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

W6/32 is a mouse monoclonal antibody that specifically recognizes a monomorphic epitope on human HLA class I molecules (i.e., HLA-A, HLA-B, HLA-C), and is widely used in human immunology for detection of these antigens. In in vivo mouse studies, its direct use is very limited due to species specificity; the W6/32 antibody reacts weakly or not at all with native mouse MHC class I molecules unless specific experimental manipulations are performed.

Key context and limitations in mouse studies:

• Species restriction: W6/32 does not recognize native murine MHC class I molecules under normal physiological conditions because its conformational epitope is dependent on both the MHC class I heavy chain and the species origin of ?2-microglobulin.
• Experimental cross-reactivity: W6/32 can bind to mouse MHC class I (particularly some alleles like H-2Db or H-2Kd), but only if the mouse class I heavy chain is associated with human or bovine ?2-microglobulin instead of mouse ?2-microglobulin. This can sometimes be achieved:
  • In vitro by culturing mouse cells with human or bovine ?2-microglobulin supplementation.
  • In vivo by injection of human or bovine ?2-microglobulin into mice, which leads to the formation of H-2 (mouse class I)–human/bovine ?2-microglobulin complexes that are recognized by W6/32.

Applications and practical caveats in mouse models:

• Direct detection of endogenous mouse class I: Not routinely feasible, as W6/32 does not robustly detect murine class I antigen under normal conditions.
• Detection of humanized or chimeric molecules: In transgenic or humanized mice expressing human HLA class I proteins, W6/32 can be used for detection and manipulation (e.g., depletion, imaging, tissue staining).
• Special experimental use: For rare studies probing mouse–human class I chimeras or investigating ?2-microglobulin exchange, W6/32 may serve as a probe to study heterologous MHC class I/?2-m alignments.

Summary Table: W6/32 Use in Mice

In summary, W6/32 is not used for general MHC class I detection in wild-type mice. It can only be used to detect HLA class I in genetically modified or xenografted mice, or in murine cells experimentally complexed with human or bovine ?2-microglobulin.

The correct storage temperature for sterile-packaged clone W6/32 (anti-human HLA-A,B,C antibody) is 2°C to 8°C (refrigerated), and the antibody should be kept undiluted and protected from prolonged exposure to light. Do not freeze this antibody, as freezing can denature or reduce its effectiveness.

• If you only need to store it for a short period (up to one month), 2–8°C is appropriate.
• For most commercial sterile packages, longer-term storage at -20°C or lower is generally NOT recommended unless specifically stated by the manufacturer—for W6/32 from major suppliers, refrigeration (2–8°C) is the standard.
• Always refer to the lot-specific data sheet accompanying your antibody for any unique instructions.

If your sample is specifically an Ultra-LEAF™ or similar preservative-free variant, maintain strict aseptic conditions and keep at 2–8°C.

Summary table:

Maintain the antibody undiluted and protected from light during storage for optimal stability.

Commonly used antibodies or proteins often paired with W6/32 in the literature include:

• ?2-microglobulin (?2m): W6/32 recognizes properly folded HLA class I molecules, which requires non-covalent association with ?2-microglobulin; many studies specifically examine this interaction or use anti-?2m antibodies alongside W6/32 to confirm formation of mature HLA class I complexes.
• Allele-specific HLA monoclonal antibodies: These include antibodies targeting particular HLA-A, HLA-B, or HLA-C alleles (e.g., BB7.2 for HLA-A2). Such antibodies are used to dissect allele-specific surface expression versus pan-class I detected by W6/32.
• Antibodies against HLA class II (e.g., L243, anti-HLA-DR): Often used as counterstains or comparative markers to distinguish class I versus class II molecules on cells within flow cytometry or immunoassays.
• Antibodies to lineage or cell markers: W6/32 is used in conjunction with markers such as CD3 (T cells), CD19 (B cells), or CD45 (leukocytes) for multiparametric cell characterization in flow cytometry and immunophenotyping.
• Isotype-matched negative controls: Mouse IgG2a isotype controls are routinely used alongside W6/32 to ensure signal specificity, since W6/32 is also an IgG2a subclass.
• Soluble or recombinant MHC/peptide complexes: For structural and biochemical studies, W6/32 is employed with peptide-loaded soluble HLA class I molecules and recombinant ?2-microglobulin to assess correct conformation and binding.
• Other pan-HLA class I antibodies: Examples include clones such as HC-10, which recognizes free HLA-B and -C heavy chains not associated with ?2m, and are used to probe different conformational or assembly states of class I MHC proteins.

In sum, W6/32 is commonly combined with anti-?2-microglobulin, allele-specific anti-HLA antibodies, class II antibodies, lineage markers, isotype controls, and other conformational antibodies to HLA class I to dissect surface expression, molecular assembly, or cell subset specificity in immunology and transplantation literature.

The monoclonal antibody W6/32 is a key reagent in immunology, widely cited for its role in detecting human HLA class I molecules (HLA-A, HLA-B, HLA-C). Key findings from its use in scientific literature include:

• Recognition of a Conserved Epitope on HLA Class I:
  W6/32 binds a conformational determinant on the alpha2 and alpha3 domains of HLA-A, -B, and -C heavy chains, specifically recognizing a surface structure requiring association with ?2-microglobulin for proper folding and stability.

• Standard Reagent for Purification and Quantitation of HLA I:
  The antibody is routinely used for the immunoaffinity purification of native HLA class I molecules from cells or tissues, often as the standard tool to measure and isolate HLA-A, -B, and -C across different cell types and preparations.

• Functional Role in Peptidome and Structural Studies:
  W6/32 enables high-confidence purification and analysis of HLA-peptide complexes, essential for mass spectrometric profiling of the HLA peptidome. It reliably isolates HLA class I molecules for structural and peptide repertoire studies, including research on antigen processing and tapasin dependence.

• Cell Surface Quantification:
  W6/32 binding has been used to quantify HLA class I copy number per cell, showing that B cell lines express significantly more HLA-A, B, and C on the surface than peripheral blood lymphocytes.

• Stability Dependent on ?2-Microglobulin:
  W6/32’s binding is enhanced or dependent on the presence of ?2-microglobulin—its recognized epitope requires the heavy chain to be non-denatured and complexed with ?2m.

• Validation Across Multiple Species and Applications:
  Although highly specific for human HLA, W6/32 partially cross-reacts with MHC class I from other primates. It's routinely validated for flow cytometry, immunohistochemistry, and immunofluorescence due to its high specificity and reproducibility.

• Trusted and Highly Cited:
  The antibody is extensively cited—over 36 times in peer-reviewed journals for just one commercial product—and recognized as a gold standard for studying HLA class I expression and function.

In summary, W6/32’s primary scientific importance lies in enabling highly sensitive, specific, and reproducible detection, quantification, and purification of native HLA class I molecules, underpinning foundational studies in immunogenetics and antigen presentation.