SWAP70 Monoclonal Antibody

Molecular Structure and Function

SWAP70 (SWAP Switching B-Cell Complex 70kDa Subunit) is a multifunctional protein initially identified as a component of an enzyme complex involved in immunoglobulin (Ig) switch region recombination in B lymphocytes . The protein contains several functional domains including an N-terminal EF-hand motif, a central Pleckstrin homology (PH) domain, and a C-terminal actin-binding domain .

SWAP70 functions as a versatile signaling molecule and cytoskeletal regulator. It can directly bind, bundle, and stabilize actin filaments through its C-terminal actin binding domain . Additionally, SWAP70 regulates cellular actin dynamics and organization via activation of RHOA and RAC1 GTPases . This activation requires SWAP70's binding to phosphoinositides, particularly phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) .

The protein has a molecular weight of approximately 70 kDa, with the antigen having a calculated molecular weight of approximately 34.25 kDa in some antibody preparations . This discrepancy likely reflects differences in the specific fragments or domains being targeted by various antibodies.

Cellular Distribution and Expression Patterns

SWAP70 expression shows a highly specific pattern, primarily restricted to mature B lymphocytes . Despite containing three nuclear localization signals, in small resting B cells, SWAP70 is mainly found in the cytoplasm . Upon B cell activation, SWAP70 undergoes dynamic changes in subcellular localization, moving between cytoplasmic, membrane, and nuclear compartments.

Immunohistochemical studies have shown that SWAP70 is not expressed in pre-B lymphocytes or T lymphocytes at any stage of development . It is absent in plasma cells, as demonstrated by the lack of co-staining with VS38c antigen . Outside the B lymphocytic compartments, SWAP70 expression has been detected in some cells in the thymic medulla, which could be either B cells or dendritic cells . Other cell types in brain, kidney, skin, prostate, and small intestine have been found negative for SWAP70 expression .

Role in Immune System Regulation

SWAP70 plays significant roles in B cell activation and development. During B cell activation, SWAP70 expression levels increase dramatically, and the protein undergoes sequential relocalization from the cytoplasm to the plasma membrane and then to the nucleus . This dynamic localization pattern suggests a multifaceted role in signaling cascades during B cell activation.

Recent research has revealed a critical role for SWAP70 in phagocytosis. SWAP70 is transiently recruited to nascent phagosomes in human monocyte-derived dendritic cells, where it remains associated shortly after phagocytic cup closure . This recruitment is mediated by specific binding of SWAP70's PH-domain to phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2) .

SWAP70 also associates with the B cell receptor (BCR) complex in activated, class-switching B cells. Interestingly, SWAP70 was found to associate with membrane IgG, but not IgM, suggesting a specific role in isotype-switched B cells . This association requires a functional pleckstrin homology domain and is controlled by the C terminus of SWAP70 .

Available Clones and Their Characteristics

Several monoclonal antibodies targeting SWAP70 have been developed for research applications. These include:

1. Clone 1A12: A mouse monoclonal antibody raised against a partial recombinant SWAP70 (amino acids 378-451) .

2. Clone 3H8: A mouse monoclonal antibody available as ascites fluid, targeting a specific epitope of SWAP70 with the sequence QTQVELQARFSTELEREKLIRQQMEEQVAQKSSELEQYLQRVRELEDMYLKLQEALEDERQARQDEETVRKLQA .

3. Clone 434CT16.5.5: A mouse monoclonal antibody conjugated with alkaline phosphatase (AP) .

4. Clone F-3: An IgG1 κ mouse monoclonal antibody that detects human SWAP70 .

These antibodies vary in their targeting epitopes, isotypes, and conjugation status. For instance, Clone 1A12 is an IgG1 isotype antibody targeting amino acids 378-451 of SWAP70 , while Clone 3H8 is an IgG2aκ isotype . These differences provide researchers with options for different experimental applications.

Production and Purification Methodologies

The SWAP70 monoclonal antibody Clone 1A12 was raised against a partial recombinant SWAP70 protein (NP_055870, amino acids 378-451) fused with a GST tag . The molecular weight of the GST tag alone is 26 kDa . This suggests that the immunogen used was a recombinant fusion protein produced in a bacterial expression system, a common approach for generating antibodies against specific protein regions.

The Clone 3H8 antibody is available as ascites fluid , indicating that it was produced by injecting hybridoma cells into the peritoneal cavity of mice and collecting the antibody-rich ascitic fluid. This production method typically yields high-titer antibody preparations suitable for various applications.

Different antibody preparations may require specific storage conditions to maintain activity. For example, the Clone 3H8 antibody should be stored at -20°C and may be shipped on dry ice to maintain optimal activity.

Epitope Specificity and Target Regions

Different SWAP70 monoclonal antibodies target different regions or epitopes of the protein:

Clone 1A12 targets amino acids 378-451 of SWAP70, corresponding to the sequence: QTQVELQARF STELEREKLI RQQMEEQVAQ KSSELEQYLQ RVRELEDMYL KLQEALEDER QARQDEETVR KLQA .

Clone 3H8 also recognizes an epitope within the same sequence region . This epitope specificity is crucial as it determines which functional domains or regions of SWAP70 the antibody recognizes.

Research has shown that different domains of SWAP70 contribute to its various functions. Truncation mutants of SWAP70 lacking its N-terminal fragment containing the EF-hand motif or its C-terminal fragment containing the putative Dbl-homology domain (DH) and actin-binding domain were recruited to phagosomes less efficiently compared to full-length SWAP70 . This indicates that not only the PH-domain but also the N- and C-terminal regions contribute to proper SWAP70 localization and function.

Western Blotting Applications

SWAP70 monoclonal antibodies have been extensively used in Western blotting (WB) applications to detect SWAP70 protein expression in various cell types and experimental conditions. The recommended dilutions for Western blotting vary among different antibody products:

• Clone 1A12 is suitable for Western blotting applications .

• Clone 3H8 is recommended for Western blotting at a dilution of 1-5 μg/mL .

• Clone 434CT16.5.5 is recommended for Western blotting at dilutions of 1:100-1000 .

Western blotting with SWAP70 antibodies has been instrumental in tracking the protein's expression and subcellular localization during B cell activation. For instance, Western blotting of nuclear and membrane fractions from activated B cells revealed that SWAP70 translocates from the cytoplasm to the membrane and then to the nucleus during activation . This technique has helped identify SWAP70 in membrane fractions at days 2 and 3 of B-cell activation, and in nuclear fractions at the same time points .

ELISA and Immunoassay Usage

Several SWAP70 monoclonal antibodies are suitable for ELISA (Enzyme-Linked Immunosorbent Assay) applications:

• Clone 1A12 is validated for ELISA applications .

• Clone 3H8 is suitable for indirect ELISA .

• Clone 434CT16.5.5 is recommended for ELISA at a dilution of 1:1000 .

ELISA applications allow quantitative measurement of SWAP70 protein levels in various samples, providing insights into its expression patterns under different conditions or in various cell types. The availability of alkaline phosphatase-conjugated antibodies like Clone 434CT16.5.5 provides additional flexibility for developing sensitive immunoassays without the need for secondary antibody detection systems.

Immunofluorescence and Microscopy Techniques

Immunofluorescence (IF) applications of SWAP70 monoclonal antibodies have revealed crucial insights into the protein's dynamic localization:

• Clone 1A12 is suitable for immunofluorescence applications .

Immunofluorescence studies using SWAP70 antibodies have shown that in resting B cells, SWAP70 is primarily cytoplasmic. Upon activation, it first associates with the plasma membrane, then translocates to the nucleus, and later in switched B cells, it associates again with the plasma membrane, particularly colocalizing with membrane IgG .

Advanced microscopy techniques, such as multi-color super-resolution STED microscopy, have utilized SWAP70 antibodies to demonstrate that SWAP70 overlaps with RAC1 and aligns with parallel F-actin filaments and concentric rings surrounding phagosomes . These sophisticated imaging approaches have been critical in revealing the precise spatial organization of SWAP70 in relation to the cytoskeleton during phagocytosis.

Other Research Applications

In addition to the applications mentioned above, SWAP70 monoclonal antibodies have been used in:

• Immunohistochemistry (IHC): To study SWAP70 expression patterns in various tissues and cell types .

• Immunoprecipitation (IP): To isolate SWAP70 protein complexes and identify interacting partners.

• Co-capping experiments: To demonstrate co-localization of SWAP70 with membrane IgG but not IgM in activated B cells .

These diverse applications highlight the versatility of SWAP70 monoclonal antibodies as research tools for investigating this multifunctional protein's roles in cellular processes. The availability of different clones with varying specificities allows researchers to select the most appropriate antibody for their specific experimental needs.

SWAP70 in B-Cell Activation and Development

Research utilizing SWAP70 monoclonal antibodies has provided significant insights into the role of SWAP70 in B-cell activation and development. Initially, SWAP70 was identified as a component of an enzyme complex that recombines Ig switch regions in vitro .

Studies tracking SWAP70 localization during B cell activation revealed a dynamic pattern: In resting B cells, SWAP70 is mainly cytoplasmic. Immediately after activation, SWAP70 expression increases dramatically and associates with the plasma membrane. Following membrane association, it translocates to the nucleus, and in switched cells that have lost IgM expression, it is detected almost exclusively in the nucleus .

Interestingly, at later stages of activation (day 7), SWAP70 expression in the nucleus decreases, and the protein associates again with the plasma membrane, specifically colocalizing with membrane IgG but not IgM . This suggests a specific role for SWAP70 in isotype-switched B cells. The association of SWAP70 with the BCR requires a functional pleckstrin homology domain and is controlled by the C terminus of the protein .

Role in Cytoskeletal Organization

SWAP70 monoclonal antibody research has revealed that SWAP70 is a critical regulator of the actin cytoskeleton. SWAP70 can directly bind, bundle, and stabilize actin filaments through its C-terminal actin binding domain .

Furthermore, SWAP70 regulates cellular actin dynamics and organization via activation of RHOA and RAC1 GTPases . This activation requires SWAP70's binding to phosphoinositides, highlighting the importance of its PH domain in mediating these interactions .

Multi-color super-resolution STED microscopy using SWAP70 antibodies has shown that SWAP70 overlaps with RAC1 and aligns with parallel F-actin filaments and concentric rings surrounding phagosomes . These F-actin structures were not observable upon siRNA knockdown of SWAP70, demonstrating its essential role in organizing phagosomal actin structures .

Involvement in Phagocytosis

A significant finding from SWAP70 antibody research is the protein's critical role in phagocytosis. SWAP70 was found to be transiently recruited to nascent phagosomes in human monocyte-derived dendritic cells .

This recruitment is mediated by specific binding of SWAP70's PH-domain to phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2) and to a lesser extent to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) . SWAP70 remains associated with these phagosomes shortly after phagocytic cup closure .

Knockdown of SWAP70 by small interfering RNA (siRNA) significantly decreased RAC1 activation, actin polymerization, and phagocytic capability . These findings establish SWAP70 as a scaffolding protein that promotes particle internalization by enabling the formation of the phagosomal actin cage .

Domain deletion studies revealed that not only the PH-domain but also the N-terminal region containing the EF-hand motif and the C-terminal region containing the putative Dbl-homology domain (DH) and actin-binding domain contribute to the efficient recruitment of SWAP70 to phagosomes . These results highlight the complex interplay between different SWAP70 domains in mediating its functions.

Potential Disease Associations

While the search results don't specifically address disease associations of SWAP70, the protein's roles in immune cell function, particularly in B cell activation and phagocytosis, suggest potential implications in immune-related disorders.

The specific expression of SWAP70 in mature B cells and its involvement in class switching could implicate it in B cell malignancies or autoimmune disorders characterized by dysregulated antibody production. Similarly, its critical role in organizing the actin cytoskeleton during phagocytosis suggests potential involvement in diseases characterized by defective clearance of pathogens or cellular debris.

Further research using SWAP70 monoclonal antibodies may help elucidate the protein's potential contributions to disease pathogenesis and identify novel therapeutic targets.