IFN g Antibody

What is the molecular target of anti-IFN-γ antibodies?

Anti-IFN-γ antibodies specifically recognize the protein encoded by the IFNG gene in humans. This 166-amino acid protein belongs to the Type II interferon family and functions as a secreted cytokine with significant immunomodulatory properties . The protein has a molecular weight of approximately 14-18 kDa and contains glycosylation sites that may affect antibody recognition depending on the epitope targeted . These antibodies are designed to detect both native and denatured forms of IFN-γ, though specificity may vary between clones.

How do anti-IFN-γ antibodies differ from anti-IFN-γ autoantibodies?

Anti-IFN-γ antibodies are laboratory-produced immunoglobulins specifically designed for research applications to detect and/or neutralize IFN-γ in experimental settings . In contrast, anti-IFN-γ autoantibodies are pathological antibodies produced endogenously by a patient's immune system that neutralize IFN-γ-mediated functions, contributing to acquired immunodeficiency . These autoantibodies have been implicated in increased susceptibility to opportunistic infections, particularly disseminated nontuberculous mycobacterial (dNTM) infections in previously healthy adults . Research has shown that >80% of patients with opportunistic infections including dNTM have neutralizing IFN-γ autoantibodies, compared to only 2% in tuberculosis patients and healthy controls .

What epitopes on IFN-γ are recognized by common research antibodies?

Research has identified specific epitopes on the IFN-γ protein recognized by antibodies. For example, the widely used B27 monoclonal antibody targets a specific determinant that includes amino acids 27-40 (TLFLGILKNWKEES) of human IFN-γ . Through mutagenesis studies, researchers have demonstrated that the residues T27, F29, and L30 are critical for B27 antibody binding . This epitope is located in the contact surface between IFN-γ and interferon gamma receptor 1, explaining the neutralizing activity of antibodies targeting this region . Understanding the specific epitope recognition is crucial for experimental design, particularly when multiple antibodies are used in the same assay.

What methods are available for detecting anti-IFN-γ antibodies or autoantibodies?

Several methods can be employed for detecting anti-IFN-γ antibodies or autoantibodies:

• Enzyme-Linked Immunosorbent Assay (ELISA): Indirect ELISA can determine the presence and concentration of anti-IFN-γ antibodies in sera. This technique uses recombinant IFN-γ (such as from R&D systems) as the coating antigen .

• Isotype and Subtype Determination: ELISA protocols with HRP-conjugated anti-human IgG1, IgG2, IgG3, IgG4, IgA, and IgM can identify the specific isotypes and subtypes of these antibodies .

• Western Blot: Used to confirm binding specificity and assess antibody recognition of denatured protein forms .

• Functional Assays: These measure the neutralizing activity of anti-IFN-γ antibodies .

• IFN-γ Release Assays: Tests like QuantiFERON-TB have been used to indirectly detect the presence of neutralizing IFN-γ autoantibodies, as these patients typically show insufficient mitogen response resulting in invalid test results .

How can I perform intracellular staining to detect IFN-γ-producing cells?

Intracellular detection of IFN-γ is a common immunological technique. The protocol using the PE-conjugated B27 antibody involves:

• Cell Preparation: Fix and permeabilize 1 × 10^6 cells to allow antibody access to intracellular cytokines.

• Staining: Resuspend cells in 20 μl of pre-titered antibody solution and 30 μl of 1X Perm/Wash Buffer. Incubate for 15 minutes (4°C, in the dark) .

• Washing: Wash twice in 100 μl of 1X Perm/Wash Buffer .

• Analysis: Perform flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations.

• Controls: Include isotype controls such as PE-MOPC-21 to assess background staining. A specificity control can be performed by pre-blocking cells with unlabeled B27 antibody prior to staining .

This technique is particularly useful for phenotyping T cells and other immune cells based on their cytokine production profile.

How can I characterize linear B cell epitopes of IFN-γ recognized by antibodies?

Linear B cell epitopes on IFN-γ can be identified using overlapping synthetic peptides spanning the protein sequence:

• Sequence Analysis: Search the amino acid sequence of IFN-γ on databases like NCBI using BLAST .

• Peptide Library Construction: Generate biotinylated peptides with 20 amino acids in length, overlapping each other by 10 amino acids. These should be of high purity (>95%) .

• Assay Setup: Coat immunoplates with streptavidin (2 μg/ml), then add biotinylated peptides after blocking .

• Detection: Incubate with test sera followed by anti-human IgG conjugated to HRP and appropriate substrate .

• Analysis: Compare binding patterns to identify specific epitope regions recognized by the antibodies.

This approach allows mapping of linear epitopes and can help identify immunodominant regions of the protein.

How can anti-IFN-γ antibodies be used to study cytokine signaling pathways?

Anti-IFN-γ antibodies are valuable tools for studying cytokine signaling in multiple contexts:

• Neutralization Studies: Neutralizing antibodies like the B27 clone can block IFN-γ signaling, allowing researchers to assess the specific contribution of this cytokine to observed biological effects .

• Receptor-Ligand Interaction Analysis: Since IFN-γ exerts its biological effects through binding to the IFN-γ receptor complex (IFN-γRα/CD119 and IFN-γRβ), antibodies targeting specific epitopes can help investigate these interactions .

• Signaling Pathway Dissection: IFN-γ activates the JAK-STAT1 signaling pathway. Anti-IFN-γ antibodies can be used in combination with phospho-specific antibodies against signaling components to dissect pathway activation .

• Functional Immunology Studies: These antibodies help investigate IFN-γ's role in upregulating immune cell functions, including antimicrobial and anti-tumor responses of macrophages, NK cells, and neutrophils .

What are the implications of IFN-γ and its antibodies in tumor immunology research?

Recent research has revealed complex roles for IFN-γ in the tumor microenvironment (TME):

• Dual Functions: While traditionally considered anti-tumorigenic, recent studies show IFN-γ can also have immunosuppressive effects on colorectal cancer (CRC) cells .

• B7H4 Expression: IFN-γ stimulation increases the expression of B7 homologous protein 4 (B7H4) in CRC tumors. B7H4 promotes CRC cell growth by inhibiting granzyme B release from CD8+ T cells and accelerating CD8+ T cell apoptosis .

• Molecular Mechanism: The IFN-γ/IRF1/B7H4 axis has been identified, where interferon regulatory factor 1 (IRF1) binds to the B7H4 promoter following IFN-γ stimulation .

• Clinical Correlations: High expression of B7H4 in cancer cells or low expression of CD8 in the microenvironment is negatively related to clinical outcomes in CRC patients .

These findings suggest that targeting the IFN-γ/IRF1/B7H4 axis might be a novel immunotherapeutic approach to restore cytotoxic killing of cancer cells.

What factors can affect the reliability of anti-IFN-γ antibody detection assays?

Several factors can influence the reliability of anti-IFN-γ antibody detection:

• Epitope Recognition: Different antibody clones recognize distinct epitopes on the IFN-γ protein. For example, specific mutations at positions T27, F29, and L30 can abolish binding of the B27 monoclonal antibody .

• Protein Conformation: Native versus denatured protein forms may be differentially recognized by antibodies, affecting detection in different assay formats (ELISA vs. Western blot) .

• Cross-reactivity: Antibodies may cross-react with related proteins, necessitating proper controls and validation.

• Sample Processing: Improper fixation or permeabilization can affect intracellular staining results .

• Antibody Titer Fluctuations: In the case of autoantibodies, studies have shown that IFN-γ autoantibody titers fluctuate with disease severity, potentially affecting detection sensitivity at different disease stages .

How can I validate the specificity of an anti-IFN-γ antibody?

To validate antibody specificity, consider these approaches:

• Pre-blocking Controls: Pre-block samples with unlabeled antibody of the same clone prior to staining with labeled antibody. Loss of signal indicates specificity .

• Isotype Controls: Use appropriate isotype controls (e.g., PE-MOPC-21 for mouse IgG1 antibodies) to assess background staining .

• Mutant Protein Testing: Testing antibody binding against wild-type versus mutant IFN-γ proteins (like the T27A, triple mutant, or T27-L33 deletion mutant) can confirm epitope specificity .

• Knockout/Knockdown Validation: Use samples from IFN-γ knockout models or cells with IFN-γ knockdown to confirm antibody specificity.

• Western Blot Analysis: Confirm binding to a protein of the expected molecular weight (14-18 kDa for IFN-γ) .

What are the best practices for determining the avidity of anti-IFN-γ antibodies?

Avidity determination is critical for characterizing antibody-antigen interactions:

• Guanidine Hydrochloride Dissociation: Treat antibody-antigen complexes with 4.0 M guanidine hydrochloride to disrupt binding. Compare OD values before and after treatment .

• Avidity Index Calculation: Calculate the avidity index as the ratio of the OD of guanidine-treated wells to the OD of untreated wells, multiplied by 100 .

• Controls: Include a well-characterized antigen-antibody interaction (e.g., tetanus toxoid) as a control for the assay .

• Comparative Analysis: Compare avidity indices between different antibody samples or patient sera to characterize binding strength differences.

High avidity antibodies resist dissociation by chaotropic agents like guanidine hydrochloride, resulting in higher avidity indices.

How do anti-IFN-γ autoantibodies contribute to adult-onset immunodeficiency?

Anti-IFN-γ autoantibodies play a significant role in acquired immunodeficiency:

• Neutralizing Activity: These autoantibodies neutralize IFN-γ-mediated functions, impairing antimicrobial immunity .

• Ethnic Predisposition: dNTM with acquired IFN-γ autoantibodies is most common in people of East Asian descent, suggesting genetic factors, though cases have been documented across diverse ethnicities .

• Correlation with Disease Activity: Higher antibody titers correlate with active infection, and levels fluctuate with disease severity while remaining detectable even during treatment-induced improvement .

• Opportunistic Infection Risk: The presence of these autoantibodies distinguishes patients with opportunistic infections from those with tuberculosis and healthy controls (>80% vs. 2%) .

• Multiple Infections: Patients often present with multiple disseminated mycobacterial infections and may develop autoimmune conditions .

This understanding has significant implications for diagnosis and management of previously healthy adults who develop unusual infections.

What role does the IFN-γ/IRF1/B7H4 axis play in cancer immunotherapy research?

The IFN-γ/IRF1/B7H4 axis represents an emerging area of cancer immunotherapy research:

• Paradoxical IFN-γ Effects: Despite IFN-γ's traditional anti-tumor role, recent research shows it can promote colorectal cancer growth through immunosuppressive mechanisms .

• Molecular Pathway: IFN-γ stimulation leads to IRF1 activation, which binds to the B7H4 promoter, increasing B7H4 expression in cancer cells .

• Immunosuppressive Mechanism: B7H4 inhibits granzyme B release from CD8+ T cells and accelerates their apoptosis, protecting cancer cells from immune elimination .

• Prognostic Value: High B7H4 expression in cancer cells or low CD8 expression in the microenvironment correlates with poor prognosis in colorectal cancer patients .

• Therapeutic Potential: Interference with this axis represents a potential novel immunotherapeutic approach to restore anti-tumor immunity .

This research highlights the complexity of cytokine functions in the tumor microenvironment and opens new avenues for targeted therapy development.

How can epitope mapping of anti-IFN-γ antibodies inform therapeutic strategies?

Detailed epitope mapping provides valuable insights for therapeutic development:

• Functional Domain Identification: The B27 monoclonal antibody targets amino acids 27-40 of human IFN-γ, which resides in the contact surface between IFN-γ and its receptor .

• Critical Residue Identification: Mutagenesis studies identified T27, F29, and L30 as critical residues for antibody binding, with mutations abolishing the interaction .

• Receptor Binding Interference: Understanding that neutralizing antibodies target the receptor-binding domain explains their mechanism of action in blocking signaling .

• Autoantibody Targeting: Knowledge of immunodominant epitopes recognized by pathological autoantibodies enables development of targeted therapies that either block these autoantibodies or restore cytokine function .

• Cross-reactivity Assessment: Epitope mapping helps predict potential cross-reactivity with related proteins or cytokines, informing therapeutic safety profiles.

This detailed molecular understanding facilitates the development of more specific therapeutic antibodies and strategies to counteract pathological autoantibodies.