CHIT1 Antibody, FITC conjugated

What is CHIT1 and why is it important in research?

CHIT1 (Chitinase 1) is an enzyme that degrades chitin, chitotriose, and chitobiose. It plays a significant role in the defense against pathogens, particularly fungi and nematodes, as these organisms contain chitin in their cell walls or exoskeletons . CHIT1 is primarily produced by activated macrophages and is involved in inflammatory cascades . Its importance in research stems from its role as a biomarker for various conditions, including metabolic dysfunction-associated steatohepatitis (MASH), systemic candidiasis, and other inflammatory disorders . The enzyme's activity can be counterproductive in some immune responses, making it an intriguing target for therapeutic intervention .

How do FITC-conjugated CHIT1 antibodies differ from unconjugated versions?

FITC-conjugated CHIT1 antibodies contain the fluorescent dye fluorescein isothiocyanate (FITC) covalently attached to the antibody molecule, whereas unconjugated versions like the 21432-1-AP antibody lack this fluorescent tag . The FITC conjugation enables direct visualization of CHIT1 in applications such as flow cytometry, immunofluorescence microscopy, and confocal imaging without requiring secondary antibodies. Unconjugated antibodies, in contrast, require additional detection steps with labeled secondary antibodies or other visualization systems. The molecular weight of the antibody increases slightly with FITC conjugation, and the conjugation process may occasionally affect binding characteristics or sensitivity compared to unconjugated versions.

What sample types are most suitable for CHIT1 detection?

CHIT1 can be detected in various biological samples, with serum and plasma being particularly suitable for quantitative analysis. According to validation data, CHIT1 concentration can be accurately measured in serum (average recovery of 98%, range 95-101%), EDTA plasma (103%, range 102-105%), heparin plasma (101%, range 98-104%), and citrate plasma (95%, range 92-97%) . For tissue samples, CHIT1 has been successfully detected in human pancreatic tissue using immunohistochemistry with appropriate antigen retrieval methods . Liver tissue samples have also proven valuable for CHIT1 detection, particularly in studies of metabolic dysfunction and fibrosis . When selecting a sample type, researchers should consider the expression level of CHIT1 in the tissue of interest and the compatibility of the sample with the selected detection method.

What are the optimal storage conditions for FITC-conjugated CHIT1 antibodies?

FITC-conjugated antibodies, including CHIT1 antibodies, require special storage considerations to maintain both antibody integrity and fluorophore activity. While specific FITC-conjugated CHIT1 antibody storage conditions aren't detailed in the search results, unconjugated CHIT1 antibodies are typically stored at -20°C in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . For FITC-conjugated antibodies, additional precautions are necessary: they should be stored protected from light to prevent photobleaching of the fluorophore, and some manufacturers recommend aliquoting to avoid repeated freeze-thaw cycles. Small aliquots (20μL) can be stored without further processing, as indicated for some CHIT1 antibody preparations . When working with FITC-conjugated antibodies, minimize exposure to bright light during experimental procedures and store the working solutions on ice and in the dark.

How can I validate the specificity of a CHIT1 antibody in my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For CHIT1 antibodies, several approaches can be employed. First, perform western blotting to confirm that the antibody detects a protein of the expected molecular weight (approximately 52-55 kDa for CHIT1) . Second, include appropriate positive controls such as human pancreatic tissue, which has been demonstrated to express CHIT1 . Third, utilize negative controls such as tissues from CHIT1 knockout models or samples treated with CHIT1-specific inhibitors like Bisdionin C, which has been shown to reduce CHIT1 activity . Fourth, perform blocking experiments with the immunizing peptide or recombinant CHIT1 protein. Fifth, if possible, use alternative detection methods such as ELISA or qRT-PCR to confirm expression patterns . Finally, for FITC-conjugated antibodies specifically, include appropriate isotype controls to distinguish between specific binding and non-specific fluorescence. The combined results from these validation steps will provide strong evidence for antibody specificity.

What are the best methods for quantifying CHIT1 levels in clinical samples?

ELISA is the gold standard for quantifying CHIT1 levels in clinical samples such as serum and plasma. The SimpleStep ELISA format allows for efficient quantification of CHIT1 in various sample types with high accuracy . Standard curves can be prepared using recombinant CHIT1 protein, with sample dilutions typically around 2.5% for serum and plasma samples . The assay can detect a range of CHIT1 concentrations: approximately 23.14 ng/mL in serum, 13.39 ng/mL in citrate plasma, 20.15 ng/mL in EDTA plasma, and 37.12 ng/mL in heparin plasma . Alternative methods include enzymatic activity assays using fluorescent substrates such as 4-Methylumbelliferyl β-D-N,N′-diacetylchitobioside (4-MU-DAC) . These assays measure CHIT1 functional activity rather than protein concentration. For tissue samples, immunohistochemistry combined with digital image analysis provides both localization and semi-quantitative data on CHIT1 expression . qRT-PCR can be used to measure CHIT1 mRNA levels, which is especially useful when protein detection is challenging or when investigating transcriptional regulation .

How can FITC-conjugated CHIT1 antibodies be utilized in single-cell analysis techniques?

FITC-conjugated CHIT1 antibodies can significantly enhance single-cell analysis techniques by enabling direct visualization of CHIT1-expressing cells. In flow cytometry, these antibodies can identify specific cell populations producing CHIT1, such as activated macrophages. This application is particularly valuable when combined with other markers to create comprehensive immunophenotyping panels . For single-cell RNA sequencing studies, index sorting with FITC-CHIT1 antibodies allows correlation between protein expression and transcriptomic profiles. Recent studies have employed single-nuclei RNA sequencing to profile macrophage populations in MASH, revealing an increase in lipid-associated macrophages . FITC-CHIT1 antibodies could be integrated into such studies to validate protein expression patterns in identified cell clusters. In imaging cytometry, these antibodies enable simultaneous visualization of cellular morphology and CHIT1 expression. For optimizing such applications, researchers should ensure proper antibody titration, include appropriate compensation controls to account for FITC spectral overlap, and validate results using alternative detection methods.

What are the challenges in detecting CHIT1 isoforms using antibodies?

Detecting specific CHIT1 isoforms presents several technical challenges. CHIT1 exists in multiple isoforms, including isoform 3 which lacks enzymatic activity . The primary challenge lies in developing antibodies with sufficient specificity to distinguish between these isoforms. Most commercial antibodies target epitopes common to multiple isoforms, making isoform-specific detection difficult. When selecting an antibody, researchers should carefully review the immunogen information to understand which regions of CHIT1 are targeted. The immunogen used for antibody 21432-1-AP, for example, is the CHIT1 fusion protein Ag16039 . Researchers interested in isoform-specific detection should consider complementary approaches such as RT-PCR with isoform-specific primers. Additionally, post-translational modifications can affect antibody binding, potentially leading to inconsistent detection across different cellular contexts. To overcome these challenges, multiple detection methods should be employed, including western blotting with appropriate controls, immunoprecipitation followed by mass spectrometry analysis, and functional assays to distinguish between enzymatically active and inactive isoforms.

How do CHIT1 expression patterns differ across inflammatory disease states?

CHIT1 expression exhibits distinct patterns across different inflammatory conditions, reflecting its multifaceted role in immune responses. In metabolic dysfunction-associated steatohepatitis (MASH), CHIT1 expression in fibrotic liver tissues correlates significantly with the extent of liver fibrosis, macrophage infiltration, and inflammation . Single-nuclei RNA sequencing has revealed that in MASH, there is a notable increase in macrophages, particularly lipid-associated macrophages that produce CHIT1 . In systemic candidiasis, plasma levels of CHIT1 parallel the extent of infection, with Chit1-deficient mice showing decreased kidney fungal burden compared to wild-type mice . This suggests that excessive CHIT1 activity may actually be counterproductive in some fungal infections. Beyond these conditions, elevated CHIT1 serum levels have been associated with atherosclerosis, COPD, and Alzheimer's disease . For researchers investigating these differential expression patterns, it is crucial to employ tissue-specific controls and standardized quantification methods. Flow cytometry with FITC-conjugated CHIT1 antibodies can identify which specific cell populations are producing CHIT1 in different disease contexts, while immunohistochemistry can reveal the spatial distribution of CHIT1-expressing cells relative to disease pathology.

What interactions between CHIT1 and other immune components should be considered in experimental design?

When designing experiments investigating CHIT1, researchers must consider its complex interactions with other immune components. A critical interaction exists between CHIT1 and the complement receptor 3 (CR3, Mac-1, CD11b/CD18) on neutrophils. The enzymatic activity of CHIT1 on fungal chitin produces chitobiose, which can interfere with CR3's lectin-like binding site, thereby inhibiting neutrophil clustering and anti-fungal functions . This interaction has significant implications for studies of fungal infections, as CHIT1 inhibition may actually enhance anti-fungal immunity in some contexts. Additionally, CHIT1 expression is closely associated with macrophage activation states, making it important to consider macrophage polarization markers (M1/M2) in experimental analyses . The relationship between CHIT1 and pro-inflammatory cytokines should also be examined, as these may have reciprocal regulatory effects. When designing flow cytometry panels with FITC-conjugated CHIT1 antibodies, researchers should include markers for relevant cell populations (CD11b, F4/80) and activation states. For inhibition studies, compounds such as OATD-01 (a chitinase inhibitor) and Bisdionin C (a CHIT1-specific inhibitor) can be valuable tools to dissect CHIT1's functional role . Finally, experimental timing is crucial, as CHIT1 expression can vary significantly throughout the course of an inflammatory response.

What are common causes of false positives/negatives when using CHIT1 antibodies?

False results with CHIT1 antibodies can arise from multiple sources that require systematic troubleshooting. False positives commonly stem from non-specific binding, particularly in tissues with high endogenous peroxidase activity or biotin. For FITC-conjugated antibodies specifically, autofluorescence from tissues like liver (which contains lipofuscin) can be misinterpreted as positive signal. To address this, proper blocking steps and inclusion of isotype controls are essential. Cross-reactivity with other chitinase family members, such as acidic mammalian chitinase (AMCase), can also generate false positives . False negatives frequently result from inadequate antigen retrieval, particularly in formalin-fixed tissues. For CHIT1 detection in human pancreatic tissue, TE buffer at pH 9.0 is recommended for antigen retrieval, though citrate buffer at pH 6.0 can serve as an alternative . Sample storage conditions can significantly impact CHIT1 detection, as the protein may degrade during improper handling. For flow cytometry applications with FITC-conjugated antibodies, excessive exposure to light can cause photobleaching, resulting in false negatives. Additionally, the 24-base pair duplication polymorphism in the CHIT1 gene, which is present in approximately 6% of the population and leads to enzyme deficiency, should be considered when interpreting negative results in human samples.

How can I optimize immunofluorescence protocols for FITC-conjugated CHIT1 antibodies?

Optimizing immunofluorescence protocols for FITC-conjugated CHIT1 antibodies requires careful attention to several parameters. Begin with thorough fixation optimization, testing both paraformaldehyde (2-4%) and methanol fixation to determine which best preserves CHIT1 epitopes while maintaining cellular architecture. For antigen retrieval, heat-induced epitope retrieval with TE buffer at pH 9.0 has proven effective for CHIT1 detection . The blocking step is particularly critical for fluorescence applications; use a combination of serum (matching the species of the secondary antibody if using a detection system) and BSA (3-5%) to minimize background. For antibody dilution, start with the manufacturer's recommended range (1:50-1:500 for unconjugated CHIT1 antibodies in IHC) and perform a titration series to determine optimal concentration. Incubation conditions significantly impact staining quality; overnight incubation at 4°C often yields better signal-to-noise ratios than shorter incubations at room temperature. Include appropriate controls: positive controls (human pancreatic tissue) , negative controls (primary antibody omission), and tissue from CHIT1-deficient models if available. To minimize photobleaching of the FITC fluorophore, keep slides protected from light during all steps, and consider using anti-fade mounting media containing DAPI for nuclear counterstaining. Finally, when imaging, capture FITC signals early in the imaging session before significant photobleaching occurs.

What considerations are important when multiplexing FITC-CHIT1 antibodies with other fluorescent markers?

Successful multiplexing with FITC-conjugated CHIT1 antibodies requires careful planning to avoid spectral overlap and cross-reactivity issues. First, consider FITC's emission spectrum (peak ~520 nm) when selecting additional fluorophores. Ideally, choose fluorophores with minimal spectral overlap such as Cy5, APC, or PE-Cy7. For flow cytometry applications, proper compensation is essential; prepare single-stained controls for each fluorophore using the same cells or compensation beads. When designing multiplexed panels, prioritize brighter fluorophores (like PE) for targets with lower expression and reserve FITC for more abundantly expressed targets like CHIT1 in activated macrophages . For immunofluorescence microscopy, sequential staining protocols may reduce cross-reactivity between antibodies, especially when multiple primary antibodies are derived from the same species. When studying CHIT1 in liver tissues, consider using Sudan Black B treatment to reduce lipofuscin autofluorescence that might interfere with FITC signal interpretation . For multicolor analysis of macrophage populations, combine FITC-CHIT1 with markers like F4/80 and CD11b, which have been used successfully in CHIT1-related research . If performing multiplexed analysis of tissue sections, spectral unmixing algorithms on confocal microscopy systems can help distinguish overlapping fluorophores. Finally, validate all multiplexed panels with appropriate controls, including FMO (fluorescence minus one) controls for flow cytometry applications.

How can CHIT1 antibodies be used to investigate therapeutic responses to chitinase inhibitors?

CHIT1 antibodies serve as essential tools for evaluating therapeutic responses to chitinase inhibitors like OATD-01 and Bisdionin C. In preclinical studies, CHIT1 antibodies can monitor changes in protein expression following inhibitor treatment. Research has demonstrated that OATD-01 treatment in MASH mice reduces CHIT1 positivity along with markers of inflammation and fibrosis (F4/80 and α-smooth muscle actin) . Flow cytometry using FITC-conjugated CHIT1 antibodies can quantify the percentage of CHIT1-expressing cells before and after inhibitor treatment, providing insights into which specific cell populations are affected. Immunohistochemistry can visualize spatial changes in CHIT1 expression patterns within tissue microenvironments following intervention . For functional assessment, combining antibody-based detection with enzymatic activity assays using substrates like 4-MU-DAC provides a comprehensive evaluation of both protein levels and functional inhibition . When designing such studies, time-course experiments are crucial, as protein expression may change more slowly than enzymatic activity. Additionally, researchers should consider downstream effects on inflammatory markers and pro-fibrotic genes, which have been shown to decrease following chitinase inhibition . For translational research, correlating tissue CHIT1 levels (detected by antibodies) with circulating CHIT1 (measured by ELISA) can help establish biomarkers for clinical monitoring of therapeutic responses.

What role does CHIT1 play in neutrophil function, and how can this be studied using FITC-conjugated antibodies?

CHIT1 plays a complex and sometimes counterintuitive role in neutrophil function, particularly in anti-fungal responses. Research has shown that CHIT1 is released from neutrophils in response to Candida hyphae, with significant increases in activity when neutrophils interact with hyphae compared to either alone . Paradoxically, CHIT1 activity may impair neutrophil anti-fungal functions through its end-product chitobiose, which interferes with the lectin-like binding site on neutrophil integrin CR3 (Mac-1, CD11b/CD18) . This interference reduces neutrophil clustering/swarming and Candida killing capabilities . FITC-conjugated CHIT1 antibodies enable direct visualization of CHIT1 production by neutrophils during pathogen interaction using live-cell imaging or flow cytometry. For studying the dynamics of CHIT1 release, time-course experiments combining FITC-CHIT1 antibodies with markers of neutrophil activation can reveal how quickly CHIT1 is mobilized during infection. Co-localization studies using confocal microscopy with FITC-CHIT1 and markers for neutrophil granules can identify the specific granule subsets containing CHIT1. To investigate the functional impacts of CHIT1, researchers can design experiments comparing wild-type neutrophils with those from CHIT1-deficient models or treated with inhibitors like Bisdionin C . Flow cytometry panels combining FITC-CHIT1 with CD11b and activation markers can assess how CHIT1 inhibition affects neutrophil activation states during fungal challenge.

What statistical approaches are most appropriate for analyzing CHIT1 expression data across different experimental conditions?

Selecting appropriate statistical approaches for CHIT1 expression data requires consideration of experimental design and data distribution characteristics. For comparing CHIT1 levels between control and experimental groups (such as inhibitor-treated versus vehicle-treated), parametric tests like Student's t-test or ANOVA are commonly used if data follow normal distribution . For non-normally distributed data, non-parametric alternatives such as Mann-Whitney U test or Kruskal-Wallis test are more appropriate. When analyzing correlations between CHIT1 expression and disease parameters (such as fibrosis extent or inflammatory markers), Pearson's correlation coefficient is suitable for linear relationships in normally distributed data, while Spearman's rank correlation is preferred for non-parametric data . For time-course experiments monitoring CHIT1 activity changes (as seen in neutrophil-Candida interaction studies), repeated measures ANOVA or mixed-effects models can account for within-subject variability . When analyzing flow cytometry data from FITC-conjugated CHIT1 antibody staining, population frequency comparisons require approaches that account for percentage data characteristics, such as arcsine transformation before parametric testing. For analyzing CHIT1 expression in single-cell RNA sequencing data, specialized methods like differential expression testing between identified cell clusters are necessary . Regardless of the chosen method, researchers should report effect sizes alongside p-values and implement appropriate multiple testing corrections when performing numerous comparisons to minimize false discovery rates.

How can researchers resolve contradictory findings between different CHIT1 detection methods?

Resolving contradictory findings between different CHIT1 detection methods requires systematic investigation of methodological variables. First, recognize that each detection method measures different aspects of CHIT1 biology: ELISA quantifies protein concentration, enzymatic assays measure functional activity, and immunostaining reveals localization patterns . Discrepancies between protein levels and enzymatic activity may indicate post-translational modifications or the presence of enzymatically inactive isoforms like isoform 3 . When antibody-based methods (such as immunohistochemistry) conflict with activity assays, examine antibody specificity—does the antibody recognize all isoforms or only specific ones? Validate findings using genetic approaches, such as CHIT1 knockdown or knockout models, to confirm specificity . Technical variables can significantly impact results; for ELISA, sample handling and storage conditions affect protein stability, while for enzymatic assays, pH and temperature influence activity measurements . When comparing flow cytometry data with tissue immunostaining, consider that disaggregation protocols for flow cytometry may alter surface epitope availability. If contradictory findings persist, design experiments that directly compare methods on split samples under identical conditions. Triangulate results using orthogonal approaches—if protein, mRNA, and activity measurements align, confidence in findings increases. Finally, consider biological context: CHIT1 expression exhibits tissue specificity and disease-state dependence, so apparent contradictions may reflect true biological variability rather than methodological error .

How can FITC-conjugated CHIT1 antibodies contribute to understanding the role of macrophage subpopulations in liver diseases?

FITC-conjugated CHIT1 antibodies offer powerful tools for dissecting the complex roles of macrophage subpopulations in liver pathology. Recent research has identified increased macrophage numbers, particularly lipid-associated macrophages, in MASH mouse models through single-nuclei RNA sequencing . FITC-CHIT1 antibodies enable direct visualization and quantification of CHIT1-expressing macrophages using flow cytometry or imaging cytometry, allowing researchers to determine what percentage of F4/80+CD11b+ cells produce CHIT1 and how this changes with disease progression. Multiparameter flow cytometry panels combining FITC-CHIT1 with markers for macrophage polarization (CD86 for M1, CD206 for M2) can reveal which functional subsets preferentially express CHIT1 during different disease stages. For spatial context, multiplex immunofluorescence microscopy using FITC-CHIT1 alongside markers for macrophage subtypes can map the distribution of CHIT1+ macrophages relative to areas of steatosis, inflammation, and fibrosis. This approach has particular value given the observed correlation between CHIT1 expression in fibrotic liver tissues and the extent of liver fibrosis . Cell sorting based on CHIT1 expression followed by transcriptomic or proteomic analysis can further characterize these subpopulations, potentially identifying novel therapeutic targets. Additionally, tracking CHIT1+ macrophages in response to treatments like chitinase inhibitor OATD-01 could provide insights into mechanisms underlying therapeutic effects, as OATD-01 treatment has been shown to reduce both CHIT1 and F4/80 positivity in MASH models .

What potential exists for developing CHIT1-targeted imaging probes for non-invasive monitoring of inflammatory conditions?

The development of CHIT1-targeted imaging probes represents a promising frontier for non-invasive monitoring of inflammatory conditions. Given CHIT1's elevated expression in various inflammatory disorders and its correlation with disease progression in conditions like MASH and systemic candidiasis , targeted imaging could provide valuable diagnostic and monitoring capabilities. Building on existing FITC-conjugated antibody technology, researchers could develop imaging probes by conjugating CHIT1 antibodies or antibody fragments (Fab, nanobodies) with contrast agents suitable for clinical imaging modalities. For PET imaging, radionuclide-labeled CHIT1 antibodies could enable whole-body visualization of inflammation sites with high sensitivity. For MRI applications, CHIT1 antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) could provide contrast enhancement at sites of macrophage accumulation. These approaches would leverage the established correlation between CHIT1 expression and macrophage infiltration in inflammatory tissues . Additionally, researchers could explore activity-based probes that become activated upon interaction with enzymatically active CHIT1, potentially distinguishing between active and inactive forms. For initial validation, animal models of MASH or fungal infection with proven CHIT1 involvement would be ideal testing grounds . Correlation studies between imaging signal intensity and traditional CHIT1 measurement methods (ELISA, IHC) would be essential for validating these novel probes. The development of such imaging technologies could eventually enable longitudinal monitoring of therapeutic responses to anti-inflammatory interventions or CHIT1 inhibitors like OATD-01, potentially reducing the need for invasive biopsies .

How might CHIT1 antibodies contribute to developing personalized medicine approaches for inflammatory disorders?

CHIT1 antibodies could significantly advance personalized medicine for inflammatory disorders by enabling patient stratification based on CHIT1 expression patterns. Research has demonstrated that CHIT1 expression in fibrotic liver tissues correlates with the extent of liver fibrosis, macrophage infiltration, and inflammation in MASH patients . By using FITC-conjugated CHIT1 antibodies to analyze patient samples through flow cytometry or immunohistochemistry, clinicians could potentially identify high CHIT1-expressing phenotypes that might benefit from targeted therapies like chitinase inhibitors. The 24-base pair duplication polymorphism in the CHIT1 gene, which causes enzyme deficiency in approximately 6% of individuals, presents an opportunity for pharmacogenomic approaches—patients with different CHIT1 genotypes might respond differently to certain treatments. In the context of fungal infections, where CHIT1 activity appears counterproductive , antibody-based testing could identify patients who might benefit from CHIT1 inhibition as an adjunct to standard antifungal therapy. For monitoring treatment response, quantitative analysis using CHIT1 antibodies could track changes in cellular expression patterns over time, potentially serving as an early biomarker of therapeutic efficacy before clinical symptoms improve. Furthermore, multiplexed analysis combining CHIT1 with other inflammatory markers could create comprehensive immune signatures for individual patients, allowing more nuanced treatment decisions. As therapeutics targeting chitinase activity (like OATD-01) continue development , CHIT1 antibody-based companion diagnostics could eventually become essential tools for selecting appropriate patients for these targeted interventions.