ITLN1 Antibody

What is ITLN1 and what are its primary biological functions?

ITLN1 (Intelectin-1, also known as Omentin) is a calcium-dependent lectin that serves multiple biological functions:

• Microbial recognition: ITLN1 specifically recognizes microbial carbohydrate chains containing terminal acyclic 1,2-diol moieties, including β-linked D-galactofuranose (β-Galf), D-phosphoglycerol-modified glycans, and specific bacterial glycans from both Gram-positive and Gram-negative bacteria .

• Metabolic regulation: ITLN1 functions as an adipokine that enhances insulin-stimulated glucose uptake in adipocytes without affecting basal glucose uptake. It increases AKT phosphorylation both in the presence and absence of insulin .

• Iron metabolism: ITLN1 may interact with lactoferrin (LTF) and increase its uptake, potentially playing a role in iron absorption .

• Defense against pathogens: ITLN1 likely contributes to innate immunity by recognizing carbohydrate structures specific to microbial pathogens rather than human glycans .

Where is ITLN1 primarily expressed in human and mouse tissues?

Expression patterns of ITLN1 show important species-specific differences:

• Human ITLN1: Primarily expressed in intestinal goblet cells of both small and large intestine. Expression is detected throughout the intestinal tract, with notable presence in rectum and stomach tissues .

• Mouse Itln1: Predominantly expressed in Paneth cells of the small intestine, with limited expression in the colon. Expression begins early in development, becoming more pronounced in the proximal small intestine and increasing with age .

• Extra-intestinal expression: ITLN1 expression has been detected in adipose tissue and other organs, though at generally lower levels than intestinal expression .

This species difference in cellular localization (goblet cells vs. Paneth cells) represents an important consideration when using mouse models to study human ITLN1 biology .

How should I optimize western blotting protocols when using ITLN1 antibodies?

For optimal western blotting results with ITLN1 antibodies:

• Sample preparation: Consider both reduced and non-reduced conditions, as ITLN1 has different migration patterns under each. Under non-reduced conditions, ITLN1 forms oligomeric structures that appear at higher molecular weights .

• Protein concentration: Use 2-20 μg/mL antibody concentration for detection, with specific optimization depending on your antibody source .

• Expected bands: The predicted molecular weight for monomeric ITLN1 is approximately 35 kDa, but oligomeric forms will appear at higher molecular weights under non-reducing conditions .

• Blocking conditions: Use standard BSA or non-fat milk in TBS-T, but optimize blocking time and concentration if background is high.

• Validation: Always include positive controls (small intestine lysate for mouse Itln1, recombinant human ITLN1 protein for human studies) .

What are the key considerations for immunohistochemistry (IHC) detection of ITLN1?

For successful IHC detection of ITLN1:

• Tissue preparation: For formalin-fixed, paraffin-embedded (FFPE) tissues, antigen retrieval is critical. Heat tissue sections in 10mM Tris with 1mM EDTA, pH 9.0, for 45 min at 95°C followed by cooling at room temperature for 20 minutes .

• Antibody concentration: Use approximately 20 μg/ml for FFPE tissues, but optimize based on your specific antibody and tissue type .

• Detection systems: DAB staining works well for colorimetric detection. For fluorescence, secondary antibodies conjugated with fluorophores like NorthernLights™ 557 have been successfully used .

• Cellular localization: For human tissues, look for goblet cell staining in intestinal sections. For mouse tissues, focus on Paneth cells at the base of the small intestinal crypts .

• Controls: Include appropriate negative controls (ITLN1 knockout tissues when available) and positive controls (small intestinal tissues known to express ITLN1) .

How do I account for species-specific differences when using mouse models to study ITLN1?

When designing experiments with mouse models to study ITLN1 biology:

• Expression pattern differences: Remember that mouse Itln1 is predominantly expressed in Paneth cells, while human ITLN1 is expressed in goblet cells. This fundamental difference may impact the interpretation of results, especially in disease models .

• Tissue distribution: Mouse Itln1 shows a proximal-to-distal gradient in the small intestine, with higher expression in proximal regions. Human ITLN1 expression is more evenly distributed throughout the intestinal tract .

• Developmental timing: Consider that Itln1 expression in mice changes during development, with expression increasing postnatally and reaching adult levels by approximately day 50 .

• Model selection: When using Itln1 knockout mice, be aware that unchallenged animals may not show obvious pathology, requiring experimental challenges (e.g., DSS-induced colitis) to reveal phenotypes .

• Translational considerations: Interpret results carefully when extrapolating from mouse models to human disease, particularly for intestinal conditions, due to these species-specific differences .

What are the best approaches to validate ITLN1 antibody specificity across species?

To ensure antibody specificity across species:

• Knockout validation: The gold standard for antibody validation is testing in knockout models. Utilize Itln1 knockout mouse tissues as negative controls for antibodies claimed to cross-react with mouse Itln1 .

• Western blot analysis: Compare protein bands between species under both reducing and non-reducing conditions. Confirm expected molecular weights (approximately 35 kDa for the monomer) .

• Immunohistochemical patterns: Verify that staining patterns match known cell-specific expression (Paneth cells in mice vs. goblet cells in humans) .

• Recombinant protein controls: Include recombinant ITLN1 proteins from relevant species as positive controls in immunoblotting experiments .

• Epitope consideration: Select antibodies raised against conserved regions when cross-species reactivity is desired. Note that many commercial antibodies target specific amino acid sequences (e.g., AA 28-270, AA 31-253) which may have varying degrees of conservation .

How is ITLN1 involved in inflammatory bowel disease (IBD) and what research approaches are most informative?

ITLN1 has been implicated in IBD through several lines of evidence:

What is the significance of ITLN1 in colorectal cancer research and how can it be studied effectively?

ITLN1 has emerging significance in colorectal cancer (CRC) research:

• Expression profile: ITLN1 is significantly underexpressed in CRC tumor tissues compared to normal tissues, suggesting potential as a diagnostic biomarker. ROC curve analysis showed an AUC of 0.894, indicating good diagnostic potential .

• Prognostic value: Higher ITLN1 expression correlates with better prognosis in CRC patients, making it potentially valuable for prognostic assessment .

• Research methodologies:

  • mRNA expression analysis via qRT-PCR to quantify expression differences

  • Immunohistochemical staining to assess protein expression in tumor vs. normal tissues

  • Risk score calculation using Cox-LASSO regression analysis incorporating ITLN1 expression

  • Construction of prognostic nomograms combining ITLN1 expression with clinicopathological factors

• Potential applications: ITLN1 shows promise as a screening tool, diagnostic marker, and prognostic indicator for CRC. The optimal threshold for ITLN1 expression has been reported as 6.6 for distinguishing tumor from normal tissue .

How do ITLN1 genetic variants affect protein function, and what experimental approaches can assess these effects?

To investigate the impact of ITLN1 genetic variants on protein function:

• Variant characterization: The missense variant V109D (rs2274907) is in linkage disequilibrium with the IBD-associated intronic SNP rs2274910. Population frequencies differ significantly between African (28%) and non-African (67%) superpopulations .

• Functional assessment approaches:

  • Glycan binding assays to test if variants affect carbohydrate recognition

  • Protein oligomerization studies to determine if variants influence quaternary structure

  • Expression analysis stratified by genotype to assess if variants alter expression levels

  • Protein-protein interaction studies to evaluate effects on binding partners like lactoferrin

• Research findings: Current research indicates that the V109D variant does not significantly alter ITLN1 glycan binding to β-D-galactofuranose or affect protein oligomerization. Additionally, intestinal expression levels do not appear to differ by genotype .

• Alternative mechanisms: The disease risk associated with ITLN1 variants may involve:

  • Changes in CD244 or Ly9 biology rather than ITLN1 directly

  • Differential expression in non-intestinal tissues

  • Alterations in protein-protein interactions not yet characterized

What are the recommended approaches for studying ITLN1's role in microbial recognition and innate immunity?

To investigate ITLN1's role in microbial recognition and innate immunity:

• Glycan binding assays: Test ITLN1 binding to specific bacterial glycans, focusing on structures containing terminal acyclic 1,2-diol moiety, β-Galf, D-phosphoglycerol-modified glycans, KO, and KDO .

• Bacterial challenge models:

  • Use defined bacterial strains with known glycan structures (K. pneumoniae, S. pneumoniae, Y. pestis, P. mirabilis, P. vulgaris)

  • Compare responses in wild-type vs. Itln1-deficient models

  • Assess microbiota differences in Itln1 knockout mice under baseline and challenged conditions

• Cellular studies:

  • For human studies, focus on goblet cell function and ITLN1 secretion

  • For mouse studies, investigate Paneth cell antimicrobial functions

  • Use calcium dependency as a control parameter, as ITLN1 binding to microbial glycans is calcium-dependent

• Limitations and considerations:

  • Species differences in expression patterns may affect interpretation

  • Luminal concentrations of ITLN1 remain to be determined

  • The biological consequences of ITLN1 binding to microbes need further characterization

What are the critical quality control measures when developing or using ELISA assays for ITLN1 detection?

For developing or using ELISA assays to detect ITLN1:

• Antibody pair selection: Choose validated antibody pairs specifically tested for ELISA applications. For example, Mouse Anti-Human Intelectin-1/Omentin Monoclonal Antibody (MAB4254) has been validated as a capture antibody when paired with MAB42541 for detection .

• Standard curve preparation: Use recombinant human ITLN1 protein for standard curve generation. Serial two-fold dilutions provide reliable standard curves for quantification .

• Detection system optimization:

  • For colorimetric detection, Streptavidin-HRP followed by appropriate substrate solutions works well

  • Ensure proper stopping of enzymatic reactions for consistent readings

  • Determine the linear range of detection and optimal sample dilutions

• Sample preparation considerations:

  • For serum/plasma samples, determine if oligomeric forms affect detection

  • Consider calcium concentration in buffers, as ITLN1 function is calcium-dependent

  • Be aware of potential matrix effects from complex biological samples

• Validation across sample types:

  • Confirm assay performance in various biological matrices (serum, tissue lysates)

  • Establish recovery rates and detection limits for each sample type

  • Verify reproducibility through intra- and inter-assay coefficient of variation measurements

How can I troubleshoot non-specific binding or weak signals when using ITLN1 antibodies?

When encountering issues with ITLN1 antibody performance:

• Non-specific binding:

  • Increase blocking time and concentration (5% BSA or milk)

  • Optimize antibody concentration - start with manufacturer's recommended range (typically 2-20 μg/mL)

  • Use more stringent washing conditions (increase wash buffer Tween-20 to 0.1-0.2%)

  • Consider using alternative blocking agents (normal serum matching secondary antibody species)

  • Verify antibody specificity using knockout controls when available

• Weak signal:

  • For FFPE tissues, ensure proper antigen retrieval (10mM Tris with 1mM EDTA, pH 9.0, for 45 min at 95°C)

  • Try signal amplification systems (e.g., biotin-streptavidin)

  • Increase primary antibody concentration and incubation time (overnight at 4°C)

  • Confirm target expression in your sample type using reference data

  • Consider sample preparation methods that preserve epitope integrity

• Antibody selection considerations:

  • Different antibodies target different epitopes within ITLN1 (e.g., AA 28-270, AA 31-253, AA 19-313)

  • For specific applications, select antibodies validated for that purpose (WB, IHC, IF)

  • Consider monoclonal antibodies for consistent performance when reproducibility is critical

What experimental controls are essential when studying the functional effects of ITLN1?

Essential controls for studying ITLN1 functional effects include:

• Expression validation controls:

  • Positive controls: tissues known to express ITLN1 (small intestine for both human and mouse)

  • Negative controls: ITLN1-knockout tissues or cell lines

  • Isotype controls: to identify non-specific binding in immunostaining experiments

• Functional study controls:

  • Calcium dependency: include EDTA conditions as negative controls for calcium-dependent binding

  • Glycan specificity: include human glycans as negative controls, as ITLN1 does not bind human glycans

  • Recombinant protein controls: include known functional and non-functional ITLN1 variants

• Experimental model controls:

  • For knockout studies: include appropriate wild-type littermates as controls

  • For disease models: include both challenged and unchallenged animals

  • For human studies: stratify by genotype to account for genetic variant effects

• Application-specific controls:

  • For Western blotting: include both reducing and non-reducing conditions

  • For IHC/IF: include secondary antibody-only controls

  • For ELISA: include standard curves on each plate and verify with recombinant protein spikes