Anti-Mouse CD32/CD16 [Clone 2.4G2] — Purified in vivo GOLD™ Functional Grade

Clone 2.4G2 is utilized in in vivo mouse studies primarily as an Fc receptor blocking antibody to prevent non-specific binding and interference from endogenous antibodies during experimental procedures.

Primary Mechanism and Purpose

The 2.4G2 antibody works by blocking the interaction between IgG antibodies and CD16 (Fc?RIII) or CD32 (Fc?RII) receptors on immune cells. These receptors are expressed on various immune cell types including B cells, monocytes, macrophages, NK cells, neutrophils, granulocytes, mast cells, and dendritic cells. By occupying these Fc receptor binding sites, the 2.4G2 antibody prevents non-specific binding of experimental antibodies that could interfere with study results.

Specialized In Vivo Formulations

For in vivo applications, specialized formulations of the 2.4G2 antibody have been developed with specific characteristics that make them suitable for use in living mice:

Low Endotoxin Content: The in vivo grade formulations contain endotoxin levels of ?0.01 EU/?g or <1 EU per 1 mg, as determined by the Limulus amebocyte lysate (LAL) test. This low endotoxin level is crucial to prevent inflammatory responses that could confound experimental results.

Azide-Free Formulation: Unlike some laboratory antibodies that contain sodium azide as a preservative, the in vivo grade 2.4G2 is formulated without azide, making it safe for injection into living animals.

Modified Variants for In Vivo Use

Several engineered versions of the 2.4G2 antibody have been developed specifically for enhanced in vivo performance:

LALA-PG Mutation: Some versions contain LALA-PG mutations in the Fc fragment, which render the antibody unable to bind to endogenous Fc? receptors. This modification prevents the blocking antibody itself from causing unwanted immune activation while still allowing it to block the target receptors.

Species Switching: Chimeric versions have been created where the variable domains remain identical to the original 2.4G2, but the constant regions are switched from rat IgG2b to mouse IgG2a. This modification can reduce immunogenicity in mouse models.

Applications in Mouse Studies

In vivo applications of 2.4G2 include blocking Fc receptors during studies investigating antibody-mediated immune responses, preventing interference in flow cytometry analysis of tissues harvested from treated mice, and neutralizing endogenous Fc receptor activity in functional assays studying biological pathways affected by CD16 and CD32 proteins. The antibody is also used in bioanalytical pharmacokinetic (PK) and anti-drug antibody (ADA) assays in mouse models.

The careful formulation and engineering of these in vivo grade 2.4G2 antibodies ensures they can be safely administered to mice while effectively blocking Fc receptors without causing adverse reactions or confounding experimental results.

Commonly Used Antibodies and Proteins with 2.4G2 in the Literature

The 2.4G2 monoclonal antibody is best known for its role in blocking mouse CD16 and CD32 (Fc?RIII and Fc?RII), preventing non-specific binding of the Fc portion of immunoglobulins during immunoassays such as flow cytometry and immunofluorescence. However, when used in research, 2.4G2 is not typically the primary immune marker; rather, it is employed as a blocking reagent to improve the specificity of other antibodies targeting cell surface markers. Below are the main categories of antibodies and proteins commonly used in concert with 2.4G2 in the literature.

Common Antibody Partners in Multiparameter Analysis

In mouse immunology and cell phenotyping studies, 2.4G2 is used alongside a variety of fluorescently labeled antibodies that target specific leukocyte subsets or activation markers. Examples from the literature include:

These antibodies are routinely used in panels for comprehensive immune cell profiling, where 2.4G2 is added as a blocking reagent to minimize non-specific Fc receptor-mediated staining before applying these primary antibodies.

Blocking with 2.4G2 in Functional Assays

• Primary Antibodies: Following Fc receptor blockade with 2.4G2, primary antibodies against specific cell surface markers (e.g., CD4, CD8, B220, CD11b, Gr1, NK1.1) are used to identify and sort cell populations.
• Secondary Antibodies: If a secondary antibody step is used (e.g., for indirect staining), it must not be anti-rat IgG2b, as 2.4G2 is a rat antibody and would otherwise be detected by such a secondary antibody.
• Functional Assays: In addition to flow cytometry, 2.4G2 is used in neutralization, pharmacokinetic (PK), and anti-drug antibody (ADA) assays to block Fc-mediated interactions that could confound results.

Other Proteins and Controls

• Isotype Controls: Mouse IgG2a (the isotype of 2.4G2) or irrelevant isotype-matched antibodies are often used as negative controls to confirm specificity in staining experiments.
• Recombinant Fc Proteins: Sometimes, purified Fc fragments or recombinant Fc proteins are used in parallel with 2.4G2 to confirm that blocking is effective and to study Fc receptor biology.
• Complement and Immune Complexes: In functional studies of phagocytosis or ADCC, 2.4G2 may be used to block Fc receptors before adding complement or immune complexes to assess receptor-specific contributions.

Summary Table of Common Antibodies Used with 2.4G2

Conclusion

In the literature, 2.4G2 is most frequently used as a blocking reagent in combination with a wide array of cell surface marker antibodies (e.g., B220, CD3, CD19, CD11b, Gr1, NK1.1) for multiparameter flow cytometry and immunofluorescence in mouse samples. Appropriate isotype controls and careful selection of secondary antibodies (avoiding anti-rat IgG2b) are also standard practice. These combinations enable precise identification and analysis of immune cell subsets by minimizing non-specific staining through Fc receptor blockade.

Clone 2.4G2 is a monoclonal antibody widely used in immunology research, especially in mouse models, and is most frequently cited for its ability to block or detect Fc gamma receptor II (CD32, Fc?RII) and Fc gamma receptor III (CD16, Fc?RIII). Key findings and scientific uses from the literature include:

• Widely Used as an Fc Blocker: The primary use of clone 2.4G2 is to block Fc?RII and Fc?RIII on mouse immune cells during experiments (such as flow cytometry and immunofluorescence) to prevent non-specific binding of antibodies via their Fc region. This increases the specificity of staining for target antigens.

• Specificity and Additional Binding: While 2.4G2 is specific for CD16 (Fc?RIII) and CD32 (Fc?RII), it has also been reported to exhibit non-specific binding to Fc?RI (CD64) via its own Fc region when the antibody is already bound to Fc?RII or Fc?RIII, making careful interpretation necessary in certain analyses.

• Blocking in Quantitative and Functional Analyses: Studies frequently employ 2.4G2 for blocking during flow cytometry, especially when quantifying or characterizing Fc? receptor expression or to avoid artifacts caused by Fc-mediated binding. The antibody is also used to block Fc receptors in vivo in animal models.

• Cell Types Affected: CD16 and CD32 are expressed on a variety of mouse immune cells, including B cells, NK cells, macrophages, granulocytes, mast cells, and dendritic cells.

• Use in Functional Studies: In addition to flow cytometry, clone 2.4G2 has been used in research to study functions regulated by Fc?RII and Fc?RIII, such as phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC), as well as in analyzing Natural Killer (NK) cell function.

• Critical Considerations:

  • Use in Immunoassays: When blocking with 2.4G2, care must be taken in subsequent detection steps, specifically avoiding secondary reagents that are anti-rat IgG2b, which may cross-react.
  • Limitations: 2.4G2 does not block all Fc gamma receptors; for example, Fc?RIV is not effectively blocked, so additional reagents (like clone 9E9) might be needed in experiments studying multiple Fc receptor types.

• Benchmark in Fc Receptor Research: The 2.4G2 clone has been cited in hundreds of publications and is considered a standard tool for Fc receptor research in immunology.

In summary, the main scientific contribution of clone 2.4G2 is its reliable and efficient blocking of Fc?RII and Fc?RIII, which underpins much of mouse immunology research by allowing for more specific analysis of immune cell markers and functions.

Dosing regimens of clone 2.4G2 (an anti-mouse Fc?RII/RIII monoclonal antibody) vary primarily by the experimental design and mouse model, with common dosages reported as a single 500?µg intraperitoneal (i.p.) injection per mouse for acute receptor blockade in immunological studies.

In published research, C57BL/6 mice received 500?µg anti-Fc?RII/RIII (clone 2.4G2, Bio X Cell) via i.p. injection 24 hours prior to antigen exposure. This regimen is designed to block Fc?RIIB (inhibitory receptor) and Fc?RIII (activating receptor) on immune cells, thus modulating responses such as antigen uptake, dendritic cell activation, and other processes sensitive to Fc?R engagement.

Regimen variables across studies and models:

• Dosage: 500?µg per mouse is the predominant dose; some protocols may titrate dose based on experimental needs, mouse strain, or desired blockade duration.
• Timing: Typically administered 24 hours before experimental challenge or antigen application, allowing sufficient receptor binding and functional inhibition.
• Mouse strain/model: While dosing tends to remain consistent across immunological mouse models (e.g., C57BL/6, BALB/c), adjustments may be necessary based on body weight, immunological background, or disease context.

Other considerations:

• Clone 2.4G2 binds both Fc?RIIB and Fc?RIII in mice. The biological specificity and impact of 2.4G2 may vary depending on the expression of these receptors in different strains or tissue locations.
• Investigators sometimes include isotype control antibodies (e.g., rat IgG2b) for specificity control in experiments.

Published protocols and vendor recommendations generally support the use of 500?µg i.p. injection for efficient blockade in most commonly used mouse models. However, dose and schedule may be adapted for chronic studies, repeated dosing, or in models with unique immune features. Direct strain-by-strain or disease model variation in published dosing is minimal, but careful titration is recommended in protocols with altered immune receptor expression or function.

Summary Table: Clone 2.4G2 dosing in mouse models

If your application involves different dosing intervals, repeated injections, alternate routes or strains, refer to primary literature or manufacturer guidelines for model-specific optimization. In all cases, dosing may be modified in accordance with receptor density, body weight, and immunological context to ensure effective Fc?R blockade.