ITIH2 Antibody

What is ITIH2 and why is it important in research?

ITIH2 (inter-alpha-trypsin inhibitor heavy chain 2) is a 106.5 kDa protein that belongs to the inter-alpha-trypsin inhibitor (ITI) family. It plays crucial roles in stabilizing the extracellular matrix (ECM) through its ability to covalently bind to hyaluronan (HA) . This protein is significant in research due to its involvement in numerous physiological and pathological processes, including inflammation, cancer progression, and tissue repair mechanisms . ITIH2 is expressed in normal brain tissues and low-grade CNS cancers but notably absent in aggressive glioblastomas, suggesting its potential anti-invasive function .

Research methodologically approaches ITIH2 by studying its expression patterns across different tissues and disease states, and by investigating its interactions with other ECM components, particularly hyaluronan.

How do ITIH2 antibodies work in experimental applications?

ITIH2 antibodies function by specifically binding to ITIH2 protein epitopes, allowing for detection and quantification of this protein in various experimental contexts. These antibodies can be conjugated or unconjugated and are designed to work across multiple applications including:

• Western Blot (WB): Allowing visualization of ITIH2 at approximately 106 kDa under reducing conditions

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of ITIH2 in biological samples with sensitivity as low as 0.78 μg/ml

• Immunohistochemistry (IHC): For visualizing ITIH2 distribution in tissue sections

• Flow cytometry (FCM): For analyzing ITIH2 expression at the cellular level

Methodologically, researchers select antibodies based on the specific application, species reactivity requirements, and conjugation needs for their particular experimental design.

What are the recommended sample types for ITIH2 antibody applications?

ITIH2 antibodies can be used with multiple sample types depending on the research question and experimental design:

• Serum and plasma samples: For measuring circulating ITIH2 levels

• Tissue homogenates: For assessing ITIH2 expression in specific organs

• Cell culture supernatants: For studying secreted ITIH2

• Urine samples: For investigating ITIH2 excretion

When working with these sample types, it's methodologically important to follow proper collection and processing protocols. For instance, serum samples should be allowed to clot for two hours at room temperature before centrifugation, while plasma samples require appropriate anticoagulants and immediate processing to preserve protein integrity.

How should I optimize Western blot conditions for ITIH2 detection?

Optimizing Western blot conditions for ITIH2 detection requires careful attention to several methodological factors:

• Sample preparation: Use reducing conditions with appropriate lysis buffers (such as Western Blot Buffer Group 1 as mentioned in the R&D Systems protocol)

• Gel percentage: Select 8-10% SDS-PAGE gels to effectively resolve the 106.5 kDa ITIH2 protein

• Transfer conditions: Use PVDF membrane for optimal protein binding

• Antibody concentration: Based on available data, 2 μg/mL of primary anti-ITIH2 monoclonal antibody has been shown to effectively detect ITIH2 in HepG2 cell lysates

• Detection system: HRP-conjugated secondary antibodies work well for visualizing ITIH2 bands

Researchers should validate these conditions in their specific experimental systems, as protein expression levels can vary across cell types and treatments.

What is the optimal protocol for ELISA-based quantification of ITIH2?

For optimal ELISA-based quantification of ITIH2, follow this methodological approach:

• Principle selection: Use a sandwich ELISA format, which offers high specificity and sensitivity (down to 0.78 μg/ml) for ITIH2 detection

• Antibody pairing: Use pre-coated plates with capture antibody specific to ITIH2, followed by an HRP-conjugated detection antibody

• Standard curve preparation: Prepare serial dilutions of ITIH2 standard (typically starting at 225 μg/ml and diluting down to 6.25 μg/ml)

• Sample preparation: Dilute samples appropriately to fall within the detection range (6.25-200 μg/ml)

• Detection methodology: Use TMB substrate solution for colorimetric detection at 450 nm

The protocol typically involves:

• Adding samples/standards to pre-coated wells

• Incubating with HRP-conjugated anti-ITIH2 antibody

• Washing to remove unbound components

• Adding TMB substrate

• Adding stop solution

• Measuring optical density at 450 nm

This approach allows for accurate quantification of ITIH2 across various biological samples.

How can I establish specificity of ITIH2 antibodies in my research?

Establishing antibody specificity is methodologically crucial. For ITIH2 antibodies, consider these approaches:

• Positive and negative controls: Use cells or tissues known to express ITIH2 (like HepG2 cells) as positive controls , and those that don't as negative controls

• Knockdown/knockout validation: Compare antibody signals between wild-type samples and those where ITIH2 has been depleted through siRNA or CRISPR techniques

• Peptide competition assays: Pre-incubate the antibody with purified ITIH2 protein or immunogen peptide before application to samples

• Western blot validation: Confirm single band detection at the expected molecular weight (~106 kDa)

• Cross-reactivity testing: Test the antibody against related proteins (other ITIH family members) to ensure specificity

Additionally, verify species cross-reactivity if working with non-human models, as some ITIH2 antibodies react with human, mouse, and rat orthologs, while others are species-specific .

How does ITIH2 expression correlate with cancer progression?

ITIH2 expression shows significant correlations with cancer progression across multiple cancer types:

• Downregulation pattern: ITIH2 is frequently downregulated in various solid tumors including lung, breast, and colon cancers, suggesting its potential role as a tumor suppressor

• Central nervous system tumors: ITIH2 is expressed in normal brain tissues and low-grade CNS cancers but notably absent in aggressive glioblastomas, particularly glioblastoma multiforme, indicating its anti-invasive function

• Breast cancer correlation: Research has established a significant association (p = 0.001) between ITIH2 expression and estrogen receptor expression in breast cancer . This is particularly relevant as estrogen has been shown to inhibit cancer invasion and motility

• Mechanistic implications: ITIH2's role in stabilizing the ECM through covalent binding to hyaluronan may explain its tumor-suppressive function, as this stabilization could inhibit tumor growth and invasion

Recent research shows that understanding ITIH2 expression patterns could provide valuable prognostic information in various cancers, with decreased expression generally associated with more aggressive disease phenotypes.

What roles does ITIH2 play in hyaluronan network remodeling in cancer?

ITIH2 serves critical functions in hyaluronan (HA) network remodeling that significantly impact cancer cell behavior:

• HA network formation: ITIH2, as an HA-binding protein, contributes to the formation and stability of the hyaluronan network in the extracellular matrix

• Transcriptional regulation: ITIH2 expression is transcriptionally upregulated by ZEB1 (an EMT-inducing transcription factor), alongside HA synthase 2 (HAS2), creating a coordinated regulation of the HA network

• Functional consequences: Research has demonstrated that depletion of ITIH2 reduces HA matrix formation, which subsequently diminishes the migration and invasion capabilities of lung cancer cells

• Therapeutic implications: Recent research using a deep learning-based drug-target interaction algorithm identified sincalide as an ITIH2 inhibitor that effectively blocks HA matrix formation and cancer cell migration, preventing metastatic colonization in mouse models

This evidence suggests that ITIH2's role in HA network remodeling represents a potential therapeutic target for inhibiting cancer progression, particularly in lung cancer.

How can ITIH2 antibodies be used to study EMT and cancer metastasis?

ITIH2 antibodies provide valuable tools for studying the relationship between epithelial-to-mesenchymal transition (EMT) and cancer metastasis:

• Co-culture experiments: Antibodies can detect ITIH2 secretion from mesenchymal-like cancer cells when co-cultured with cancer-associated fibroblasts, allowing for the study of tumor microenvironment interactions

• ZEB1-ITIH2-HAS2 axis: ITIH2 antibodies enable researchers to investigate the regulatory relationship between ZEB1 (EMT-inducing factor), ITIH2, and HAS2, revealing how this axis controls HA matrix formation and cancer cell motility

• CD44 isoform detection: Combined with CD44 antibodies, ITIH2 antibodies can help elucidate how ZEB1 facilitates alternative splicing and isoform expression of CD44 (an HA receptor) in relation to ITIH2 expression

• Therapeutic response monitoring: ITIH2 antibodies can be used to monitor changes in ITIH2 expression and localization following treatment with potential EMT-targeting therapies

Methodologically, researchers can employ immunohistochemistry, Western blotting, and immunofluorescence with ITIH2 antibodies to visualize changes in protein expression and distribution during EMT and metastatic progression.

How do post-translational modifications affect ITIH2 antibody recognition?

Post-translational modifications (PTMs) of ITIH2 can significantly impact antibody recognition and experimental outcomes:

• Glycosylation effects: ITIH2 contains glycosylation sites that may affect epitope accessibility. Researchers should consider using antibodies raised against different regions of ITIH2 if glycosylation is suspected to interfere with detection

• Proteolytic processing: ITIH2 undergoes proteolytic processing during its interaction with hyaluronan, potentially creating neo-epitopes or masking existing ones. This processing can affect antibody binding, particularly for antibodies targeting the C-terminal region involved in HA binding

• Cross-linking considerations: The covalent binding between ITIH2 and hyaluronan involves an ester exchange reaction , which may alter antibody recognition in tissues where this cross-linking has occurred

When selecting antibodies for specific applications, researchers should review the immunogen information and epitope mapping data to understand whether the antibody targets regions susceptible to PTMs.

What are the methodological considerations for studying ITIH2-hyaluronan interactions?

Studying ITIH2-hyaluronan interactions requires specialized methodological approaches:

• Isothermal Titration Calorimetry (ITC): This technique can quantify binding parameters between ITIH2 and its binding partners, similar to approaches used for related proteins . Proper buffer selection (e.g., phosphate-buffered saline without KCl) is crucial for accurate measurements

• Pull-down assays: Hyaluronan-coated beads can be used to pull down ITIH2 from biological samples, followed by Western blot detection with ITIH2 antibodies

• Particle exclusion assays: To visualize the HA matrix, researchers can use fixed red blood cells or beads that are excluded from HA-rich pericellular matrices, allowing assessment of how ITIH2 contributes to matrix formation

• ELISA-based interaction studies: Coating plates with hyaluronan and detecting bound ITIH2 using specific antibodies can provide quantitative data on these interactions

• Imaging approaches: Fluorescently labeled hyaluronan combined with ITIH2 immunostaining allows visualization of co-localization and potential interaction sites within tissues or cell cultures

When analyzing data from these studies, researchers should consider the Hill transformation for adhesion curves and other appropriate statistical methods to accurately interpret interaction kinetics .

How can ITIH2 antibodies be integrated into multi-omics cancer research approaches?

Integration of ITIH2 antibodies into multi-omics research strategies can provide comprehensive insights into cancer biology:

• Proteogenomic correlation: ITIH2 antibodies can validate protein expression findings from mass spectrometry, correlating with genomic data on ITIH2 gene expression or mutations

• Spatial transcriptomics integration: Combining ITIH2 immunohistochemistry with spatial transcriptomics can reveal relationships between ITIH2 protein localization and regional gene expression patterns in tumors

• Single-cell analysis: ITIH2 antibodies can be incorporated into single-cell protein profiling techniques to understand heterogeneity in ITIH2 expression across different cell populations within tumors

• Extracellular vesicle (EV) studies: ITIH2 antibodies can detect ITIH2 in tumor-derived EVs, potentially revealing novel mechanisms of tumor-stroma communication

• Functional genomics validation: Following CRISPR screens targeting ECM-related pathways, ITIH2 antibodies can validate the impact of gene perturbations on ITIH2 protein levels and localization

An integrated approach combining these methods provides a more comprehensive understanding of ITIH2's role in cancer biology than any single method alone.

What are common sources of variability in ITIH2 antibody-based assays?

Several factors can introduce variability in ITIH2 antibody-based assays:

To minimize these variables, researchers should implement rigorous quality control procedures, including positive and negative controls with each experiment, and regular antibody validation tests .

How should I interpret discrepancies between ITIH2 mRNA and protein expression data?

Discrepancies between ITIH2 mRNA and protein levels are common and should be interpreted with these methodological considerations:

• Post-transcriptional regulation: ITIH2 may be subject to microRNA regulation or RNA-binding protein interactions that affect translation efficiency without changing mRNA levels

• Protein secretion dynamics: As a secreted protein, cellular ITIH2 levels may not correlate with mRNA expression if secretion rates vary between conditions

• Protein stability factors: ITIH2 protein stability may be affected by its interaction with hyaluronan or other ECM components, leading to differential turnover rates

• Technical limitations: Different sensitivities between RT-qPCR (for mRNA) and antibody-based methods (for protein) can create apparent discrepancies

• Temporal disconnection: Time lags between transcription and translation may explain some discrepancies, especially in dynamic processes like EMT

When faced with such discrepancies, researchers should conduct time-course experiments and consider using protein synthesis or degradation inhibitors to determine the mechanistic basis of the observed differences.

How can I validate ITIH2 antibody results in tissue microarrays for clinical research?

Validating ITIH2 antibody results in tissue microarrays (TMAs) for clinical applications requires rigorous methodology:

Following these validation steps ensures that ITIH2 antibody results from TMAs provide reliable data for clinical research applications, particularly in cancer studies where ITIH2 has shown potential as a prognostic biomarker .