FGFBP2 Antibody

What is FGFBP2 and why is it a significant research target?

FGFBP2, also known as killer-specific secretory protein of 37 kD (Ksp37), is a 37 kDa Th1-specific secretory protein produced primarily by natural killer (NK) cells, γ/δ T cells, a subset of effector CD8 T cells, and Th1 cells. It binds fibroblast growth factor and is secreted into serum. Most FGFBP2-expressing cells coexpress perforin, suggesting its involvement in essential processes of cytotoxic lymphocyte-mediated immunity . FGFBP2 has been associated with various conditions, including atopic asthma, mild extrinsic asthma, certain infectious diseases, and more recently, its genetic variants have been implicated in IgG4-related disease (IgG4-RD) . Recent observations have also shown that FGFBP2 correlates strongly with histology, stage, and outcomes in ovarian cancer, making it a valuable research target .

How do I select the appropriate FGFBP2 antibody for my specific research application?

When selecting an FGFBP2 antibody, consider these critical parameters:

• Experimental application: Different applications require different antibody properties:

  • Flow cytometry: Consider conjugated antibodies (PE, FITC) such as the TDA3 clone

  • Western blot: Polyclonal antibodies often perform well (dilutions typically 1:1000-1:4000)

  • Immunohistochemistry: Both monoclonal and polyclonal options are available (dilutions typically 1:200-1:500)

• Epitope recognition: Consider what region of FGFBP2 you need to target:

  • N-terminal (AA 20-223)

  • Middle region (AA 121-220)

  • C-terminal region

• Species reactivity: Most FGFBP2 antibodies are reactive with human samples, but some also recognize monkey FGFBP2 .

• Clonality: Monoclonal antibodies like TDA3 offer high specificity, while polyclonal antibodies may provide broader epitope recognition .

• Host species: Available in mouse (monoclonal) or rabbit (polyclonal) options, which affects secondary antibody selection .

• Validation data: Review the validation data for your application (Western blots, IHC images, flow cytometry plots) .

How should I validate FGFBP2 antibody specificity for my experiments?

Comprehensive validation of FGFBP2 antibody specificity should include:

• Positive and negative controls:

  • Positive control: Tissues or cell lines known to express FGFBP2 (NK cells, cytotoxic T lymphocytes, SH-SY5Y cells, BxPC-3 cells)

  • Negative control: Tissues or cell lines with minimal FGFBP2 expression, or vector-only transfected cells

• Blocking peptide experiments: Use a synthetic FGFBP2 peptide (such as ABIN940365) to pre-adsorb the antibody and verify signal disappearance .

• Molecular weight verification: Confirm the observed molecular weight matches the expected size (FGFBP2 has a calculated molecular weight of approximately 28 kDa but is typically observed at 37 kDa due to post-translational modifications) .

• Multiple antibody comparison: When possible, test multiple antibodies targeting different FGFBP2 epitopes to confirm consistent staining patterns .

• Genetic approaches: Use FGFBP2 knockdown or knockout samples as definitive negative controls if available.

What are the optimal fixation and staining protocols for detecting FGFBP2 in different sample types?

Optimal protocols vary by application and sample type:

For flow cytometry (intracellular staining):

• Fix cells in 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1% saponin or commercial permeabilization buffer

• Block with 5% normal serum from the same species as the secondary antibody

• Incubate with PE-conjugated anti-FGFBP2 (TDA3 clone) at 5 μL per million cells

• Analyze by flow cytometry

For immunohistochemistry (FFPE sections):

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with 5% normal serum

• Incubate with primary antibody at 1:200-1:500 dilution overnight at 4°C

• Apply appropriate detection system and counterstain

For Western blot:

• Extract proteins using a buffer containing protease inhibitors

• Separate proteins by SDS-PAGE

• Transfer to PVDF or nitrocellulose membrane

• Block with 5% non-fat milk in TBST

• Incubate with primary antibody at 1:1000-1:4000 dilution overnight at 4°C

• Incubate with HRP-conjugated secondary antibody

• Develop using ECL substrate

What considerations are important when detecting FGFBP2 in cytotoxic lymphocytes?

When studying FGFBP2 in cytotoxic lymphocytes, consider these technical aspects:

• Cell type identification: Include appropriate markers to identify specific lymphocyte subsets:

  • NK cells: CD56, CD16

  • Cytotoxic T cells: CD8, perforin

  • γ/δ T cells: TCR γ/δ

  • Th1 cells: CD4, IFN-γ

• Cell activation status: FGFBP2 expression may vary depending on lymphocyte activation state. Consider analyzing both resting and activated cells (using stimuli like PMA/ionomycin or IL-2) .

• Subcellular localization: FGFBP2 is a secreted protein but can be detected intracellularly before secretion. Staining for both intracellular and surface FGFBP2 may provide complementary data .

• Colocalization studies: Since FGFBP2 is often coexpressed with perforin, dual staining can provide valuable insights into cytotoxic lymphocyte function .

• Isolation method effects: Different lymphocyte isolation methods may affect FGFBP2 detection; consider comparing density gradient separation, magnetic bead isolation, and flow sorting .

How can I distinguish between specific and non-specific staining when using FGFBP2 antibodies?

To distinguish between specific and non-specific staining:

• Control experiments:

  • Isotype controls: Use the same concentration of non-specific antibody of the same isotype and host species

  • Secondary-only controls: Omit primary antibody to assess background from the detection system

  • Blocking peptide competition: Pre-incubate antibody with synthetic FGFBP2 peptide (ABIN940365) to confirm signal specificity

• Pattern analysis:

  • Specific FGFBP2 staining should show cytoplasmic localization in cytotoxic lymphocytes

  • In immunohistochemistry, strong cytoplasmic positivity is expected in cells expressing FGFBP2, such as glandular cells in colon tissue

• Signal intensity correlation:

  • FGFBP2 expression levels should correlate with known biological parameters (e.g., higher in cytotoxic lymphocytes, lower in non-immune cells)

  • Signal intensity should decrease in dose-dependent manner when using blocking peptides

• Multi-method validation:

  • Confirm FGFBP2 detection using multiple techniques (e.g., validate IHC findings with Western blot)

  • Use multiple antibodies targeting different epitopes to confirm specificity

What are common pitfalls in Western blot analysis of FGFBP2, and how can they be addressed?

Common Western blot issues with FGFBP2 detection include:

• Molecular weight discrepancies:

  • Calculated MW: 28 kDa (223 amino acids)

  • Observed MW: 37 kDa

  • This difference is likely due to post-translational modifications. Use positive control lysates to confirm the correct band

• Multiple bands:

  • May represent splice variants, degradation products, or non-specific binding

  • Validate using overexpression lysates compared to vector-only controls

  • Optimize antibody concentration and washing steps to reduce non-specific binding

• Weak signal:

  • FGFBP2 is secreted; consider using concentrated culture supernatants or serum samples

  • Optimize protein extraction methods to preserve FGFBP2 integrity

  • Use more sensitive detection methods (e.g., enhanced chemiluminescence substrates)

• High background:

  • Increase blocking time/concentration (5% milk or BSA in TBST)

  • Reduce primary antibody concentration (test dilutions from 1:1000 to 1:4000)

  • Increase washing duration and number of washes

• Sample preparation:

  • Include protease inhibitors in lysis buffers to prevent FGFBP2 degradation

  • Avoid repeated freeze-thaw cycles of samples

How can FGFBP2 antibodies be utilized to investigate its role in disease mechanisms?

FGFBP2 antibodies can be employed in multiple sophisticated approaches to study disease mechanisms:

• IgG4-Related Disease (IgG4-RD):

  • Investigate FGFBP2 expression in tissue biopsies from patients with IgG4-RD, particularly those with the common variant rs758329, which is enriched in IgG4-RD patients (found in 73% of cases vs. 40% in the general population)

  • Correlate FGFBP2 expression with CD4+ cytotoxic T cell infiltration, as IgG4-RD patients with FGFBP2 variants had 5-10 fold higher numbers of circulating cytotoxic CD4+ T cells

• Cancer research:

  • Analyze FGFBP2 expression in tumor samples and correlate with histology, stage, and clinical outcomes, particularly in ovarian cancer

  • Investigate potential interactions between FGFBP2 and FGF2 in cancer progression, as FGF2 neutralization by antibodies like GAL-F2 has shown anti-tumor effects in hepatocellular carcinoma xenograft models

• Asthma and inflammatory diseases:

  • Quantify FGFBP2 levels in bronchoalveolar lavage fluid or serum from asthma patients compared to controls

  • Correlate FGFBP2 expression with specific immune cell infiltrates and inflammatory mediators

• Mechanistic studies:

  • Use FGFBP2 antibodies in chromatin immunoprecipitation (ChIP) experiments to identify transcription factors regulating FGFBP2 expression

  • Perform co-immunoprecipitation to identify FGFBP2 binding partners beyond FGF

What approaches can be used to study the structural and functional consequences of FGFBP2 genetic variants?

The study of FGFBP2 genetic variants, such as those identified in IgG4-RD, requires specialized approaches:

• Structural analysis:

  • The frameshift variant in FGFBP2 found in IgG4-RD changes the C-terminal sequence from NEEAKKKAWEHCWKPFQALCAFLISFFRG to AKKRPGNIVGNPSRPCAPFSSASSEGDR, disrupting the helical-turn-helix structure stabilized by a disulfide bond

  • Use antibodies recognizing wild-type vs. variant-specific epitopes to examine expression patterns and subcellular localization

• Functional assays:

  • Compare FGF binding capacity between wild-type and variant FGFBP2 using co-immunoprecipitation or surface plasmon resonance

  • Assess effects on T cell function (cytotoxicity, cytokine production) when expressing wild-type vs. variant FGFBP2

• Cell-based models:

  • Generate cell lines expressing wild-type or variant FGFBP2 using CRISPR/Cas9 genome editing

  • Compare secretion levels, protein stability, and downstream signaling effects

• Animal models:

  • Create humanized mouse models expressing the common homozygous variant (rs758329) or the rare frameshift variant

  • Assess immune phenotypes and susceptibility to relevant disease models

• Patient-derived samples:

  • Compare FGFBP2 expression, localization, and function in cells from patients with different FGFBP2 genotypes

  • Correlate findings with clinical parameters and treatment responses

How can multiplexed imaging approaches be optimized for studying FGFBP2 in the tissue microenvironment?

Advanced multiplexed imaging techniques offer powerful tools for studying FGFBP2 in complex tissue contexts:

• Multiplex immunofluorescence:

  • Combine FGFBP2 antibodies with markers for specific cell types (CD8, CD4, CD56), activation states (perforin, granzyme B), and tissue structures

  • Use tyramide signal amplification (TSA) or similar methods to enable multiple rounds of staining

  • Consider spectral unmixing systems to separate closely overlapping fluorophores

• Mass cytometry imaging (IMC):

  • Label FGFBP2 antibodies with rare earth metals for detection by imaging mass cytometry

  • Combine with up to 40 additional markers to comprehensively characterize the immune microenvironment

  • Particularly valuable for analyzing FGFBP2+ cells in tumor or inflammatory disease tissues

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions between FGFBP2 and potential binding partners in situ

  • Useful for studying FGFBP2-FGF interactions or other molecular associations

• RNA-protein co-detection:

  • Combine immunofluorescence for FGFBP2 protein with RNA in situ hybridization for FGFBP2 mRNA or other genes of interest

  • Helps distinguish cells actively producing FGFBP2 versus those potentially taking up secreted protein

• Digital spatial profiling:

  • Use antibody panels including FGFBP2 with spatial transcriptomics to correlate protein expression with gene expression signatures

  • Particularly valuable for understanding FGFBP2's role in diverse microenvironments

How might FGFBP2 antibodies be used in developing potential diagnostic or therapeutic approaches?

FGFBP2 antibodies hold promise for diagnostic and therapeutic applications:

• Diagnostic applications:

  • Development of sensitive ELISAs or other immunoassays to quantify FGFBP2 in serum or other body fluids

  • Potential biomarker for cytotoxic lymphocyte activation in inflammatory diseases or cancer

  • Diagnostic marker for IgG4-RD, particularly when combined with genetic testing for FGFBP2 variants

• Therapeutic considerations:

  • While direct targeting of FGFBP2 is not yet established as a therapeutic approach, understanding its role in binding and potentially regulating FGF availability suggests possible applications

  • The success of anti-FGF2 antibodies like GAL-F2 in cancer models suggests that modulating the FGF pathway, potentially including FGFBP2, could have therapeutic value

  • Potential applications in autoimmune conditions where cytotoxic lymphocyte dysfunction is implicated, particularly IgG4-RD

• Companion diagnostics:

  • FGFBP2 expression levels might predict response to therapies targeting the FGF pathway or cytotoxic lymphocyte function

  • The common FGFBP2 variant (rs758329) could be investigated as a predictive marker for treatment response

What are the current technical limitations in FGFBP2 research, and how might they be overcome?

Current technical challenges in FGFBP2 research include:

• Antibody cross-reactivity:

  • Challenge: Ensuring antibody specificity across applications and species

  • Solution: Rigorous validation using multiple controls, including genetic knockout/knockdown approaches and multiple antibodies targeting different epitopes

• Detection of secreted vs. intracellular FGFBP2:

  • Challenge: Distinguishing between different pools of FGFBP2

  • Solution: Combine cell surface staining, intracellular staining, and analysis of culture supernatants/body fluids

• Functional readouts:

  • Challenge: Connecting FGFBP2 expression to functional outcomes

  • Solution: Develop robust assays measuring FGF binding, signaling pathway activation, and cellular effects in relevant models

• Variant-specific detection:

  • Challenge: Distinguishing wild-type from variant FGFBP2 proteins

  • Solution: Generate epitope-specific antibodies targeting regions affected by genetic variants

• Tissue heterogeneity:

  • Challenge: Analyzing FGFBP2 in complex tissues with varied cell populations

  • Solution: Implement single-cell approaches and spatial profiling technologies to provide cellular resolution