 quantification requires attention to:

• Standard curve optimization:

  • For ELISA, prepare fresh standards for each experiment

  • Ensure the standard curve covers the physiological range of ITIH1 in the sample type

  • Use appropriate curve-fitting methods (typically 4-parameter logistic regression)

• Normalization strategies:

  • For Western blotting, normalize ITIH1 signal to appropriate housekeeping proteins

  • For tissue samples, account for cellular heterogeneity when interpreting results

  • Consider using multiple reference genes for RT-qPCR normalization

• Biological variability:

  • Include sufficient biological replicates (minimum n=3)

  • Consider diurnal or other temporal variations in ITIH1 expression

  • Account for age, sex, and disease status variables in human samples

How does ITIH1 contribute to insulin resistance and what methodologies best demonstrate this relationship?

ITIH1's contribution to insulin resistance has been characterized through several methodological approaches:

• Liver-specific gene deletion models: Studies using liver-specific deletion of the Gna13 gene in mice resulted in systemic glucose intolerance, with comparative secretome analysis identifying ITIH1 as responsible for the systemic insulin resistance in these models .

• Structural analysis: Glycosyl modification of ITIH1 facilitated its deposition on hyaluronan surrounding adipose tissue and skeletal muscle, creating a physical barrier between insulin and its receptor . This barrier mechanism can be demonstrated through:

  • Immunofluorescence microscopy to visualize ITIH1 deposition

  • Co-immunoprecipitation to assess ITIH1-hyaluronan interactions

  • Functional assays measuring insulin receptor signaling in the presence/absence of ITIH1

• Antibody neutralization experiments: Neutralization of secreted ITIH1 ameliorated glucose intolerance in obese mice , suggesting a therapeutic approach that can be further explored through:

  • Dose-response studies with neutralizing antibodies

  • Combination therapy approaches with established diabetes treatments

  • Long-term efficacy and safety evaluations

What signaling pathways does ITIH1 modulate in cancer cells and how can researchers effectively study these interactions?

Research has identified several key signaling pathways modulated by ITIH1 in cancer cells:

• NF-κB pathway: In renal cell carcinoma, ITIH1 knockdown significantly increased phosphorylation levels of NF-κB while decreasing IκB expression and increasing IKK, Cyclin D1, proliferating cell nuclear antigen, and α-smooth muscle actin expression . To study these interactions, researchers can:

  • Use phospho-specific antibodies to track activation status of pathway components

  • Employ pathway inhibitors (like JSH-23) in combination with ITIH1 manipulation

  • Perform chromatin immunoprecipitation to identify NF-κB binding to target genes following ITIH1 modulation

• DNA methylation mechanisms: ITIH1 expression showed significant negative correlation with methylation in multiple cancers, with the highest correlation observed in LIHC . This relationship can be studied through:

  • Bisulfite sequencing of ITIH1 promoter regions

  • Correlation analysis between ITIH1 expression and DNA methyltransferases (DNMT1, DNMT2, DNMT3A, and DNMT3B)

  • Treatment with demethylating agents to assess ITIH1 re-expression

• Immune microenvironment interactions: ITIH1 expression has been associated with immune components in the tumor microenvironment . This can be investigated through:

  • Multiplex immunohistochemistry to visualize ITIH1 in relation to immune cell populations

  • Co-culture experiments with immune cells and cancer cells with variable ITIH1 expression

  • Analysis of cytokine/chemokine profiles following ITIH1 modulation

What is the significance of ITIH1 as a biomarker in hepatocellular carcinoma and how can antibody-based approaches improve its clinical utility?

ITIH1 shows significant potential as a biomarker in hepatocellular carcinoma (HCC):

How can researchers address contradictory findings regarding ITIH1 expression between tissue samples and cell culture models?

The discrepancy between ITIH1 expression in tissues versus cell lines (as noted in RCC research) presents a methodological challenge. Researchers can address this through:

• Comprehensive experimental design:

  • Parallel analysis of matched tissue samples and derived cell lines

  • Inclusion of primary cell cultures alongside established cell lines

  • Three-dimensional culture systems to better recapitulate tissue architecture

• Microenvironmental considerations:

  • Co-culture experiments incorporating stromal components

  • Manipulation of culture conditions (oxygen levels, growth factors, extracellular matrix)

  • In vivo validation of cell culture findings using xenograft or orthotopic models

• Multi-omics approach:

  • Integration of transcriptomic, proteomic, and epigenomic data

  • Analysis of post-translational modifications affecting ITIH1 function but not expression

  • Investigation of ITIH1 protein stability and turnover rates in different contexts

What are the methodological challenges in studying ITIH1's role across different disease contexts, and how can they be overcome?

ITIH1 functions differently across disease contexts, presenting several methodological challenges:

• Tissue-specific regulation:

  • Challenge: ITIH1 shows variable expression and function across tissues

  • Solution: Develop tissue-specific conditional knockout models rather than global deletion

  • Solution: Use tissue-specific promoters for targeted ITIH1 overexpression or knockdown

• Temporal dynamics:

  • Challenge: ITIH1's role may vary during disease progression

  • Solution: Implement inducible gene manipulation systems for temporal control

  • Solution: Design longitudinal studies with serial sampling at defined disease stages

• Translational relevance:

  • Challenge: Findings in model systems may not translate to human disease

  • Solution: Validate findings across multiple species and model systems

  • Solution: Establish collaborations with clinical researchers for access to human samples

  • Solution: Develop humanized animal models for more relevant disease modeling

How should researchers optimize ITIH1 antibody-based therapeutic approaches based on current evidence?

To develop ITIH1 antibody-based therapeutic approaches:

• Epitope selection strategy:

  • Target functional domains of ITIH1 identified through structure-function studies

  • For metabolic disorders, target regions involved in hyaluronan binding

  • For cancer applications, consider targeting regions that affect ITIH1's interaction with signaling pathways

• Delivery optimization:

  • Evaluate tissue-specific delivery methods to target ITIH1 in relevant compartments

  • Consider antibody format (full IgG vs. fragments) based on required tissue penetration

  • Test combination with tissue-targeting moieties for enhanced specificity

• Efficacy evaluation framework:

  • Establish clear, disease-relevant endpoints (e.g., glucose tolerance for metabolic applications)

  • Determine dose-response relationships and optimal treatment schedules

  • Evaluate combination approaches with standard-of-care treatments

• Safety assessment strategy:

  • Consider ITIH1's physiological roles when designing safety studies

  • Monitor for immune-related adverse events with prolonged antibody administration

  • Develop biomarkers of target engagement to correlate with efficacy and toxicity