Anti-mouse/rat MHC Class II (I-Ek/RT1-D) (Clone 14-4-4S (HB-32)) – Purified in vivo PLATINUM™ Functional Grade

Clone 14-4-4S is a well-characterized monoclonal antibody widely used in mice to target the I-Ek epitope of MHC Class II molecules, with several established in vivo applications. The main uses in live animal studies are:

• Blockade of Antigen Presentation: 14-4-4S can be administered in vivo to block MHC class II-mediated antigen presentation, thereby inhibiting certain T cell responses. This approach is frequently used to study the role of class II MHC in immune recognition and activation, as well as in models of autoimmune disease or transplantation.
• Depletion or Modulation of Antigen-Presenting Cells: Through complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, 14-4-4S can be used to partially deplete I-E-expressing antigen-presenting cells (APCs), such as specific B cell populations, in vivo. This enables assessment of different APC contributions in immune processes.
• In Vivo Immunophenotyping and Cell Tracking: While more commonly performed ex vivo, in some studies fluorescently labeled 14-4-4S variants have been administered to live animals to label MHC-II-expressing cells, which are then tracked or isolated from tissues for further analysis.
• Functional Blocking in Disease Models: By interfering with antigen presentation, 14-4-4S is used to dissect immune mechanisms in various models—autoimmunity, transplantation tolerance/rejection, and infectious disease—by transiently disabling a major APC pathway.

Supporting details and considerations:

• The antibody is effective only in mouse strains expressing the I-Ek MHC molecule (notably H-2d, H-2p, H-2r haplotypes).
• Whole antibody or F(ab')₂ fragments can be used, with the latter reducing Fc-dependent effects.
• The antibody is sometimes used at high doses to achieve sustained blocking or depletion, often necessitating repeated injections for lasting effects.
• Safety and strain compatibility must be considered, since not all mouse strains express the I-E molecule.

Summary Table: Main In Vivo Applications of Clone 14-4-4S in Mice

References for these uses include vendor and technical product descriptions as well as reviews of common functional assays using 14-4-4S.

When using 14-4-4S, which is a monoclonal antibody specific for the mouse (and rat) MHC class II I-Ek subregion glycoprotein, researchers commonly use it in combination with antibodies to characterize or distinguish among immune cell populations, especially in flow cytometry, immunoprecipitation, or immunohistochemistry experiments. The most frequently co-used antibodies and proteins are:

• Anti-MHC Class II (I-Ak, I-Ad, or other I-E variants): To define or distinguish different mouse MHC class II haplotypes, especially to separate I-E-positive from I-A-positive subtypes or from negative controls.
• Anti-CD3, anti-CD4, anti-CD8: These T cell markers are often used in conjunction with 14-4-4S to phenotype T cell subsets or to gate T cell populations during flow cytometry.
• Anti-CD19 or anti-B220: These B cell markers identify B cells, which also express MHC class II, typically co-analyzed in immunophenotyping.
• Macrophage and dendritic cell markers (e.g., anti-CD11b, anti-F4/80, anti-CD11c): Used to identify professional antigen-presenting cells that express MHC class II.
• Viability dyes or lineage cocktail antibodies: Used to exclude dead cells or non-relevant cell types as part of multiparametric analysis.

Thus, any comprehensive immunophenotyping or antigen presentation study will typically pair 14-4-4S with other MHC-class II antibodies, T and B cell lineage markers, and myeloid cell-specific antibodies for accurate characterization of immune subsets involved in antigen presentation and immune regulation.

If you are interested in 14-3-3 proteins (a ubiquitous family involved in phosphorylation-dependent signaling and not related to MHC), these are studied alongside a very different set of partners, typically:

• Kinases (e.g., Raf, BAD, Cdc25)
• Phosphatases
• Synaptopodin, as an actin-binding protein, has been highlighted as a specific partner in the context of 14-3-3 interactions.

Summary Table: Commonly used antibodies/proteins with 14-4-4S (MHC II I-Ek)

If you meant 14-3-3 proteins instead of 14-4-4S, please clarify for a detailed list of their protein partners and frequently used detection antibodies.

Key Scientific Findings on Clone 14-4-4S

Molecular Specificity and Binding

• Clone 14-4-4S is a monoclonal antibody that specifically recognizes the MHC class II molecule encoded by the I-E subregion in mice, particularly the Ia.7 determinant.
• It binds to mature I-E heterodimers on the cell surface of B cells, macrophages, and dendritic cells, but does not recognize unassembled or invariant chain-associated forms of I-E.
• The antibody cross-reacts with rat I-E molecules and is widely used as a reagent to detect and quantify MHC class II (I-E) expression in research.

Idiotype and Shared Recognition

• 14-4-4S expresses a shared idiotype—an antigenic determinant unique to the variable region of the antibody—that is also found in certain allogeneic antisera, but not in unrelated immunoglobulins. This was the first demonstration of a shared idiotype in an anti-MHC antibody system.
• The presence of this shared idiotype suggests that some structural features of the antibody’s variable region are conserved across immune responses, analogous to shared idiotypes seen in responses to haptens and other proteins.

Functional Assays and Applications

• 14-4-4S is a useful tool for flow cytometry, immunoprecipitation, cytotoxicity assays, and immunohistochemistry for MHC class II–expressing cells in mice (and rats).
• It has been reported to block antigen presentation in vitro, indicating its ability to interfere with the interaction between MHC class II and T cell receptors.
• Quantitative assays using radiolabeled 14-4-4S have shown that activated B cells express high levels of mature I-E molecules on their surface (approximately 1.5–2.0 × 10⁵ molecules per cell).
• The antibody's binding is specific to the I-E subregion and does not react with haplotypes that lack I-E expression (e.g., H-2ᵇ, H-2ˢ, H-2ᶠ, H-2ᵍ).

Research Applications and Limitations

• 14-4-4S is widely used to study MHC class II antigen presentation and has been instrumental in demonstrating that only mature, surface-expressed MHC class II molecules are efficiently recognized by T cells.
• The antibody does not bind to invariant chain-associated or intracellular I-E molecules, making it a specific probe for functional, mature MHC class II complexes.
• Titer and purity: The antibody has a high cytotoxic titer (1:128 on C3H spleen cells) and can be purified to high purity, as evidenced by a single band in immunoelectrophoresis.

Summary Table: Key Properties of Clone 14-4-4S

Conclusion

Clone 14-4-4S is a foundational reagent in murine immunology, specifically detecting mature MHC class II I-E molecules and helping to elucidate the biology of antigen presentation. Its unique idiotypic properties and high specificity have made it indispensable for research on immune recognition, MHC class II trafficking, and the functional consequences of MHC class II blockade.

Dosing regimens of clone 14-4-4S, an anti-mouse MHC class II (I-Ek) monoclonal antibody, vary depending on experimental purpose and mouse model MHC haplotype rather than explicit, universally established in vivo dosing schedules. The majority of published protocols and vendor data emphasize in vitro applications, with limited standardized information on in vivo dosing regimens.

Key context for 14-4-4S dosing:

• Most common applications are flow cytometry, immunohistochemistry, immunoprecipitation, antigen presentation blockade, and complement-mediated cell depletion.
• Mouse strain specificity: Only strains with H-2^k, H-2^d, H-2^p, or H-2^r haplotypes express the relevant I-Ek antigen and are therefore appropriate for in vivo 14-4-4S studies.

Reported and recommended doses:

• In vitro (e.g., flow cytometry): ≤0.5 μg/test (with ~10^6 cells), often sufficient for cell marker identification or antigen presentation inhibition.
• In vivo dosing: Reliable, explicit peer-reviewed dosing regimens for clone 14-4-4S are lacking.
  • Some sources state use "has been reported" for complement-dependent cytotoxicity and cell depletion in mice, but do not specify dose or schedule.
  • For antibodies with similar Fc and functional properties, typical in vivo regimens are 100–250 μg per mouse via intraperitoneal injection, every 3–4 days, but these are general guidelines drawn from checkpoint inhibitors or depletion antibodies, not 14-4-4S specifically.

Experimental considerations that affect dosing:

• Purpose: Depletion, functional blockade, and cell labeling may require different amounts.
• Strain and tissue target expression: Only target-positive strains (see above) will respond.
• Mouse size, age, and immunocompetence: Standardization per body weight (e.g., μg/g) may be used in some protocols.
• Route of administration: Typically intraperitoneal or intravenous for systemic effects in mice.

Summary Table: 14-4-4S Dosing by Setting

Conclusion:
There is no universally standardized in vivo dosing regimen for clone 14-4-4S across mouse models; protocols are typically extrapolated from similar antibody formats and applications. For in vivo use, consult peer-reviewed reports with the same application, consider starting with general depletion doses for murine IgG2a antibodies (100–250 μg per injection every 3–4 days intraperitoneally), and carefully match mouse strain haplotype. Always optimize dose and frequency for the specific model, application, and experimental endpoint, confirming efficacy and specificity in preliminary experiments.