Anti-Human CD16 [3G8] – Purified in vivo PLATINUM™ Functional Grade

The clone 3G8, a monoclonal antibody specific for human CD16, is used in various applications, but its direct use in in vivo mouse studies is not extensively detailed in the search results. However, here are some insights into how such antibodies could be utilized in mouse models:

1. Specificity and Cross-Reactivity: The 3G8 clone is known to specifically bind to human and non-human primate CD16, which includes Fc?RIII receptors found on natural killer cells, neutrophils, monocytes, macrophages, and certain T cells. In mouse models, if the focus is on studying human CD16 or its functions, the antibody might be used in xenograft models or humanized mice that express human CD16.

2. Functional Assays: In vivo applications could involve blocking or modulating antibody-dependent cellular cytotoxicity (ADCC), which is mediated by CD16. The 3G8 antibody can block neutrophil phagocytosis and stimulate NK cell proliferation, which might be relevant in some mouse models of disease.

3. Challenges and Considerations: When using human-specific antibodies like 3G8 in mouse studies, researchers must consider the potential for an immune response against the human antibody. This issue can be mitigated by using mouse models that are tolerant to human antibodies, such as those described in studies involving human IgG antibodies in knock-in mice.

Overall, while the 3G8 clone is not explicitly mentioned for in vivo mouse studies in the search results, it could be used in research contexts where human CD16 function or ADCC needs to be studied or manipulated in a mouse model, particularly if the model is designed to accommodate human proteins.

For the clone 3G8 monoclonal antibody (anti-human CD16), the correct storage temperature is consistently specified as 2–8°C.

Key storage instructions:

• Store undiluted at 2–8°C.
• Do not freeze the antibody.
• Protect from prolonged exposure to light.
• Avoid repeated freeze-thaw cycles, so aliquot if possible for repeated use.
• Once diluted, use immediately; do not store diluted reagent for later use.

These directions apply whether the antibody is labeled with FITC, PE, APC, biotin, or other common fluorophores, and are consistent across multiple suppliers and product formats. Room temperature storage or freezing is not recommended for clone 3G8, as it may compromise the stability and performance of the antibody.

Commonly Used Antibodies and Proteins with 3G8 in the Literature

3G8 is a well-known monoclonal antibody (mAb) directed against the human Fc?RIII (CD16) receptor and is widely used in immunological research. Below are some antibodies and proteins commonly cited in conjunction with 3G8, along with their primary contexts of use.

Comparative Anti-CD16 Antibodies

CB16, B73.1, MEM-154: These are additional anti-CD16 (Fc?RIII) monoclonal antibody clones often used alongside 3G8 in studies comparing NK cell responses and expansion. While 3G8 is used due to its binding to both Fc?RIIIa (CD16a) and Fc?RIIIb (CD16b), these clones may differ in their binding profiles, cell type specificity, and functional effects on NK cells.

Antibodies Targeting Related Fc? Receptors

Anti-Fc?RIIa (CD32a) mAbs: Studies investigating the synergistic activation of neutrophils by 3G8 and Fc?RIIa use antibodies specific to Fc?RIIa. The 3G8 mAb induces neutrophil activation only when its Fc part interacts with Fc?RIIa, highlighting the cooperative role of Fc?RIIa in mediating cellular responses in the presence of 3G8. While specific other clones are not named in the available literature, the context implies the use of anti-Fc?RIIa antibodies in such experiments.

Non-Antibody Inhibitors and Reference Proteins

Affimer Proteins (e.g., AfF4): These are engineered, non-antibody binding proteins used as specific inhibitors of Fc?RIIIa (CD16a). In contrast to 3G8, which cross-reacts with both Fc?RIIIa and Fc?RIIIb, Affimer proteins like AfF4 can selectively block IgG binding to Fc?RIIIa without affecting Fc?RIIIb or Fc?RIIa, providing a tool for more specific blockade in functional studies.

Bispecific Antibodies (e.g., 2B1): The bispecific antibody 2B1, which contains a single 3G8 Fab arm, has been used to dissect the mechanism of neutrophil activation. Studies show that 2B1 does not induce activation, indicating that bivalent engagement of Fc?RIIIb by 3G8 and simultaneous Fc engagement of Fc?RIIa are required for neutrophil triggering.

Summary Table

Key Points

• 3G8 is frequently used in comparative studies with other anti-CD16 antibodies such as CB16, B73.1, and MEM-154 to evaluate differential effects on NK cells and other immune responses.
• In neutrophil activation studies, 3G8 is used alongside anti-Fc?RIIa antibodies to demonstrate receptor cooperation.
• Non-antibody tools like Affimer proteins (e.g., AfF4) and bispecific antibodies (e.g., 2B1) are used to dissect receptor specificity and activation mechanisms, often in contrast to 3G8’s broader reactivity.

These combinations are employed to explore the functional differences, receptor specificity, and downstream signaling of Fc?RIII and related receptors in immunology research.

Key Findings from Clone 3G8 (Anti-CD16) Citations

Clone 3G8 is a well-characterized monoclonal antibody targeting human CD16, specifically the Fc?RIII (Fc fragment of IgG receptor III) isoforms predominantly expressed on natural killer (NK) cells, neutrophils, and macrophages. The scientific literature highlights several important functional and practical findings regarding 3G8:

Functional Effects

• Neutrophil Activation and Aggregation: Clone 3G8 interacts with Fc?RIIa and Fc?RIIIb, leading to neutrophil activation and aggregation. This interaction can cause a reduction in the number of granulocytes or alter their scatter profile during whole blood staining, which is important to consider for flow cytometry analyses.
• Blockade of Neutrophil Phagocytosis: 3G8 blocks phagocytosis mediated by neutrophils, which has implications for understanding Fc receptor-mediated immune cell functions.
• NK Cell Stimulation: 3G8, along with other anti-CD16 clones, is commonly used to stimulate NK cell proliferation and activation in vitro. However, systematic comparisons reveal that while 3G8 is effective, other clones such as CB16 can induce a stronger NK cell response in terms of degranulation (CD107a) and cytokine (IFN-?, TNF-?) production.
• Epitope Recognition: The epitope for 3G8 (and CB16) is located on the FG loop of the membrane-proximal Ig-like domain of CD16, the major binding site for IgG. This differs from other clones like B73.1 and MEM-154, which target regions closer to the IgG binding site and the distal Ig domain.

Technical and Diagnostic Applications

• Flow Cytometry and Immunophenotyping: 3G8 is widely used for the identification and enumeration of CD16-positive cells (e.g., NK cells, neutrophils) in clinical and research settings. It is quality-tested for immunofluorescent staining and flow cytometry, with specific recommendations for optimal use.
• Immunohistochemistry and Immunoprecipitation: Beyond flow cytometry, 3G8 has applications in immunohistochemical staining of acetone-fixed frozen tissue sections and immunoprecipitation.
• Functional Assays: The Ultra-LEAF™ purified form of 3G8 is recommended for functional assays due to its low endotoxin and azide-free formulation.

Comparative Studies

• Clone-Specific Differences: Not all anti-CD16 clones behave identically. In head-to-head comparisons, CB16 was the most effective at inducing NK cell responses, followed by 3G8, then B73.1 and MEM-154. These differences are attributed to distinct epitope recognition patterns and potentially differential signaling outcomes.
• Affinity Profiling: Flow cytometry using 3G8 and B73.1 can differentiate NK cell populations based on their CD16a affinity for IgG, providing a tool for "affinity profiling" of NK cell subsets.

Summary Table: Functional and Technical Properties of Clone 3G8

Conclusion

Clone 3G8 is a foundational tool in immunology for studying CD16 (Fc?RIII), with well-documented effects on neutrophil and NK cell function. Its use in diagnostics and research is broad, but recent comparative studies highlight that not all anti-CD16 clones are equivalent—specific functional outcomes depend on the clone’s epitope and signaling properties. Careful consideration of clone choice is necessary, especially in functional assays and when interpreting flow cytometry data in whole blood.