Anti-Mouse MHC Class I (H-2K^b) (Clone AF6-88.5) – Purified in vivo GOLD™ Functional Grade

The AF6-88.5 clone is a mouse monoclonal antibody that specifically recognizes the H-2K^b MHC class I alloantigen on mouse cells, and it is widely used in in vivo mouse studies for several immunological applications.

Key uses in in vivo mouse studies

• Depletion or masking of MHC Class I molecules, typically to block antigen presentation to CD8^+ T cells and study the role of MHC-I in immune responses.
• Functional assays and neutralization, such as investigating how interference with MHC-I affects immune pathways, rejection, or tolerance in transplantation and tumor immunity models.
• Characterization of cell populations by flow cytometry, especially to confirm MHC haplotype (H-2K^b) expression on mouse cells, often in strains like C57BL/6.
• Bioanalytical assays, including pharmacokinetics (PK) and anti-drug antibody (ADA) assessments in the context of MHC-I involvement.

Usage specifics

• The antibody is formulated for in vivo use with low endotoxin levels and is often supplied in bulk amounts for experimental dosing.
• AF6-88.5 is highly specific for the H-2K^b alloantigen, which is ubiquitous on all nucleated cells in H-2K^b haplotype mice, but does not cross-react with other MHC class I haplotypes.
• The recommended applications and optimal dilutions may vary depending on the specific use case and experimental protocol, with flow cytometry and direct functional in vivo administration being common modalities.

In summary:
AF6-88.5 is primarily used to block, deplete, label, or functionally interrogate MHC Class I H-2K^b molecules in C57BL/6 and related mouse strains in both in vivo and ex vivo settings.

Commonly used antibodies or proteins in combination with AF6-88.5 (anti-mouse MHC class I H-2K^b) in the literature include those targeting major cell surface markers and other MHC molecules, especially in immunological assays and mouse model studies.

Key antibodies/proteins frequently used alongside AF6-88.5

• CD4: Used to identify helper T cells in flow cytometry and immunohistochemistry.
• CD8: Used to identify cytotoxic T cells, which interact directly with MHC class I molecules such as H-2K^b.
• CD3/TCR: AF6-88.5 is often paired with antibodies against CD3 or the T-cell receptor to study antigen presentation and T cell activation, as H-2K^b presents antigens to CD3/TCR-expressing cells.
• Other MHC Class I antibodies: Such as anti-H-2D^b and anti-H-2L^d, which help distinguish between MHC class I haplotypes in mouse models.
• β2-microglobulin (β2M): This protein non-covalently associates with the MHC class I heavy chain and is essential for its stability; antibodies against β2M are sometimes used to study MHC class I structure and function.
• Markers for cell subsets: Antibodies targeting B220 (B cells), CD45 (leukocytes), CD11b (monocytes/macrophages), and NK1.1 (NK cells) are frequently used in multi-color panels to characterize immune cell populations in combination with MHC-I detection.

Typical applications

These combinations are common in:

• Flow cytometry
• Immunofluorescence
• Immunohistochemistry

where multiparametric analysis of immune cell populations and their activation status is required.

For optimal experimental design, researchers also use:

• Various fluorochrome conjugates (e.g., FITC, PE, APC) of these antibodies to facilitate multiplexing.
• Reagents detecting cell activation/proliferation markers (e.g., Ki-67, CD69) or apoptotic markers (Annexin V), depending on study focus.

In summary: Antibodies against CD8, CD4, other MHC-I proteins (H-2D^b, H-2L^d), CD3/TCR, and lineage markers are most commonly used alongside AF6-88.5 in immunological studies and mouse model research.

Clone AF6-88.5 is a well-characterized monoclonal antibody that specifically recognizes the mouse MHC class I H-2K^b alloantigen and is widely used in immunology research for detecting and analyzing H-2K^b expression. Key findings from scientific literature and product citations include:

• Epitope Specificity: AF6-88.5 binds to a unique epitope on H-2K^b and does not cross-react with other H-2K haplotypes (such as d, f, j, k, p, q, r, s, u, v), ensuring high specificity in mouse models of the H-2K^b haplotype.
• Binding Affinity: Studies using Surface Plasmon Resonance (SPR) consistently show that AF6-88.5 and its subclone (AF6-88.5.5.3) possess potent and selective binding to H-2K^b, more so than comparative antibodies (e.g., 34-1-2S shows only weak cross-reactivity and C1.18.4 does not bind).
• Functional Applications: It is extensively utilized for:
  • Flow cytometry (typically at ~0.25 µg per million cells).
  • Immunoprecipitation and immunohistochemistry on acetone-fixed frozen sections but not on formalin-fixed, paraffin-embedded sections.
  • Studying antigen presentation, as H-2K^b is an essential molecule for presenting peptides to CD8^+ T cells, making AF6-88.5 valuable for T-cell mediated immune response analysis.
• Biological and Structural Insights: AF6-88.5 has been used to differentiate functional and structural aspects of murine MHC class I molecules, including the study of binding domains, peptide-MHC stability, and Fcγ receptor interactions.
• Fcγ Receptor Binding: AF6-88.5 shares FcγR-binding properties with other anti-MHC class I antibodies but with nuanced differences in binding affinities (statistically significant though minor when compared across several FcγR types).

Notable Scientific Citations:

• Early and continued references (including Loken et al., J. Immunol. Methods 1982; Wall et al., J. Immunol. 1983; Andersson et al., J. Immunol. 1998; Shao et al., J. Immunol. 2005; Zhou et al., Cytotherapy 2010) document its use in both basic and translational immunology studies.

In summary, clone AF6-88.5 is a standard, highly specific antibody for detecting murine H-2K^b in immunological contexts, with well-documented performance in cell staining, antigen presentation studies, and Fc receptor analysis in mice of the H-2K^b haplotype.

Dosing regimens for clone AF6-88.5 (an anti-mouse MHC class I H-2K^b monoclonal antibody) vary primarily by application and are generally not highly dependent on specific mouse models, but rather on the experimental use and strain's H-2K^b expression. The most common dosing information available concerns in vitro and ex vivo assay applications, with limited published data on in vivo dosing in mouse models.

Essential context and supporting details:

• Flow Cytometry: The recommended working dilution for staining is 1:10 to 1:20, typically using 10 µl of diluted antibody per 1 million cells in 100 µl volume. Alternatively, a guide suggests ~0.25 µg per 10^6 cells in 100 µl for flow cytometry. These recommendations are for cell-based assays and not for systemic administration in live mice.
• Immunohistology and Immunoprecipitation: Specific working dilutions are not standardized and should be optimized per investigator depending on tissue type, fixation method, and detection system.
• Target Population: AF6-88.5 is specific for mice expressing the H-2K^b determinant, primarily C57BL/6 and related strains. It does not cross-react with other H-2K haplotypes, so its use is only valid in mouse models carrying the correct genetic background.

Additional relevant information:

• No standardized in vivo dosing regimens are publicly documented for clone AF6-88.5. Most literature and supplier guidance focus on its use in flow cytometry, immunohistochemistry, and immunoprecipitation.
• Typical in vivo antibody dosing for mouse research (for unrelated antibodies) ranges from about 50 to 300 µg per mouse by intraperitoneal or intravenous injection, but there is no specific affirmation that AF6-88.5 is routinely used in this way. If in vivo depletion or blocking is required, dosing should be determined empirically by pilot studies and guided by comparable antibody protocols.

In summary, dosing regimens of clone AF6-88.5 are driven by application type (mostly in vitro/ex vivo assays) and H-2K^b expression, not by mouse disease model, since its use depends on the presence of the correct MHC haplotype, and there is no standard in vivo dosing protocol available for this clone. Investigators must optimize working concentrations for each assay and mouse strain, and consult up-to-date datasheets for lot-specific instructions.