CHI3L2 Antibody

What is CHI3L2 and why is it significant in biomedical research?

CHI3L2 (Chitinase 3-like 2), also known as Chondrocyte Protein 39, YKL-39, or YKL39, is a 39 kDa glycoprotein belonging to the glycosyl hydrolase 18 family. Despite sharing structural similarity with bacterial chitinases, CHI3L2 lacks chitinase activity. It functions primarily as a lectin that binds chitooligosaccharides and various glycans with high affinity but not heparin. The protein is secreted by synovial fibroblasts and chondrocytes, particularly in osteoarthritis, and likely contributes to extracellular matrix stability and cartilage biogenesis . Its significance in research has increased substantially since it was identified as one of the most overexpressed genes in glioblastoma, suggesting its potential role as a biomarker or therapeutic target in cancer research .

How does CHI3L2 differ structurally and functionally from other chitinase-like proteins?

The human CHI3L2 precursor comprises 390 amino acids with a 26-amino acid signal sequence and a 364-amino acid mature region. It has potential alternate start sites at Met24 and Met80, with a possible isoform showing deletion of amino acids 15-24 . Unlike functional chitinases that hydrolyze chitin polymers, CHI3L2 belongs to a family of chitinase-like proteins that maintain structural elements of chitinases but lack enzymatic activity due to substitutions in the catalytic domain. This distinguishes it from chitotriosidase (a functional human chitinase) while aligning it functionally with other chitinase-like proteins such as CHI3L1 (YKL-40). Human CHI3L2 shares approximately 89% amino acid identity with bovine CHI3L2 over the region spanning amino acids 27-390, but interestingly, no rodent ortholog of CHI3L2 has been reported in the literature .

What types of antibodies are available for CHI3L2 detection and what are their characteristics?

Several monoclonal antibodies have been developed for CHI3L2 detection, including:

These antibodies have been validated for specificity against recombinant CHI3L2 and can detect the native protein in human tissue samples, including lung tissue and glioblastoma lysates .

What are the optimal conditions for Western blot detection of CHI3L2?

For optimal Western blot detection of CHI3L2, researchers should use reducing conditions with appropriate immunoblot buffers (such as Immunoblot Buffer Group 1 as mentioned in the literature). A typical protocol includes using 2 μg/mL of mouse anti-human CHI3L2 monoclonal antibody (such as MAB5116) followed by HRP-conjugated anti-mouse IgG secondary antibody . The protein typically appears as a specific band at approximately 40 kDa. For tissue samples, proper lysate preparation is critical; documented success has been achieved with human lung tissue and glioblastoma tissue lysates. Potential cross-reactivity with other chitinase family members should be controlled for, particularly in complex biological samples, by including appropriate negative controls and validation with recombinant protein standards .

How can I design a reliable ELISA protocol for quantitative CHI3L2 measurement?

For quantitative measurement of CHI3L2 in human samples, a sandwich ELISA approach is recommended. Commercial kits offer detection ranges of approximately 0.31-20 ng/mL with minimum detection limits around 0.125-0.31 ng/mL . When designing your own ELISA protocol:

• Plate Coating: Use a validated capture antibody at optimized concentration (typically 1-5 μg/mL) in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C.

• Sample Preparation: Dilute serum/plasma samples appropriately (often 1:2 to 1:10) in sample diluent containing blocking agents to minimize matrix effects.

• Standards: Prepare a standard curve using recombinant CHI3L2 protein (ranging from 0.31-20 ng/mL).

• Detection: Employ a detection antibody recognizing a different epitope than the capture antibody, followed by enzyme-conjugated secondary antibody.

• Substrate Reaction: Use TMB substrate with appropriate incubation time before adding stop solution.

• Validation: Include positive and negative controls, and assess intra- and inter-assay variability to ensure reproducibility.

For tissue homogenates, additional optimization may be required, including determination of appropriate extraction buffers and protein normalization strategies .

What are the key considerations when using CHI3L2 antibodies for immunohistochemistry?

While specific immunohistochemistry protocols for CHI3L2 weren't detailed in the provided search results, general principles for antibody-based tissue staining apply. Researchers should consider:

• Fixation: Optimize fixation methods (formalin, paraformaldehyde) and duration to preserve antigen integrity while maintaining tissue morphology.

• Antigen Retrieval: Test different methods (heat-induced epitope retrieval using citrate or EDTA buffers) to maximize antibody accessibility to the target.

• Antibody Concentration: Titrate antibody concentration to determine optimal signal-to-noise ratio.

• Controls: Include positive controls (tissues known to express CHI3L2, such as osteoarthritic cartilage or glioblastoma samples) and negative controls (antibody isotype controls and tissues not expected to express CHI3L2).

• Detection Systems: Select appropriate secondary antibody and visualization systems based on required sensitivity and available equipment.

• Counterstaining: Choose counterstains that don't interfere with the primary staining pattern.

Given CHI3L2's elevated expression in glioblastoma tissues, brain tumor sections could serve as positive controls for protocol optimization .

How can I address nonspecific binding when using CHI3L2 antibodies in Western blot applications?

Nonspecific binding in Western blot applications using CHI3L2 antibodies can be mitigated through several strategies:

• Blocking Optimization: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers) to determine which provides the best signal-to-noise ratio.

• Antibody Dilution: Titrate primary antibody concentration; higher concentrations can increase background while too low may reduce specific signal.

• Washing Protocol: Implement more stringent washing steps (increasing duration, number of washes, or detergent concentration in wash buffer).

• Buffer Composition: Use appropriate immunoblot buffer systems; the literature indicates success with Immunoblot Buffer Group 1 for CHI3L2 detection .

• Sample Preparation: Ensure proper sample denaturation and reduction; CHI3L2 detection has been successfully performed under reducing conditions .

• Membrane Selection: PVDF membranes have been successfully used for CHI3L2 detection .

• Pre-absorption: For antibodies showing consistent cross-reactivity, consider pre-absorbing with recombinant proteins of closely related family members.

If multiple bands appear near the expected 40 kDa size, this could represent glycosylation variants or alternate isoforms of CHI3L2, as the protein has potential alternate start sites at Met24 and Met80 .

What strategies can resolve inconsistent CHI3L2 detection in clinical samples?

Inconsistent detection of CHI3L2 in clinical samples may stem from multiple factors:

• Sample Handling: Standardize collection, processing, and storage protocols to minimize pre-analytical variability.

• Protein Degradation: Include protease inhibitors in extraction buffers and avoid repeated freeze-thaw cycles ("Use a manual defrost freezer and avoid repeated freeze-thaw cycles") .

• Extraction Methods: Compare different extraction buffers and protocols to optimize CHI3L2 recovery from tissues.

• Sample Type Differences: Recognize that detection protocols may require optimization for different sample types (serum vs. plasma vs. tissue homogenates).

• Biological Variability: Consider potential biological variations in CHI3L2 expression related to patient characteristics, disease state, or treatment effects.

• Antibody Selection: Different antibody clones may recognize distinct epitopes that could be differentially affected by sample processing or biological modifications.

• Internal Controls: Include appropriate housekeeping proteins or recombinant CHI3L2 spikes to normalize results across samples.

For longitudinal studies, consistency in analytical methodology is crucial for reliable interpretation of CHI3L2 expression patterns .

How should I store and handle CHI3L2 antibodies to maintain optimal activity?

Based on the storage recommendations for CHI3L2 antibodies:

• Long-term Storage: Store at -20°C to -70°C for up to 12 months from the date of receipt in the supplied formulation .

• Working Stock: After reconstitution, antibodies can be stored at 2-8°C under sterile conditions for approximately 1 month .

• Aliquoting: For longer storage after reconstitution (up to 6 months), prepare small aliquots and store at -20°C to -70°C under sterile conditions .

• Freeze-Thaw Cycles: Minimize repeated freeze-thaw cycles by preparing appropriate working aliquots .

• Buffer Conditions: Some CHI3L2 antibodies are supplied in formulations containing PBS (pH 7.4), 10% glycerol, and 0.02% sodium azide as preservative .

• Sterility: Maintain sterile conditions when handling reconstituted antibodies to prevent microbial contamination.

• Temperature Transitions: Allow frozen antibodies to thaw completely at appropriate temperatures before use; rapid temperature changes can affect antibody activity.

Proper storage and handling are essential for preserving antibody specificity and sensitivity in experimental applications .

How can CHI3L2 antibodies be utilized to investigate the protein's role in glioblastoma progression?

CHI3L2 has been identified as one of the most overexpressed genes in glioblastoma, making it a promising target for cancer research . Researchers can leverage CHI3L2 antibodies to:

• Expression Profiling: Quantify CHI3L2 protein levels across glioblastoma subtypes, grades, and in comparison to normal brain tissue using Western blot, ELISA, or immunohistochemistry.

• Prognostic Value Assessment: Correlate CHI3L2 expression with patient survival, treatment response, and disease progression through retrospective and prospective clinical studies.

• Functional Studies: Use antibodies for neutralization experiments in cell culture models to assess the functional contribution of secreted CHI3L2 to glioblastoma cell proliferation, migration, and invasion.

• Signaling Pathway Analysis: Combine CHI3L2 antibodies with phospho-specific antibodies in multiplex assays to identify downstream signaling pathways activated by CHI3L2.

• Extracellular Matrix Interactions: Employ co-immunoprecipitation with CHI3L2 antibodies to identify binding partners in the tumor microenvironment.

• In vivo Imaging: Develop labeled CHI3L2 antibodies for experimental tumor imaging in preclinical models.

• Therapeutic Targeting: Evaluate CHI3L2 antibodies as potential therapeutic agents for blocking CHI3L2 function in glioblastoma.

The strong interaction of CHI3L2 antibodies with the protein in glioblastoma tissue lysates provides a solid foundation for these research applications .

What methodological approaches can distinguish between CHI3L2 and other chitinase-like proteins in complex biological samples?

Distinguishing CHI3L2 from other chitinase-like proteins requires careful methodological approaches:

• Epitope-Specific Antibodies: Select antibodies raised against regions with minimal sequence homology to other family members. The epitope mapping data for available antibodies should be consulted.

• Knockout/Knockdown Controls: Include samples from CHI3L2 knockout models or knockdown experiments as negative controls to confirm antibody specificity.

• Recombinant Protein Panels: Test antibody cross-reactivity against a panel of recombinant chitinase and chitinase-like proteins under identical conditions.

• Mass Spectrometry Validation: Confirm antibody-detected proteins through immunoprecipitation followed by mass spectrometry analysis.

• Sequential Immunodepletion: Deplete samples of related proteins using specific antibodies before CHI3L2 analysis.

• Differential Expression Systems: Utilize expression systems where human CHI3L2 is expressed but related proteins are absent (considering that no rodent CHI3L2 has been reported, mouse models might provide a clean background) .

• Multiple Antibody Approach: Use multiple antibodies recognizing different epitopes to increase confidence in specific detection.

The reported 89% amino acid identity between human and bovine CHI3L2 suggests that species-specific detection might also require careful antibody selection .

How can researchers accurately quantify CHI3L2 in osteoarthritis research and correlate with disease progression?

For accurate quantification of CHI3L2 in osteoarthritis research:

• Sample Collection Standardization:

  • Establish protocols for consistent collection of synovial fluid, cartilage tissue, and serum samples

  • Document clinical parameters using validated scoring systems (e.g., WOMAC, Kellgren-Lawrence)

• Multi-modal Detection:

  • Implement complementary techniques (ELISA, Western blot, immunohistochemistry) for comprehensive analysis

  • Correlate protein levels with mRNA expression through RT-qPCR

• Comparative Analysis:

  • Include age-matched controls without osteoarthritis

  • Compare CHI3L2 levels across disease stages and in different joint compartments

• Longitudinal Monitoring:

  • Establish baseline CHI3L2 levels and track changes over time

  • Correlate fluctuations with clinical progression and therapeutic interventions

• Functional Correlation:

  • Assess relationship between CHI3L2 levels and cartilage degradation markers (MMPs, COMP, CTX-II)

  • Evaluate CHI3L2's association with inflammatory mediators (IL-1β, TNF-α)

• Methodological Considerations:

  • Normalize tissue measurements to total protein or specific housekeeping proteins

  • Account for potential confounding factors (age, BMI, comorbidities)

  • Consider diurnal variations in secreted protein levels

Since CHI3L2 is secreted by chondrocytes and synovial fibroblasts, particularly in osteoarthritis, these approaches can help establish its utility as a biomarker for disease activity and progression .

What are the considerations for developing multiplex assays that include CHI3L2 detection?

Developing multiplex assays incorporating CHI3L2 detection requires addressing several technical challenges:

• Antibody Compatibility: Ensure that antibodies used for CHI3L2 detection are compatible with other antibodies in the multiplex panel in terms of species origin, isotype, and working concentrations.

• Signal Optimization: Balance signal intensities across all analytes to prevent dominant signals from one marker masking detection of others.

• Cross-reactivity Assessment: Thoroughly test for cross-reactivity between detection antibodies and non-target analytes in the multiplex panel.

• Buffer Compatibility: Develop common sample dilution and assay buffers that maintain optimal conditions for all antibody-antigen interactions in the panel.

• Dynamic Range Considerations: Accommodate the different concentration ranges of CHI3L2 and other analytes in biological samples.

• Platform Selection: Choose appropriate technology platforms (bead-based, planar array, proximity extension assay) based on required sensitivity, sample volume constraints, and equipment availability.

• Validation Strategy: Validate the multiplex assay against singleton assays for each analyte to ensure comparable performance.

Multiplex approaches could be particularly valuable for investigating CHI3L2 alongside other chitinase-like proteins or inflammatory mediators in disease contexts like osteoarthritis or glioblastoma .

How can CHI3L2 antibodies be utilized in developing therapeutic approaches targeting this protein?

CHI3L2 antibodies offer several potential routes for therapeutic development:

• Neutralizing Antibody Screening:

  • Test existing antibody clones for their ability to block CHI3L2 function in cell-based assays

  • Identify epitopes critical for CHI3L2's interaction with binding partners

• Antibody Engineering:

  • Develop humanized or fully human versions of effective neutralizing antibodies

  • Explore antibody fragments (Fab, scFv) for improved tissue penetration

• Antibody-Drug Conjugates:

  • Utilize CHI3L2's elevated expression in glioblastoma to deliver cytotoxic payloads

  • Optimize drug-to-antibody ratios and linker chemistry for specific disease contexts

• Bispecific Antibodies:

  • Design constructs targeting both CHI3L2 and immune effector cells for enhanced tumor targeting

  • Develop bispecifics addressing multiple disease mediators simultaneously

• Mechanism Elucidation:

  • Use antibodies to identify the precise molecular mechanisms through which CHI3L2 contributes to disease

  • Characterize binding interactions with potential receptors or signaling molecules

• Companion Diagnostics:

  • Develop standardized assays to identify patients with elevated CHI3L2 expression who might benefit from targeted therapies

  • Establish clinically relevant cutoff values for therapeutic decision-making

The strong interaction of CHI3L2 antibodies with the protein in glioblastoma tissue provides a foundation for exploring these therapeutic approaches .

What methodological adaptations are required when investigating CHI3L2 in different tissue and cell types?

Investigating CHI3L2 across diverse tissue and cell types requires methodological adaptations:

• Extraction Protocol Optimization:

  • Adjust lysis buffers based on tissue composition (detergent concentration, mechanical disruption methods)

  • Optimize protein extraction from cartilage tissue, which can be particularly challenging due to dense extracellular matrix

  • Test different homogenization techniques for preserving protein integrity in various tissue types

• Cell Type-Specific Considerations:

  • For chondrocytes: Culture in three-dimensional systems to maintain phenotype

  • For synovial fibroblasts: Ensure appropriate passage number to prevent phenotypic drift

  • For glioblastoma cells: Consider heterogeneity within tumor cell populations

• Detection Method Adjustments:

  • Western blot: Modify sample preparation and loading amounts based on expected expression levels

  • ELISA: Adjust sample dilutions to fall within the assay's linear range

  • Immunocytochemistry: Optimize fixation and permeabilization for different cell types

• Background Reduction Strategies:

  • Employ tissue-specific blocking agents (normal serum matching tissue origin)

  • Adjust antibody concentrations based on target abundance in specific tissues

  • Consider autofluorescence quenching for certain tissues in fluorescence-based detection

• Validation Approaches:

  • Include appropriate positive control tissues (osteoarthritic cartilage, glioblastoma)

  • Utilize orthogonal detection methods to confirm expression patterns

  • Consider genetic manipulation (siRNA, CRISPR) to validate antibody specificity in cell culture models