CXE9 Antibody

What is CXCL9 and what are its primary biological functions?

CXCL9 (C-X-C motif chemokine 9) is a chemokine that affects the growth, movement, and activation state of cells participating in immune and inflammatory responses. It is chemotactic for activated T-cells and specifically binds to CXCR3 receptor. Also known as MIG (Monokine Induced by Interferon-gamma), CXCL9 is primarily induced by interferon-gamma and plays critical roles in T-cell trafficking and immune surveillance .

The protein functions in multiple biological contexts:

• Recruitment of activated T-cells to sites of inflammation

• Regulation of tumor microenvironment and potential anti-tumor activity

• Involvement in autoimmune conditions

• Participation in antimicrobial responses

Understanding these functions is essential for proper experimental design when using CXCL9 antibodies in research applications.

What sample types can be analyzed using CXCL9 antibodies?

CXCL9 antibodies have been validated for detection in multiple sample types:

• Cell lines: Particularly effective in THP-1 human acute monocytic leukemia cell line, with stronger detection following IFN-gamma treatment

• Tissue sections: Successfully applied in formalin-fixed paraffin-embedded (FFPE) human spleen and mouse brain tissues

• Primary cells: Effective in detecting CXCL9 in macrophages, including those in hepatitis liver tissue

• Biological fluids: Used in ELISA applications for detecting CXCL9 in serum samples from both humans and non-human primates

When selecting sample types, consider that CXCL9 expression is highly inducible and may require stimulation with IFN-gamma or other inflammatory mediators for optimal detection in certain experimental systems.

What are the main applications of CXCL9 antibodies in research?

CXCL9 antibodies have been validated for several key research applications:

Researchers should note that optimal dilutions should be determined for each specific laboratory application, as conditions may vary based on sample type, fixation methods, and detection systems.

How can dual-specificity of anti-CXCL9/CXCL10 antibodies be characterized?

The dual-specificity of antibodies that recognize both CXCL9 and CXCL10 presents a fascinating research topic. To characterize such antibodies:

• Cross-species reactivity analysis: Test antibody binding against CXCL9 and CXCL10 from different species (human, cynomolgus, mouse, rat, rabbit). This approach can reveal critical binding determinants, as demonstrated by scFvs that bind to human and cynomolgus CXCL10 but not mouse CXCL10, while binding to mouse but not cynomolgus CXCL9 .

• Sequence alignment and epitope mapping: Align sequences of proteins that do and do not bind to identify candidate epitope residues. For example, despite 83% sequence identity between human and rabbit CXCL10, certain scFvs bind only to human CXCL10, allowing identification of eleven potentially critical residues for antibody binding .

• Site-directed mutagenesis: Generate mutants of CXCL9, CXCL10, and CXCL11 to identify specific residues critical for antibody binding. This approach identified serine 13 as a key residue for dual-specific scFv binding to both CXCL9 and CXCL10 .

• Functional neutralization assays: Determine if the antibody blocks chemokine-receptor interactions by measuring the inhibition of chemotaxis or calcium flux in CXCR3-expressing cells.

This methodological approach not only characterizes the antibody but provides insight into structural mimicry between chemokines that may have evolutionary and functional significance.

What methodologies are optimal for detecting CXCL9 expression in stimulated versus unstimulated cells?

When investigating CXCL9 expression under different stimulation conditions, several methodological considerations are important:

• Cell stimulation protocol:

  • IFN-gamma is the primary inducer of CXCL9. For optimal results in THP-1 cells, treatment with IFN-gamma significantly increases CXCL9 expression compared to untreated controls .

  • Document treatment duration and concentration of stimulants.

• Immunofluorescence detection:

  • Fixed cell preparation: Use immersion fixation rather than cross-linking fixatives for optimal epitope preservation.

  • Primary antibody incubation: Extend to 3 hours at room temperature using 25 μg/mL of anti-CXCL9 antibody for optimal signal-to-noise ratio .

  • Secondary detection: Fluorophore-conjugated secondary antibodies (such as NorthernLights™ 557-conjugated Anti-Mouse IgG) provide excellent signal with low background.

  • Counterstaining: DAPI nuclear counterstain helps visualize all cells for accurate comparative analysis.

• Controls:

  • Include both stimulated and unstimulated cells from the same cell line.

  • Use isotype controls to confirm specificity.

• Quantification:

  • Employ image analysis software to quantify fluorescence intensity.

  • Normalize to cell number based on nuclear counterstain.

This methodology enables reliable detection of differential CXCL9 expression, which is predominantly localized to the cytoplasm in responsive cells .

How can CXCL9 immunohistochemistry be utilized to distinguish immunodeficiency syndromes from lymphomas?

Recent research has established CXCL9 as a valuable biomarker for differentiating adult-onset immunodeficiency syndrome associated with anti-IFN-γ autoantibodies (AIGA) from nodal T follicular helper cell lymphoma, angioimmunoblastic type (nTFHL-AI) . The methodological approach includes:

• Specimen preparation:

  • Use formalin-fixed paraffin-embedded lymph node specimens.

  • Employ standard antigen retrieval techniques.

• Immunohistochemistry protocol:

  • Apply validated anti-CXCL9 antibodies.

  • Utilize appropriate detection systems.

  • Include positive and negative controls.

• Interpretation criteria:

  • Quantify density of CXCL9-positive cells.

  • Apply established cutoff values: AIGA specimens show significantly lower density of CXCL9-positive cells compared to nTFHL-AI.

• Diagnostic metrics:

  • Sensitivity: 92.3% for distinguishing AIGA from lymphomas

  • Specificity: 100% for distinguishing AIGA from lymphomas

This approach provides a robust diagnostic tool that can prevent misdiagnosis of AIGA as lymphoma, especially in cases where clinical suspicion of immunodeficiency might not be initially present. The methodology offers clinicians a reliable means to avoid unnecessary treatments and ensure appropriate management of immunodeficiency conditions.

What are common issues in CXCL9 antibody applications and how can they be resolved?

Researchers may encounter several challenges when working with CXCL9 antibodies:

• Low signal intensity in Western blot applications:

  • Problem: CXCL9 detection may be compromised under reducing conditions.

  • Solution: Use non-reducing conditions as some CXCL9 antibodies work optimally under non-reducing conditions only . This preserves the tertiary structure important for antibody recognition.

• Variable results in ELISA applications:

  • Problem: Inconsistent standard curves or sensitivity.

  • Solution: Optimize antibody concentrations. For sandwich immunoassays, typical concentrations are 0.5-4 μg/mL for the capture antibody in the presence of 0.25 μg/mL recombinant human CXCL9 .

• Negative results in ICC/IF applications:

  • Problem: No detection of CXCL9 in unstimulated cells.

  • Solution: Stimulate cells with appropriate cytokines (particularly IFN-gamma) to induce CXCL9 expression. Comparative analysis of treated versus untreated cells can serve as internal controls .

• Cross-reactivity concerns:

  • Problem: Uncertainty about antibody specificity across related chemokines.

  • Solution: Validate antibody specificity using ELISA against recombinant CXCL9, CXCL10, and CXCL11 proteins. For dual-specific antibodies, characterize binding epitopes through mutagenesis studies .

• Species cross-reactivity limitations:

  • Problem: Antibody may not recognize CXCL9 from all species despite sequence homology.

  • Solution: Test antibody against CXCL9 from multiple species or select antibodies specifically validated for your species of interest .

How should controls be designed for CXCL9 antibody validation in immunohistochemistry?

Proper control design is critical for validating CXCL9 antibody performance in immunohistochemistry:

• Positive tissue controls:

  • Human spleen tissue serves as an excellent positive control, consistently showing CXCL9 expression .

  • IFN-gamma stimulated tissues or cells with known CXCL9 upregulation.

  • For mouse studies, mouse brain tissue has been validated for CXCL9 detection .

• Negative controls:

  • Primary antibody omission: Replace primary antibody with buffer or isotype-matched non-specific immunoglobulin.

  • Tissues known to lack CXCL9 expression.

  • Unstimulated THP-1 cells show minimal CXCL9 expression compared to IFN-gamma stimulated cells .

• Antibody validation controls:

  • Peptide blocking: Pre-incubate antibody with the immunizing peptide before application to tissue to confirm specificity.

  • Dual antibody approach: Use two different antibodies targeting different epitopes on CXCL9.

• Protocol validation controls:

  • Dilution series: Test a range of antibody dilutions (e.g., 1:50, 1:100, 1:200, 1:500) to determine optimal signal-to-noise ratio .

  • Multiple detection systems: Compare different visualization methods (e.g., DAB chromogen versus fluorescence).

Careful documentation of all control results provides critical validation for research findings and ensures reproducibility across studies.

What is the significance of CXCL9 as a biomarker in clinical immunodeficiency research?

Recent research has identified CXCL9 as an important biomarker with significant clinical applications, particularly in distinguishing immunodeficiency conditions from lymphomas:

• Diagnostic application in adult-onset immunodeficiency:

  • CXCL9 immunohistochemistry offers remarkable diagnostic accuracy (92.3% sensitivity, 100% specificity) for differentiating adult-onset immunodeficiency syndrome associated with neutralizing anti-interferon γ autoantibodies (AIGA) from nodal T follicular helper cell lymphoma, angioimmunoblastic type (nTFHL-AI) .

  • This application is particularly valuable because AIGA often clinically resembles lymphoma, leading to potential misdiagnosis and inappropriate treatment.

• Molecular mechanism insights:

  • The downregulation of CXCL9 gene expression in AIGA provides insight into the immunopathology of this condition.

  • The lower density of CXCL9-positive cells in lymph node specimens from AIGA patients compared to lymphoma patients reflects underlying differences in immune activation and cytokine signaling pathways .

• Translational impact:

  • Implementation of CXCL9 immunohistochemistry in diagnostic algorithms could prevent unnecessary lymphoma treatments.

  • This approach enables timely and accurate diagnosis of immunodeficiency conditions that might otherwise be misclassified.

Future research directions may include investigating whether serum CXCL9 levels correlate with tissue expression and could serve as a less invasive diagnostic marker, and examining whether CXCL9 expression patterns could predict response to specific immunotherapies.

How do CXCL9 antibodies contribute to tumor immunology research?

CXCL9 antibodies have become valuable tools in tumor immunology research, revealing important roles for this chemokine in cancer immunity:

• Tumor microenvironment characterization:

  • CXCL9 antibodies enable identification of "M1hot" tumor-associated macrophages that influence tissue-resident memory T cell infiltration and survival in human lung cancer .

  • Immunohistochemical analysis of CXCL9 expression patterns in tumor tissues can help classify tumor immune microenvironments.

• CXCL9-CXCR3 axis in cancer immunity:

  • Neutralizing antibodies against CXCL9 allow functional studies examining the consequences of disrupting CXCL9-CXCR3 signaling in tumor models.

  • Such studies have revealed that pathogen evasion can occur through suppression of chemokine responses, including CXCL10, which may have implications for understanding how tumors evade immune surveillance .

• Therapeutic potential assessment:

  • Antibodies that specifically target CXCL9 help evaluate its role independent of other CXCR3 ligands (CXCL10, CXCL11).

  • Dual-specific antibodies targeting both CXCL9 and CXCL10 offer potential therapeutic approaches for modulating immune responses in various conditions, including tumors .

• Biomarker applications:

  • CXCL9 detection in patient samples may serve as a prognostic or predictive biomarker for immunotherapy response.

  • Correlation of CXCL9 expression with immune cell infiltration patterns and clinical outcomes informs patient stratification strategies.

As cancer immunotherapy continues to evolve, CXCL9 antibodies will likely play increasingly important roles in both basic and translational research.