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

Common in vivo applications of clone 3G8 in mice include blocking human FcγRIII (CD16)-mediated antibody-dependent cellular cytotoxicity (ADCC), inhibiting neutrophil phagocytosis, and stimulating NK cell proliferation in mouse models engineered to express human CD16 or with engrafted human immune cells.

Key applications and contexts:

• Blocking ADCC: 3G8 is frequently used to inhibit CD16-mediated effector functions (such as ADCC) in humanized mouse models, facilitating studies of immune cell interactions and therapeutic antibody mechanisms.
• Modulating neutrophil and NK cell activity: 3G8 can block neutrophil phagocytosis and enhance NK cell proliferation when administered in vivo, relevant for research on inflammation, tumor immunology, and infectious disease models using human immune system mice.
• Functional assays: The antibody is also used to neutralize or modulate CD16-dependent signaling pathways, including cell depletion experiments and assessment of CD16's role in immune responses.
• Fc receptor blocking: In mouse models with human CD16-expressing immune cells, 3G8 serves as an Fc blocking reagent to reduce background binding and increase specificity in downstream immunological assays.

Important context:

• Clone 3G8 is specific for human and non-human primate CD16 and is not cross-reactive to mouse CD16; thus, its in vivo application in mice typically involves humanized (transgenic or engrafted) models.
• Some applications may be referred to as in vivo functional grade but are generally limited to models containing human cells, as wild-type mice do not possess the target epitope.
• Investigators should determine optimal dosing and application procedures based on their specific mouse strain and experimental setup; blocking efficiency and functional modulation are context-dependent.

Summary of main functions in vivo (humanized mouse models):

• Blockade of CD16 to study ADCC and immune effector pathways.
• Inhibition of phagocytosis by neutrophils.
• Stimulation/depletion or modulation of NK cell activity.
• Fc receptor blocking to enhance assay specificity in flow cytometry and related functional studies.

If clarification is needed, please specify if the focus is on wild-type, transgenic, or engrafted mouse models, as 3G8's application is restricted to contexts where human CD16 is expressed.

In the literature, several antibodies and proteins are commonly used alongside the 3G8 antibody, particularly in studies related to immune responses and cell activation. Here are a few examples:

1. Other Anti-CD16 Antibodies:

   • CB16, B73.1, and MEM-154: These antibodies are often compared with 3G8 in studies evaluating their effects on NK cells and other immune responses.

2. Anti-FcγRIIa Antibodies:

   • These are used in conjunction with 3G8 to demonstrate receptor cooperation in neutrophil activation studies.

3. Bispecific Antibodies:

   • For example, the 2B1 antibody, which combines specificity with FcγR interactions, is used to study synergistic effects on neutrophil activation.

4. FcγR-Targeting Constructs:

   • Fusion proteins like 3G8 scFv-HSA and 2.4G2 scFv-MSA are being developed to inhibit FcγR interactions without the adverse effects of traditional antibodies.

These antibodies and proteins are used in various experimental settings to explore immune mechanisms, cell signaling, and therapeutic applications.

Clone 3G8 is a widely used monoclonal antibody against CD16 that has generated substantial scientific insights across multiple functional domains. This antibody has proven instrumental in understanding NK cell biology, neutrophil function, and receptor interactions.

Functional Activities and Mechanisms

Clone 3G8 exhibits multiple functional activities that have been extensively documented. The antibody inhibits cytotoxic ability, activates cell signaling, and achieves NK cell depletion in vivo. A particularly important finding is that 3G8 blocks neutrophil phagocytosis while simultaneously stimulating NK cell proliferation. The antibody interacts with both FcγRIIa and FcγRIIIb receptors, causing neutrophil activation and aggregation. This interaction has practical implications for experimental design, as staining in whole blood may cause a reduction in the number of granulocytes or alter their scatter profile.

Epitope Recognition and Binding Characteristics

Clone 3G8 recognizes an epitope located on the putative FG loop of the membrane-proximal Ig-like domain of CD16, which represents the major binding site for IgG. Cross-blocking studies have demonstrated that 3G8 recognizes the same epitope as CB16, though 3G8 and B73.1 antibody clones bind distinct epitopes. The antibody shows sensitivity to IgG competition, with binding to CD16a on NK cells being significantly affected by the presence of human IgG. This characteristic distinguishes 3G8 from other clones and reflects its binding site overlap with the Fc region of immunoglobulins.

NK Cell Response and Expansion

Comparative studies evaluating multiple anti-CD16 antibody clones have revealed important differences in their ability to activate and expand NK cells. When measuring CD107a degranulation and IFN-γ and TNF-α cytokine production, 3G8 demonstrated intermediate effectiveness among four tested monoclonal antibodies, ranking second after CB16 but ahead of B73.1 and MEM-154. The NK cell expansion rate also differed depending on which anti-CD16 antibody was coated on microbeads, with these variations likely attributable to distinct epitope recognition patterns.

Cross-Reactivity and Applications

Clone 3G8 demonstrates remarkable cross-reactivity across multiple non-human primate species, including capuchin monkey, chimpanzee, common marmoset, cynomologous monkey, hamadyras baboon, olive baboon, pigtailed macaque, rhesus, and squirrel monkey. This broad reactivity has made it valuable for comparative immunology studies. The antibody has been successfully employed in flow cytometric analysis, functional assays, immunohistochemical staining of acetone-fixed frozen tissue sections, immunoprecipitation, and blocking of immunoglobulin binding to FcγRIII.

Affinity Profiling Applications

Recent innovations have utilized 3G8 in combination with other antibody clones for "affinity profiling" approaches. By exploiting differences in binding between 3G8 and B73.1 antibodies, researchers can distinguish NK cells based upon IgG-binding affinity to CD16a using flow cytometry bivariate plots. This method provides the ability to differentiate cell populations with varying affinities for immunoglobulin binding.

Historical Significance

Clone 3G8 played a pivotal role in understanding NK cell receptor interactions, revealing how low-affinity Fc receptors mediate critical immune responses. Its development contributed foundational knowledge to the field of natural killer cell biology and antibody-dependent cellular cytotoxicity mechanisms.

The dosing regimens of clone 3G8, an anti-human CD16 monoclonal antibody, in mouse models can vary significantly based on the specific application and target cell population being studied. However, detailed information on how these regimens differ across various mouse models is not explicitly provided in the available search results.

Here are some general considerations for using clone 3G8 in mouse models:

1. Application Dependency: The dosing regimen can depend on whether the antibody is used in flow cytometry, immunohistochemistry, or in vivo studies. For example, in flow cytometry, specific dilutions are recommended to optimize staining performance.

2. Target Cell Population: The dose may also be influenced by the specific cells being targeted, such as NK cells or granulocytes, as the antibody's effects can vary between these populations.

3. Experimental Design: Factors like the duration of the study, the route of administration, and the overall experimental design can also impact dosing decisions.

For precise dosing regimens in specific mouse models, it is essential to consult detailed experimental protocols or publications related to those models.

If you're planning experiments, it's advisable to titrate the antibody to find the optimal dose for your particular study conditions. This approach ensures that you achieve the desired effects while minimizing potential side effects or off-target results.