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

Use of Clone W6/32 in In Vivo Mouse Studies

Background on W6/32
Clone W6/32 is a mouse monoclonal antibody (IgG2a) raised against human HLA class I (HLA-A, -B, -C) major histocompatibility complex (MHC) molecules. It recognizes a conformational epitope shared among these HLA-A, B, and C loci, primarily targeting the alpha-2 and alpha-3 domains of the HLA class I heavy chain. Its classical applications—including flow cytometry, immunofluorescence, ELISA, immunohistochemistry, and immunoprecipitation—are predominantly used in human or primate systems. However, its use in mice, especially in vivo, is highly context-dependent due to a lack of endogenous HLA.

Cross-Reactivity with Mouse MHC

• In Vitro Findings: W6/32 has been shown to cross-react with certain mouse H-2 class I antigens, specifically H-2Db and H-2Kd, but only when the native mouse beta-2 microglobulin (?2m) is exchanged for human or bovine ?2m. This cross-reactivity is not observed with murine ?2m and is dependent on the formation of a complex between the mouse H-2 heavy chain and the exogenous ?2m.
• Mapping the Epitope: The W6/32 epitope in mouse cells is mapped to specific residues in the alpha-2 domain of H-2 class I genes, and its detection is structurally dependent on the species of ?2m associated with the heavy chain.
• In Vivo Relevance: The critical detail for in vivo use is that, under normal physiological conditions, mouse cells express only mouse ?2m. This means W6/32 does not bind endogenously expressed mouse MHC I molecules in untreated mice.

Practical Use in Mouse Studies

Expression of Human HLA in Mice
W6/32 is not used to detect or target native mouse MHC I. Instead, it is employed to study humanized mouse models—for example, mice genetically engineered to express human HLA molecules (e.g., HLA-A2 or HLA-B27 transgenic mice). In these models, W6/32 can be used to detect human HLA expression on the surface of mouse cells by flow cytometry, immunofluorescence, or other imaging methods in vivo or ex vivo.

?2m Substitution Experiments
Studies have demonstrated that if human or bovine ?2m is injected into mice, it can associate with mouse H-2 heavy chains and, in some strains (e.g., H-2Db or H-2Kd), create a conformational epitope recognizable by W6/32. In such experiments, W6/32 could theoretically be used as a probe to track ?2m exchange in vivo, though this is not a standard or routine application.

Key Limitations and Considerations

• No Binding to Native Mouse MHC I: W6/32 does not bind mouse MHC I when only mouse ?2m is present.
• Transgenic or Humanized Models Required: Its in vivo utility in mice is limited to models expressing human HLA class I, not wild-type or unmodified mice.
• Cross-Reactivity Artifact: The reported cross-reactivity with mouse H-2 antigens is an experimental artifact dependent on ?2m exchange, which does not typically occur under physiological conditions.

Summary Table: W6/32 Use in Mouse Studies

Conclusion

In standard in vivo mouse studies, W6/32 is not used to target or detect native mouse MHC I due to a lack of cross-reactivity under physiological conditions. Its primary in vivo application in mice is limited to humanized or transgenic models expressing human HLA class I molecules, where it serves as a specific probe for human HLA expression. Any reported cross-reactivity with mouse H-2 depends on artificial conditions (?2m substitution) and is not relevant to normal mouse physiology.

Storage Temperature Recommendations for Unconjugated, Sterile Packaged Clone W6/32

Most suppliers of unconjugated, sterile functional-grade monoclonal antibody clone W6/32 specify short-term storage at 2–8°C (refrigerator temperature) and do not recommend freezing the product. For example, BioLegend and Origene both state the antibody should be stored undiluted between 2°C and 8°C and explicitly instruct users "DO NOT FREEZE". This guidance is typical for many liquid antibody formulations to avoid denaturation and precipitation.

Leinco Technologies offers a more flexible approach for their sterile, functional-grade preparations: store at 2–8°C for up to 1 month as received. For longer term storage, they recommend aseptically aliquoting the antibody (without diluting) and freezing at (leq-70^circ)C, avoiding repeated freeze-thaw cycles. This is an exception to the "do not freeze" guidance and is specific to their formulation and packaging.

Comparison Table

*Only for Leinco’s functional-grade, sterile, animal-free product. For most standard W6/32, do NOT freeze.

Summary and Best Practice

• Most standard W6/32 antibodies (liquid, unconjugated, non-functional grade): Store undiluted at 2–8°C; do not freeze. Shelf life is typically up to one year from despatch.
• Leinco functional-grade, sterile antibody: Store at 2–8°C for up to 1 month as received; for longer storage, aseptically aliquot and freeze at (leq-70^circ)C to avoid repeated freeze-thaw cycles.
• Always check the manufacturer’s datasheet for your specific product lot, as formulations and recommendations can vary.

If you have a standard, unconjugated W6/32 clone—do not freeze. Refrigerate at 2–8°C. For Leinco’s special functional-grade product, follow their specific guidance for sterile, long-term storage at ultra-low temperature. When in doubt, consult the product insert or contact the manufacturer directly.

Based on the available search results, information about commonly used antibodies or proteins in combination with W6/32 is limited. However, several key molecular components and experimental controls are frequently mentioned in the literature.

Core Molecular Components

Beta-2 microglobulin (?2m) emerges as the most critical protein used alongside W6/32. The antibody's reactivity is absolutely dependent on the association between HLA class I heavy chains and ?2m, with the W6/32 epitope requiring proper three-dimensional configuration maintained through this association. Interestingly, the antibody shows species-specific sensitivity, reacting with HLA class I antigens only when complexed with human or bovine ?2m, but not with ?2m from other species like goat, sheep, rabbit, or mouse.

HLA class I heavy chains (HLA-A, HLA-B, and HLA-C) are the primary target antigens that W6/32 recognizes. The antibody binds to a conformational epitope involving the alpha2 and alpha3 domains of these heavy chains.

Experimental Controls and Comparisons

In flow cytometry applications, mouse IgG2a, ? isotype control antibodies are routinely used as negative controls when working with W6/32. This provides a baseline for determining specific binding versus background staining.

Functional Applications Context

W6/32 has been utilized in studies involving NK cell-mediated lysis, where it can function as both an inhibitor and activator. The antibody has also been employed in immunoprecipitation experiments and Western blotting under non-reducing conditions.

The search results indicate that W6/32 is frequently used as a standard reagent for detecting and assaying HLA-A,B,C antigens, but specific information about other commonly co-used antibodies in multiplex panels or comparative studies is not extensively detailed in the available literature. The primary focus appears to be on the critical requirement for proper ?2m association and the antibody's utility as a pan-HLA class I detection tool.

The W6/32 monoclonal antibody has been extensively cited in scientific literature as a key reagent for detecting, purifying, and quantifying HLA class I antigens (HLA-A, -B, -C) across diverse human cells and tissues. Its main findings and uses include:

• Broad Specificity: W6/32 specifically recognizes a conformational epitope present on all major HLA class I molecules (A, B, and C) by binding to the complex of the heavy chain associated with ?2-microglobulin. The determinant it recognizes is retained even after certain biochemical extractions, allowing its use on both native and solubilized HLA molecules.

• Structural and Quantitative Studies: W6/32 has enabled detailed studies of HLA class I molecular structure and quantification of cell-surface expression. For instance, it was used to show that B cell lines have, on average, nine times more HLA-A,B,C molecules on their surface than peripheral lymphocytes.

• Purification Tool: Due to its high specificity, W6/32 is used to create immunoaffinity columns that can purify HLA class I molecules in a single step from cell lysates—facilitating biochemical and serological analyses.

• Epitope Dependency: The binding of W6/32 demonstrates that the intact conformation of HLA class I, including the presence of ?2-microglobulin, is essential; binding drops with the dissociation of ?2-microglobulin. This helps distinguish folded, functional HLA class I from free or misfolded ? chains.

• Versatile Applications: The antibody is validated for use in flow cytometry, immunoprecipitation, immunohistochemistry, immunofluorescence, and Western blotting, making it a standard across immunological and clinical labs.

• Reactivity and Batch Consistency: W6/32 cross-reacts with HLA class I in several primate species and, thanks to recombinant production, current versions offer high lot-to-lot consistency and are animal-free.

• Foundational Reagent: Scientifically, W6/32’s introduction enabled many discoveries regarding immune recognition, HLA diversity, and transplantation immunology, as it defines a universal “pan-HLA class I” marker.

In summary, W6/32’s foundational citations demonstrate its critical role in immunology as a reliable and versatile tool for studying HLA class I antigen expression, purification, and function.