FUT4 Monoclonal Antibody

What is FUT4 and what are its key molecular properties?

FUT4, encoded by the FUT4 gene on chromosome 11q21, is a 314 amino acid protein with a molecular weight of approximately 37 kDa. The protein undergoes several post-translational modifications including phosphorylation, glycosylation, and formation of disulfide bonds, which are critical to its function. FUT4 plays a significant role in cell adhesion and signaling processes, particularly in leukocyte migration and inflammation. The protein facilitates cell-cell interactions and mediates immune responses and host defense mechanisms, making it an important target for immunological research . The enzyme belongs to the fucosyltransferase family and is officially designated as Alpha-(1,3)-fucosyltransferase 4, with the EC number 2.4.1.65, highlighting its enzymatic function in transferring fucose residues .

Where is FUT4/CD15 normally expressed in the human body?

FUT4/CD15 is primarily expressed on the surface of specific immune cells, most notably neutrophils (present on >95% of granulocytes), eosinophils, and to a lesser extent on monocytes . Beyond these leukocytes, FUT4 expression can also be detected in various tissues including bone marrow, lymph nodes, and certain epithelial cells . The differential expression patterns across tissues make FUT4 an important marker for distinguishing cell types in mixed populations. The regulation of FUT4 expression involves various factors including cytokines, growth factors, and cellular signaling pathways that are activated during immune responses and inflammation . This regulated expression pattern makes FUT4 particularly valuable as a marker for studying immune cell activation and inflammatory conditions.

What are the optimal protocols for using FUT4 antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with FUT4/CD15 monoclonal antibodies, researchers should use formalin-fixed, paraffin-embedded (FFPE) sections at an antibody concentration of 0.5-1.0 μg/mL. This concentration has been validated specifically on human Hodgkin's lymphoma tissues, where Reed-Sternberg cells serve as positive controls . For immunofluorescence applications, similar concentrations (0.5-1.0 μg/mL) are recommended on either 4% PFA-fixed or acetone-fixed tissue sections . When performing IHC, standard antigen retrieval methods are typically required, though specific optimization may be necessary depending on tissue type and fixation conditions. Signal development systems should be selected based on the antibody's conjugate status, with appropriate secondary antibodies for unconjugated primary antibodies. Validation of staining should include known positive controls such as neutrophil-rich tissues or Hodgkin's lymphoma samples containing Reed-Sternberg cells.

How should researchers optimize flow cytometry protocols for FUT4/CD15 detection?

For flow cytometry applications, the recommended concentration of FUT4/CD15 monoclonal antibodies is 0.5-1 μg per million cells in a volume of 0.1 mL . Positive controls should include U937 cells or Reed-Sternberg cells from Hodgkin's lymphoma samples . Researchers should consider the following optimization steps: (1) Titration of antibody concentration to determine optimal signal-to-noise ratio; (2) Appropriate blocking to minimize non-specific binding; (3) Selection of proper fluorochrome conjugates based on the cytometer configuration and experimental design; and (4) Inclusion of isotype controls to establish gating strategies. When using antibodies with different fluorophore conjugates, researchers should refer to specific excitation/emission profiles. For example, CF®488A conjugated antibodies have excitation/emission at 490/515nm and are compatible with GFP/FITC detection channels, while CF®647 conjugates have excitation/emission at 650/665nm and work with Cy®5 channels .

What strategies should be employed for validating FUT4 antibody specificity?

Validating FUT4/CD15 antibody specificity requires a multi-faceted approach. First, researchers should confirm the antibody reacts with the expected molecular weight protein (approximately 220 kDa for CD15/FUT4 expressed on Reed-Sternberg cells) . Second, positive and negative control samples should be included in each experimental run – neutrophils, U937 cells, and Reed-Sternberg cells serve as excellent positive controls . Third, blocking experiments with recombinant FUT4 protein or competitive binding assays can help confirm specificity. Fourth, cross-reactivity testing against related proteins, particularly other fucosyltransferases, should be performed to ensure target selectivity. Finally, consistent results across multiple detection methods (IHC, ICC, flow cytometry) provide additional validation of specificity. For research requiring absolute specificity, validation in FUT4-knockout or knockdown models offers the most stringent confirmation of antibody performance.

How do FUT4 expression patterns aid in distinguishing Hodgkin's lymphoma from other lymphomas?

FUT4/CD15 expression on Reed-Sternberg cells represents a critical diagnostic marker for Hodgkin's lymphoma . The distinguishing feature is that while FUT4 is consistently expressed in Hodgkin's lymphoma, it is only occasionally expressed in large cell lymphomas of B and T phenotypes . This differential expression pattern aids pathologists in accurate classification. Advanced research protocols leverage dual or triple immunostaining approaches combining FUT4/CD15 with other markers like CD30 to increase diagnostic specificity. Quantitative image analysis of FUT4 staining patterns (intensity, distribution, and co-localization with other markers) can further enhance diagnostic accuracy. Researchers should be aware that while FUT4 is a valuable marker, it must be interpreted in the context of morphological features and additional immunophenotypic markers. Recent studies have explored the relationship between FUT4 expression levels and Hodgkin's lymphoma prognosis, suggesting potential value beyond mere diagnostic applications.

What is the relationship between FUT4 function and IgG antibody fucosylation in immune research?

Fucosylation plays a critical role in antibody function, with profound implications for immune research using monoclonal antibodies. IgG1 antibodies lacking Fc core α-1,6-fucose show a 10-100 fold increased binding to FcγRIIIA and FcγRIIIB on immune cells including NK cells, monocytes/macrophages, and polymorphonuclear leukocytes (PMNs) . This enhanced binding results in increased antibody-dependent cellular cytotoxicity (ADCC) by NK cells and enhanced competition with plasma IgGs . Low Fc core fucose can also increase the release of cytokines such as IL-6, TNF-α, and IL-8 both in vitro and in vivo . When designing experiments using FUT4 monoclonal antibodies for functional studies, researchers must consider the fucosylation status of these antibodies, as it may significantly impact their binding properties and downstream effector functions. The interplay between FUT4 as a target and the fucosylation status of anti-FUT4 antibodies adds a layer of complexity that researchers must account for when interpreting experimental results.

How does FUT4 contribute to neutrophil function in immunological research models?

FUT4/CD15 plays essential roles in mediating phagocytosis, bactericidal activity, and chemotaxis in neutrophils, which are present on >95% of granulocytes . In research models, FUT4 functions as both a marker and functional molecule for studying neutrophil biology. Researchers investigating neutrophil recruitment, activation, and effector functions should consider FUT4's role in these processes. Experimental approaches may include using anti-FUT4 antibodies to track neutrophil migration in inflammatory models, assess neutrophil activation states, or even modulate neutrophil function through FUT4 targeting. The expression of FUT4 on neutrophils can be regulated by inflammatory cytokines, making it a dynamic marker that reflects the activation state of these cells. Advanced imaging techniques combining FUT4 antibodies with functional readouts (reactive oxygen species production, neutrophil extracellular trap formation) can provide deeper insights into how this molecule contributes to neutrophil-mediated immunity in both health and disease.

What are common challenges when using FUT4 antibodies across different applications?

Researchers frequently encounter several challenges when working with FUT4/CD15 antibodies. First, the glycosylation status of the FUT4 protein can affect epitope accessibility, leading to variable antibody binding across different tissue preparation methods. Second, the presence of FUT4 on multiple cell types may complicate interpretation in heterogeneous samples. To address these challenges, researchers should: (1) Validate antibody performance in their specific sample types; (2) Include appropriate positive controls (e.g., neutrophils or Reed-Sternberg cells) ; (3) Consider using multiple antibody clones targeting different epitopes; and (4) Implement rigorous blocking steps to minimize background staining. For flow cytometry applications, additional considerations include properly compensating for spectral overlap when using multiple fluorochromes and utilizing viability dyes to exclude dead cells that may bind antibodies non-specifically. Finally, batch testing of antibodies is recommended for longitudinal studies to ensure consistent performance over time.

How can researchers differentiate between FUT4 and related fucosyltransferases in their studies?

Distinguishing FUT4 from other fucosyltransferases requires careful experimental design. FUT4 (Alpha-(1,3)-fucosyltransferase 4) is one member of a family of enzymes with similar catalytic functions but different substrate preferences and expression patterns . To ensure specificity, researchers should: (1) Select antibodies that have been validated against multiple fucosyltransferases to confirm target selectivity; (2) Use genetic approaches (siRNA, CRISPR) to knock down FUT4 specifically and confirm antibody specificity; (3) Employ mass spectrometry or enzymatic activity assays to distinguish between different fucosyltransferases based on their products; and (4) Consider the molecular weight differences – FUT4 has a molecular weight of approximately 37 kDa as a protein , but the antibody targets a 220 kDa glycoprotein complex on Reed-Sternberg cells . Additionally, comparative expression analysis across tissues known to differentially express various fucosyltransferases can provide supporting evidence for specific FUT4 detection.

What factors affect the reproducibility of FUT4 antibody-based experiments?

Multiple factors can impact the reproducibility of FUT4 antibody-based experiments. First, antibody source and clone variability: different clones may recognize distinct epitopes on the FUT4 protein, affecting binding characteristics. Second, sample preparation methods: fixation protocols, antigen retrieval conditions, and cell permeabilization techniques can all alter epitope availability. Third, experimental conditions: variations in incubation times, temperatures, and buffer compositions may affect antibody binding kinetics. To maximize reproducibility, researchers should: (1) Document detailed protocols including specific antibody catalog numbers and lot numbers; (2) Establish consistent sample processing workflows; (3) Include standardized positive and negative controls in each experiment; (4) Implement quality control measures such as regular antibody validation; and (5) Consider the post-translational modifications of FUT4, which include phosphorylation, glycosylation, and disulfide bond formation . Additionally, researchers should be aware that cytokines and cellular activation can alter FUT4 expression levels, potentially introducing variability in dynamic cellular systems.

How is FUT4 being investigated as a potential therapeutic target?

FUT4/CD15 has emerged as a promising therapeutic target due to its association with disease states including inflammatory disorders and cancer . Current research approaches include: (1) Development of specific anti-FUT4 monoclonal antibodies for targeted therapy, similar to brentuximab vedotin which targets CD30 but shows cross-reactivity with CD15 on Reed-Sternberg cells in Hodgkin's lymphoma ; (2) Investigation of small molecule inhibitors targeting FUT4 enzymatic activity; (3) Exploration of FUT4's role in tumor progression and metastasis to identify intervention points; and (4) Assessment of FUT4 as part of combination therapy approaches, particularly for Hodgkin's lymphoma treatment. Research challenges include ensuring sufficient specificity to avoid affecting normal neutrophil function, as FUT4 is expressed on >95% of granulocytes . Future directions may involve developing antibody-drug conjugates specifically targeting FUT4 on malignant cells or creating bispecific antibodies that recognize both FUT4 and tumor-specific antigens to enhance specificity.

What recent advances have been made in understanding FUT4 biology in developmental processes?

Recent research has expanded our understanding of FUT4's role beyond the immune system to developmental processes. FUT4, also known as SSEA-1 (stage-specific embryonic antigen 1), plays important roles in embryonic development . Current investigations focus on: (1) FUT4's contribution to stem cell biology, particularly in maintaining pluripotency or directing differentiation; (2) The role of FUT4 in tissue development and regeneration; (3) Comparative analysis of FUT4 function across species to identify evolutionarily conserved developmental pathways; and (4) Exploration of FUT4's interaction with other developmental signaling molecules. Advanced research techniques including single-cell RNA sequencing, CRISPR-based gene editing, and high-resolution imaging are revealing FUT4's dynamic expression patterns during development. These studies are shedding light on how fucosylation contributes to cellular identity and function during embryogenesis and may open new avenues for regenerative medicine applications where modulating FUT4 activity could potentially direct cell fate decisions.