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

The clone 3G8 is a monoclonal antibody widely used to bind human CD16 (Fc?RIII) and is commonly utilized in in vitro studies with human cells. In the context of in vivo mouse studies, 3G8 is primarily used under the following conditions and with significant considerations:

• Species specificity: 3G8 binds specifically to human and non-human primate CD16, but does not cross-react with murine (mouse) CD16. Therefore, typical use of this clone in standard mice is not productive unless the mice express human CD16 through genetic modification.

• Humanized mice: For in vivo mouse studies, 3G8 is used in humanized mice models—mice engineered to express human CD16 on their immune cells. In these models, 3G8 can:

  • Block human CD16 function: It is used to block binding of IgG to CD16, thereby suppressing Fc-mediated activation such as antibody-dependent cellular cytotoxicity (ADCC) in human immune cells reconstituted in these mice.
  • Assess immune cell function: It enables functional studies on human NK cell activity, phagocytosis, and granulocyte function within the mouse, since it has been shown to both block phagocytosis and stimulate NK cell proliferation.

• In vivo grade antibody: When used for in vivo administration, 3G8 is formulated to be endotoxin low and free of stabilizers and preservatives that could cause toxicity in mice. This is critical for preclinical safety and immunology studies to avoid confounding effects.

• Limitations:

  • 3G8 will not recognize or block mouse CD16, and thus has no effect in non-humanized mice.
  • Use in humanized mice may provoke anti-human antibody responses, so careful monitoring and optimized mouse strains are often recommended to reduce immune rejection or rapid antibody clearance.

In summary, in vivo use of 3G8 in mouse studies is restricted to humanized mouse models expressing human CD16, where it serves as a powerful tool for blocking, functional, and mechanistic studies of human Fc receptor biology. It is not effective in wild-type mice due to strict species specificity.

The correct storage temperature for sterile packaged clone 3G8 (anti-human CD16 monoclonal antibody) is 2°C to 8°C. The antibody should be stored undiluted at this refrigerated temperature range and protected from prolonged exposure to light.

Important Storage Guidelines

Temperature Requirements: Clone 3G8 must be kept refrigerated between 2°C and 8°C and should not be frozen. This temperature range maintains the antibody's stability and binding capacity while preventing degradation.

Light Protection: The antibody solution should be protected from prolonged exposure to light during storage. This helps preserve the integrity of any fluorescent conjugates and prevents photodegradation.

Freezer Considerations: It's specifically recommended to use a manual defrost freezer and avoid repeated freeze-thaw cycles if any freezing is necessary. However, the primary storage instruction is to avoid freezing altogether.

Stability Period: When stored properly at 2°C to 8°C, clone 3G8 maintains its stability for 12 months from the date of receipt. The antibody should be stored undiluted in its original formulation to maintain optimal performance for flow cytometry applications.

This storage protocol ensures maximum antibody performance and longevity for flow cytometry applications while preventing common storage-related issues that could compromise experimental results.

Commonly, 3G8 (an anti-CD16 monoclonal antibody) is used with other antibodies or proteins targeting CD3, HER2, Fc?RIIa, and Fc?RIIIb in immunological studies and therapeutic antibody engineering.

Key co-used antibodies/proteins:

• OKT3 (anti-CD3): Frequently paired with 3G8 in bispecific and trispecific antibody constructs to engage both NK cells (via CD16) and T cells (via CD3), enhancing cytotoxic responses.
• Trastuzumab (anti-HER2): Used in bispecific constructs with 3G8 to simultaneously target tumor antigens (HER2) and recruit NK cell effector functions (via CD16).
• 2B1 bispecific antibody: Contains a single 3G8 Fab and is used to dissect cooperative activation of neutrophil Fc receptors; particularly in combination studies analyzing Fc?RIIa and Fc?RIIIb interactions.
• Affimers against Fc?RIIIa: Engineered non-antibody proteins (e.g., AfF4) are tested alongside 3G8 to distinguish effects specifically mediated by Fc?RIIIa versus Fc?RIIIb, since 3G8 binds both.
• Other Fc?R-targeting antibodies: Investigations often combine 3G8 with antibodies or proteins specific for Fc?RIIa or Fc?RIIIb to elucidate synergistic activation pathways in neutrophils.

Study designs and rationale:

• Multispecific antibody formats exploit 3G8’s specificity for CD16 to redirect effector cell activity, while antibodies like OKT3 (CD3) or trastuzumab (HER2) define target cell populations.
• Functional immune cell assays often require 3G8 in combination with markers for other Fc? receptors (such as Fc?RIIa) to monitor receptor cross-talk and cell activation.
• Affimer proteins and other molecular tools are deployed with 3G8 to improve selectivity or dissect receptor-specific functions in the immune response.

3G8 thus appears in literature primarily in cocktails or constructs with anti-CD3, anti-HER2, and other Fc?R-targeting agents, especially in studies involving NK cell or neutrophil activation, bispecific antibodies, and mechanistic immunology.

Clone 3G8 is a monoclonal antibody widely cited in scientific literature as a tool to identify, enumerate, and characterize CD16 (Fc?RIII)–expressing cells, notably natural killer (NK) cells and some myeloid cells. Key findings from scientific citations involving 3G8 include:

• Functional Modulation of Immune Cells: Clone 3G8 can block neutrophil phagocytosis, stimulate NK cell proliferation, and interact with Fc?RIIa and Fc?RIIIb, leading to neutrophil activation and aggregation.
• Epitope and Functional Specificity:
  • The 3G8 epitope is located on the FG loop of the membrane-proximal Ig-like domain of CD16a, a region essential for IgG binding. This localization underlies its use in studies evaluating receptor-ligand interactions, antibody-dependent cellular cytotoxicity (ADCC), and immune effector functions.
  • 3G8, compared with other anti-CD16 clones (e.g., B73.1, MEM-154), often produces distinct functional outcomes, particularly in NK cell stimulation and cytokine production; CB16 is generally more effective, but 3G8 is a standard for robust NK cell activation.
• Methodological Applications:
  • Flow cytometry: 3G8 is commonly employed for the identification and enumeration of CD16+ cell populations in blood and tissues.
  • Immunohistochemistry, immunoprecipitation, and blocking studies: 3G8 is used to block phagocytosis, study receptor-ligand binding, and in functional assays probing Fc receptor activity.
  • Affinity profiling: When paired with other anti-CD16 mAbs like B73.1, 3G8 enables discrimination of different IgG-binding affinities on NK cells, aiding in detailed immune cell subset analysis.
• Impact on Experimental Readouts: Staining with 3G8 in whole blood can activate and aggregate granulocytes, sometimes altering cell frequencies or scatter profiles—an important consideration for assay design.
• Pivotal Role in Immunology: The development of 3G8 has been instrumental in elucidating how low-affinity Fc receptors mediate immune responses and has seeded the foundation for key discoveries in NK cell biology and therapeutic antibody mechanisms.

In summary, 3G8 is a foundational reagent for immunological research, enabling the detailed study of Fc?RIII functions, immune cell phenotyping, and the mechanisms underlying antibody-dependent immune responses.