Anti-Human HLA-A, B, C (MHC Class I) [Clone W6/32] — Purified in vivo PLATINUM™ Functional Grade

Clone W6/32 is a mouse monoclonal antibody specific for human MHC Class I molecules (HLA-A, B, and C), and in mice, its common in vivo applications mainly involve studies using humanized mouse models rather than wild-type or standard laboratory mice.

Key in vivo applications of W6/32 in mice:

• Human Immune System Engraftment: In humanized mice, W6/32 is used to detect and characterize human MHC Class I expression, which is critical for profiling human hematopoietic or immune cell engraftment and reconstitution.
• Functional Assays: The antibody is applied to verify the presence and distribution of human cells in mouse tissues by flow cytometry or immunohistochemistry, enabling researchers to monitor human cell populations and immune responses after engraftment.
• Immune Response Studies: W6/32 is used to investigate mechanisms of antigen presentation, test immune cell activation, and track development of antibody responses following vaccination, infection, or immunotherapy in humanized mice.
• Autoimmunity and Disease Modeling: The antibody assists in models of autoimmunity (e.g., lupus), transplantation, or tumor immunology using humanized mice, providing insight into human immune cell interactions and pathology.
• NK Cell Function and Inhibition: Clone W6/32 helps study NK cell-mediated lysis and its inhibition by blocking human MHC class I on engrafted cells.

Important Considerations:

• Species Specificity: W6/32 does not recognize mouse MHC Class I molecules; its reactivity is strictly with human antigens. Therefore, standard in vivo experiments in wild-type or immunocompetent mice do not employ this antibody unless human cells are present.
• Endotoxin-Free Formulations: In vivo studies require low-endotoxin or endotoxin-free antibody preparations (sometimes marketed as "in vivo GOLD™" or "in vivo PLATINUM™"), which are essential to avoid inflammatory artifacts in mouse experiments.

Summary Table: W6/32 In Vivo Use in Mice

Clone W6/32 is not functional for direct mouse MHC detection; its in vivo use in mice is exclusively tied to experimental systems incorporating human cells or tissues such as humanized mouse models, where it provides a critical tool for validating and analyzing human immune processes.

Other commonly used antibodies or proteins alongside W6/32 in the literature include those that recognize different epitopes or subclasses of HLA class I, HLA class II molecules, or associate with immunological reference markers such as β2-microglobulin, CD8, and NK cell ligands.

Key examples often found in the literature:

• β2-microglobulin antibodies: Since W6/32 binding depends on intact MHC class I–β2-microglobulin complexes, β2-microglobulin–specific antibodies are often co-used for epitope mapping, validation of folding, or for flow cytometry and immunoprecipitation controls.
• Allele-specific or locus-specific HLA class I antibodies: These include antibodies that selectively recognize HLA-A2, HLA-B7, etc., allowing discrimination between pan-HLA reactivity (W6/32) and allele-limited detection for refined phenotyping or biochemical confirmation.
• CD8 antibodies: When studying T cell interactions with MHC class I, anti-CD8 antibodies are often included to label or functionally interrogate cytotoxic T cells, since CD8 is the coreceptor binding MHC class I at a site overlapping the W6/32 epitope.
• Leukocyte Ig-like receptor (LILR) antibodies: These are relevant when exploring interactions of HLA class I molecules not only with T cells but with innate immune cells.
• Killer-cell Ig-like receptor (KIR) antibodies: For studies involving NK cell ligands, such as modulation of HLA class I–KIR interactions, these antibodies are often included as experimental controls or read-outs.
• Other pan-HLA antibodies: Alternative clones such as G46-2.6 or HC-10 (which detects denatured/classical heavy chains) are sometimes used in parallel for cross-validation and to detect conformation-specific versus denatured class I molecules.

Research context also frequently determines additional marker selection:

• In immunopeptidomics and MHC ligandome analyses, anti-HLA-DR, anti-HLA-DQ, and anti-HLA-DP (for class II) antibodies are co-used with W6/32 to capture or differentiate class I and class II complexes.
• In functional assays (NK cell inhibition, blocking experiments), antibodies blocking activating/inhibitory receptors such as anti-CD94/NKG2 or anti-LILRB1 are combined with W6/32 to map specific receptor-ligand interactions.

HLA-E–specific antibodies such as 3D12 and 4D12 are also commonly cited, particularly in contexts where non-classical class I molecules are relevant.

In summary, common pairs or panels with W6/32 include:

• β2-microglobulin
• Allele-specific HLA class I antibodies
• HLA class II antibodies (DR, DQ, DP)
• CD8
• KIR and LILR family antibodies
• Non-classical class I antibodies (HLA-E, HLA-G, HLA-F) such as 3D12, 4D12

Selection depends on the experimental goal: detection, functional analysis, or specificity mapping.

Clone W6/32 is a monoclonal antibody that has been extensively used in immunological research for over six decades, with several key findings emerging from its widespread application in scientific literature.

Recognition of a Conserved HLA Class I Epitope

The most fundamental discovery is that W6/32 recognizes a conformational epitope that is conserved across diverse HLA-I allotypes, effectively bypassing the extreme polymorphism that typically confounds HLA class I studies. This monomorphic epitope is expressed on native β2-microglobulin (β2m)-associated Major Histocompatibility Complex (MHC) class I molecules, specifically HLA-A, HLA-B, and HLA-C. The antibody binds to a discontinuous epitope comprised of the α1, α2, and α3 domains of the HLA-I heavy chain along with β2-microglobulin.

Structural Characterization

Crystal structure analysis has revealed that W6/32 binds beneath the antigen-binding groove of HLA class I molecules. The epitope comprises a region of low polymorphism, which explains the pan-HLA-I nature of its binding capability. This structural positioning is particularly significant because it does not overlap with binding sites for T cell antigen receptors or killer cell immunoglobulin-like receptors (KIRs).

Functional Implications for Immune Cell Interactions

A critical finding is that the W6/32 epitope coincides with binding sites for leukocyte immunoglobulin-like receptors (LILRs) and CD8 coreceptors. Functional studies demonstrated that using W6/32 to block the interaction between NK cells and HLA-I only weakly impaired inhibition mediated by KIR3DL1, but significantly impacted HLA-LILR recognition. This selective blocking pattern provides important insights into differential receptor-ligand interactions on HLA class I molecules.

Clinical and Research Applications

The antibody has proven valuable for detecting variations in HLA class I expression across different cell types and disease states. Research has documented that HLA class I expression is upregulated on activated cells or those responding to proinflammatory cytokines, while reduced expression is found on certain virus-infected or tumor cells. Studies have identified cases where tumors show complete loss of HLA class I expression, with some pancreatic carcinomas testing negative for W6/32.

The ability of W6/32 to recognize its epitope only on properly folded, β2m-associated HLA class I molecules makes it particularly useful for assessing the native conformation and stability of these proteins in various experimental contexts.

Dosing regimens of clone W6/32 are not standardized across mouse models, because this monoclonal antibody is primarily used for ex vivo or in vitro applications—such as flow cytometry and immunofluorescence detection of human HLA class I molecules—rather than for systemic administration in live mice. Most suppliers and reviews confirm that no established in vivo dosing protocol exists for W6/32 in typical mouse strains, as it is not intended for immunomodulation or depletion in murine models.

Key context and details:

• W6/32 targets human HLA-A, -B, -C class I molecules and is widely used for detection of these antigens in human samples or xenografted tissues, not for functional modulation in mice.
• According to major antibody suppliers, it is "not routinely administered as a systemic dose in mouse models," and therefore, "does not have established dosing regimens across mouse strains".
• Recommended guidelines are available for other functional antibodies in mice (such as anti-CTLA-4 or anti-CD3), with specified doses and schedules, but these do not apply to W6/32.
• For in vitro labeling (e.g., flow cytometry), recommendations do exist: typically, 10 µl of a 1/50–1/100 dilution to label 1×10^6 cells in 100 µl buffer.

Exception—rare in vivo use:

• There are isolated reports in humanized or xenograft mouse models where W6/32 was administered in vivo. For instance, one preclinical immunomodulation protocol involved giving mice three intraperitoneal doses of W6/32 (1 mg/kg) on days -7, -5, and -3 before organ transplantation. These are highly context-dependent and not generalizable, as the antibody does not bind to native murine MHC and is only functionally relevant in models expressing human HLA.

Summary Table: W6/32 Use Contexts

Conclusion:
There are no standard W6/32 dosing regimens for in vivo use in typical mouse models, as its principal application is in detecting human HLA class I in ex vivo or in vitro assays. In rare experimental settings involving humanized mice, specific ad hoc dosing protocols may be reported, but these are not widely validated or standardized.