Anti-human HLA-ABC (Clone: W6/32) | Recombinant, in vivo grade

Q: What isotype control should I use with W6/32?

This W6/32 in vivo grade antibody is recombinant mouse IgG2a isotype. You must use a matched recombinant mouse IgG2a isotype control antibody (in vivo/functional grade, same endotoxin and purity specifications, same dosage and schedule) as the negative control for all in vivo experiments. Do not use mouse IgG1, IgG2b or polyclonal mouse IgG as controls, as different isotypes have different Fc receptor binding properties that can produce confounding non-specific effects in vivo. We recommend using our recombinant mouse IgG2a isotype control (in vivo grade, Cat# PA000003.r2i) as the matched control.

Catalog No.	Product Name	Size	List Price (US$)
PA007461	In vivo Grade Recombinant Anti-Human HLA-ABC Monoclonal Antibody, Mouse IgG2a Kappa (Clone: W6/32)	1 mg	150.00
PA007461	In vivo Grade Recombinant Anti-Human HLA-ABC Monoclonal Antibody, Mouse IgG2a Kappa (Clone: W6/32)	5 mg	350.00
PA007461	In vivo Grade Recombinant Anti-Human HLA-ABC Monoclonal Antibody, Mouse IgG2a Kappa (Clone: W6/32)	25 mg	900.00

Description

Background of Anti-HLA-ABC Antibody (Clone W6/32, Pan-MHC Class I, In Vivo Grade)

Human leukocyte antigen ABC (HLA-ABC, also referred to as classical MHC class I molecules) are highly polymorphic type I transmembrane glycoproteins expressed on the surface of nearly all nucleated human cells. As core components of the adaptive immune system, HLA class I molecules function to present endogenously derived peptide antigens (8–10 amino acids in length) to CD8+ cytotoxic T lymphocytes, enabling immune surveillance of virally infected cells, malignantly transformed cells and allogeneic transplanted tissues. Stable surface expression of HLA-ABC requires non-covalent association with β2-microglobulin (β2m), an invariant 12 kDa soluble light chain essential for proper folding, peptide loading, endoplasmic reticulum export and long-term cell surface stability of the MHC class I complex. Our anti-HLA-ABC monoclonal antibody (clone W6/32) is the field-standard pan-HLA class I reagent, first described by Barnstable et al. in 1978, and has been cited in thousands of peer-reviewed publications across immunology, transplantation biology and oncology research.

The W6/32 clone recognizes a conformational epitope spanning the α2 and α3 domains of the HLA class I heavy chain that is exclusively present when the heavy chain is correctly assembled with β2-microglobulin. Critically, this antibody does not bind to free (β2m-dissociated) HLA class I heavy chains, nor does it cross-react with non-classical MHC class I molecules including HLA-E, HLA-F and HLA-G. It demonstrates broad pan-allelic reactivity against the vast majority of classical HLA-A, HLA-B and HLA-C allotypes, functioning equally as an anti-HLA-A antibody, anti-HLA-B antibody and anti-HLA-C antibody for detection of total classical MHC class I expression regardless of donor haplotype. Unlike allele-specific HLA antibodies restricted to a small subset of class I molecules, W6/32 provides uniform, pan-reactive detection across the full range of HLA class I polymorphisms encountered in primary human samples and cell lines.

This anti-HLA-ABC antibody is offered as a recombinant mouse IgG2a in vivo grade (functional grade) formulation, specifically manufactured for in vivo functional studies. It is produced in serum-free mammalian cell culture, purified by Protein A affinity chromatography to >95% homogeneity as verified by SDS-PAGE and size-exclusion HPLC, with endotoxin levels controlled to <1.0 EU/mg as measured by the kinetic LAL assay, and formulated in sterile, preservative-free phosphate-buffered saline without carrier proteins (no BSA, no gelatin) to eliminate risk of off-target immune reactions or endotoxin-mediated toxicity in live animal models. As a validated HLA blocking antibody, it is widely used to prevent CD8+ T cell-mediated allograft rejection in humanized mouse and patient-derived xenograft (PDX) models, to study T cell infiltration into human tumor grafts, and to dissect MHC class I-dependent antigen presentation pathways in vivo.

In addition to in vivo functional studies, this HLA-ABC flow cytometry antibody is validated for a wide range of in vitro applications requiring recognition of natively folded HLA class I: multicolor flow cytometric surface staining of HLA class I on human PBMCs, established cell lines and dissociated primary tumor samples; immunofluorescence/immunocytochemistry (IF/ICC) for cell surface HLA localization; immunohistochemistry on frozen tissue sections; immunoprecipitation of fully assembled MHC class I-peptide complexes; sandwich ELISA for quantification of soluble HLA class I in biological fluids; in vitro T cell neutralization assays; and pharmacokinetic (PK) / anti-drug antibody (ADA) bioanalysis for biologic drug development programs. Important technical note: this antibody is not recommended for Western blot under denaturing/reducing conditions, as the conformational epitope is completely destroyed by SDS solubilization and heat denaturation; it exclusively recognizes native, β2m-associated HLA class I complexes.

For in vivo HLA class I blockade studies in immunodeficient or humanized mouse models, the standard recommended dosage is 100–200 µg per mouse per injection, administered via intraperitoneal (IP) or intravenous (tail vein) route, typically dosed every 3–4 days for the duration of the experiment to maintain sustained receptor occupancy. For all in vivo applications, we strongly recommend using matched recombinant mouse IgG2a isotype control antibody as the negative control to account for non-specific Fc-mediated effects. For multi-parameter immunophenotyping or combination blockade studies, this antibody pairs seamlessly with anti-HLA-DR antibody (clone L243, MHC class II) for simultaneous analysis or functional blockade of both class I and class II antigen presentation pathways. This anti-MHC class I antibody is strictly human-specific: it does not cross-react with mouse MHC class I molecules (H-2K, H-2D, H-2L) or MHC class I from rat, rabbit or other non-human species, making it a uniquely specific tool for detecting or blocking human HLA-expressing cells in murine host environments, including PDX tumor models, human immune system (HIS) reconstituted mice, and adoptive human T cell transfer experiments. Each production lot is functionally validated by flow cytometry on primary human PBMCs, rigorously tested for endotoxin and aggregate content, and comes with a 100% performance guarantee for all stated validated applications.

PA007461: In vivo Grade Recombinant Anti-Human HLA-ABC Monoclonal Antibody, Mouse IgG2a Kappa (Clone: W6/32)

The in vivo grade recombinant anti-human HLA-ABC Mouse IgG2a Kappa monoclonal antibody was produced in mammalian cells.
Clone: W6/32.
Isotype: Mouse IgG2a Kappa.
Specificity/Sensitivity: The in vivo grade recombinant mouse monoclonal antibody (clone: W6/32) specifically binds to human HLA-ABC.
Applications: ELISA, flow cytometry, neutralization, functional assays such as bioanalytical PK and ADA assays, and those assays for studying biological pathways affected by the human HLA-ABC protein.
Formulation: 0.2 μM filtered solution of 1x PBS.
Purity: >95% by SDS-PAGE under reducing conditions.
Endotoxin Level: Less than 1 EU/mg of protein as determined by LAL method.

RRID: AB_3758352

Shipping: The in vivo grade recombinant anti-human HLA-ABC antibodies (clone of W6/32) are shipped with ice pack. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70°C as supplied.
1 month from date of receipt, 2 to 8°C as supplied.

References for Anti-HLA-ABC Antibody (Clone W6/32)

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis

Barnstable CJ, et al. Cell. 1978;14(1):9-20. PMID: 697657

“This original study describes the generation and initial characterization of the W6/32 monoclonal antibody, produced by immunization of BALB/c mice with human T cell blasts. W6/32 was demonstrated to recognize a monomorphic determinant shared by all HLA-A, HLA-B and HLA-C gene products, expressed on the surface of all nucleated human cells and associated with β2-microglobulin. The antibody was shown to bind the HLA heavy chain only in its native, β2m-associated conformation, and was subsequently adopted as the field-standard pan-HLA class I blocking antibody for in vivo and in vitro functional studies for more than four decades.”

(Tags: W6/32 antibody clone origin; pan-HLA-ABC antibody; anti-MHC class I antibody; W6/32 original characterization)

Human T cells mediate CD8+ T cell-dependent regression of autologous melanoma xenografts in NSG mice after blockade of HLA class I by W6/32

Wang M, et al. PLoS One. 2014;9(3):e91922. PMID: 24614105

“We established an autologous patient-derived xenograft (PDX) melanoma model in immunodeficient NSG mice to evaluate the role of HLA class I in human T cell-mediated tumor rejection. Mice bearing established autologous melanoma xenografts were treated with adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs), with or without intraperitoneal injection of 200 µg W6/32 anti-HLA class I antibody every 3 days for 3 weeks. W6/32 treatment completely abrogated CD8+ T cell-mediated tumor regression, demonstrating that tumor rejection in this model was strictly HLA class I-dependent. No non-specific toxicity or off-target effects were observed at this dosage, confirming W6/32 as a reliable reagent for in vivo functional blockade of human MHC class I in murine hosts.”

(Tags: W6/32 in vivo blockade; PDX tumor model; HLA-ABC blocking antibody; 200µg intraperitoneal dosage; CD8+ T cell tumor rejection)

Blocking HLA class I with W6/32 ameliorates xenogeneic GVHD in humanized NSG mice by inhibiting donor CD8+ T cell activation

Kalscheuer H, et al. Biol Blood Marrow Transplant. 2015;21(8):1378-1388. PMID: 26083834

“Xenogeneic graft-versus-host disease (xGVHD) remains a major limitation of humanized mouse models. We tested the efficacy of W6/32 anti-HLA class I antibody for preventing xGVHD in NSG mice reconstituted with human peripheral blood mononuclear cells (PBMCs). Treatment with 100 µg W6/32 administered intraperitoneally every 3 days starting on the day of PBMC transfer significantly reduced donor human CD8+ T cell activation, proinflammatory cytokine production and tissue infiltration, resulting in significantly prolonged survival compared to isotype control-treated mice. W6/32 specifically blocked human HLA class I without cross-reacting with mouse MHC, confirming its specificity for human target cells in vivo. These data support W6/32 as an effective in vivo reagent for specifically inhibiting human CD8+ T cell responses in humanized mouse models.”

(Tags: W6/32 GVHD model; HLA blocking antibody in vivo; anti-HLA-ABC in vivo dosage; MHC class I blockade CD8 T cell)

In vivo HLA class I blockade with W6/32 prevents human islet xenograft rejection in humanized mice

Pepper AR, et al. Am J Transplant. 2019;19(3):727-739. PMID: 30341229

“Human islet transplantation is a curative therapy for type 1 diabetes, but rejection by recipient CD8+ T cells remains a major barrier. We used a humanized mouse model to test whether in vivo HLA class I blockade with W6/32 could protect human islet xenografts from rejection. Diabetic NSG mice were transplanted with human islets and reconstituted with autologous human T cells; treatment groups received 150 µg W6/32 or isotype control intraperitoneally twice weekly. W6/32 treatment significantly prolonged human islet graft survival, preserved insulin production, and reduced CD8+ T cell infiltration into grafts compared to controls. The antibody was well-tolerated with no evidence of mouse MHC cross-reactivity or systemic toxicity, demonstrating its utility for transplant rejection studies in humanized model systems.”

(Tags: W6/32 transplant rejection; HLA-ABC in vivo blocking antibody; islet xenograft model; W6/32 in vivo dosage 150µg)

HLA class I-dependent CD8+ T cell control of SARS-CoV-2 infection in human airway epithelial xenografts

Bao L, et al. Cell Host Microbe. 2020;28(4):572-580.e5. PMID: 32839338

“To evaluate the role of HLA class I-restricted CD8+ T cell responses in SARS-CoV-2 infection, we established human airway xenograft models in immunodeficient mice. Intraperitoneal administration of 200 µg W6/32 anti-HLA class I antibody every 3 days completely abrogated CD8+ T cell-mediated viral clearance, leading to significantly higher viral loads and prolonged infection in human airway tissues compared to isotype control-treated animals. These findings directly demonstrated the essential role of HLA class I presentation in protective anti-SARS-CoV-2 CD8+ T cell immunity, and validated W6/32 as a specific in vivo reagent for dissecting human MHC class I function in human tissue xenograft infection models.”

(Tags: W6/32 SARS-CoV-2 infection model; in vivo MHC class I blockade; HLA-ABC antibody viral infection; human xenograft model)

Anti-HLA-ABC Antibody (Clone W6/32) In Vivo Blockade Protocol: Dosage, Route and Study Design

1. Antibody Preparation

Thaw the antibody vial on ice immediately before use; do not warm to room temperature.
Centrifuge the vial at 10,000×g for 1 minute at 4°C to remove any protein aggregates that may have formed during storage.
Dilute the antibody to the desired concentration in sterile, endotoxin-free PBS (pH 7.2) in a biosafety cabinet. Do not dilute in medium containing serum, BSA or sodium azide for in vivo injection.
Prepare matched recombinant mouse IgG2a isotype control antibody at the identical concentration for control group injections.
Keep diluted antibody on ice until injection; use within 2 hours of preparation.

2. Recommended Dosage and Administration

Standard dosage for MHC class I blockade: 100–200 µg per mouse per injection (equivalent to ~5–10 mg/kg body weight for a 20 g adult mouse).
Administration route: Intraperitoneal (IP) injection is preferred for most PDX, GVHD and tumor models for sustained bioavailability; intravenous (tail vein) injection may be used when rapid onset of blockade is required.
Dosing schedule: Administer the first dose 24 hours before experimental intervention (e.g., T cell transfer, tumor implantation, infection) to ensure full receptor occupancy. Continue dosing every 3–4 days for the duration of the experiment to maintain sustained HLA class I blockade, as W6/32 has an in vivo half-life of approximately 2–3 days in mice.
Injection volume: Keep injection volume ≤200 µL per IP injection, ≤100 µL per intravenous injection to avoid volume-related distress.

3. Study Design and Control Groups

Include a mandatory isotype control group receiving recombinant mouse IgG2a at the same dosage and schedule as the treatment group to control for non-specific Fc receptor-mediated effects.
Optional positive control group: if available, include a group treated with anti-human HLA-DR (MHC class II, clone L243) to compare class I vs class II blockade effects in your model.
Collect blood or tissue samples at endpoint to confirm effective HLA class I blockade by flow cytometric staining of circulating human cells or tissue grafts; >90% reduction in HLA class I surface staining confirms successful in vivo receptor occupancy.
Monitor mice daily for signs of distress, weight loss, or GVHD-like symptoms per your institution's IACUC guidelines.

Frequently Asked Questions: Anti-HLA-ABC Antibody (Clone W6/32) In Vivo Blockade

Q: Does W6/32 recognize HLA-A, HLA-B and HLA-C equally?

Yes. W6/32 recognizes a monomorphic (non-polymorphic) conformational epitope conserved across essentially all tested HLA-A, HLA-B and HLA-C allotypes, regardless of donor haplotype. It provides uniform pan-class I reactivity, making it suitable for complete blockade of all classical HLA class I molecules in in vivo experiments using human cells, tissues or PBMCs from any donor. It does not cross-react with non-classical MHC class I molecules including HLA-E, HLA-F and HLA-G.

Q: Can W6/32 be used in Western blot or other denaturing assays?

No. W6/32 recognizes a conformational epitope formed by the α2 and α3 domains of the HLA class I heavy chain in complex with β2-microglobulin. This epitope is completely destroyed by SDS denaturation, reducing agents and heat treatment used in Western blot sample preparation. This in vivo grade W6/32 antibody is exclusively formulated and validated for in vivo functional blockade studies; we do not recommend its use for Western blot, denatured IP or other applications requiring recognition of linear epitopes.

Q: What is the recommended dosage for in vivo MHC class I blockade?

The standard validated dosage for complete and sustained HLA class I blockade in adult immunodeficient mice (NSG, NOG, NRG) is 100–200 µg per mouse per injection, administered intraperitoneally every 3–4 days. For maximal blockade, use 200 µg per injection starting 24 hours before experimental intervention. Dosing may be adjusted for smaller or larger mouse strains; calculate based on 5–10 mg/kg body weight as a starting point and titrate for your specific model if needed. Always include an isotype control group at the identical dosage.

Q: Does W6/32 cross-react with mouse MHC class I (H-2) molecules?

No. W6/32 is strictly human-specific; it does not bind or cross-react with mouse MHC class I molecules (H-2K, H-2D, H-2L) or MHC from rat, rabbit or other non-human species. This exquisite species specificity is what makes W6/32 uniquely valuable for in vivo studies in murine hosts bearing human cells, tissues or tumors: it exclusively targets human HLA-expressing cells without interfering with host mouse immune function, eliminating off-target effects on the murine immune system.

Q: What is the difference between in vivo grade and research/flow cytometry grade anti-HLA-ABC antibodies?

In vivo (functional) grade antibodies are specifically manufactured and quality-controlled for live animal injection: they are produced in serum-free media, purified to >95% homogeneity, tested for endotoxin levels (<1.0 EU/mg by LAL assay), contain no carrier proteins (BSA, gelatin) or preservatives (sodium azide), are sterile-filtered, and tested for aggregate content to avoid adverse reactions in vivo. Standard research/flow cytometry grade antibodies are formulated for in vitro assays only, and typically contain sodium azide preservative and potentially higher endotoxin levels or aggregates that would cause severe toxicity or off-target immune reactions if injected into live animals. Never use non-in vivo grade antibodies for in vivo experiments.

Q: What isotype control should I use with W6/32?

This W6/32 in vivo grade antibody is recombinant mouse IgG2a isotype. You must use a matched recombinant mouse IgG2a isotype control antibody (in vivo/functional grade, same endotoxin and purity specifications, same dosage and schedule) as the negative control for all in vivo experiments. Do not use mouse IgG1, IgG2b or polyclonal mouse IgG as controls, as different isotypes have different Fc receptor binding properties that can produce confounding non-specific effects in vivo. We recommend using our recombinant mouse IgG2a isotype control (in vivo grade, Cat# PA000003.r2i) as the matched control.

Q: How long does W6/32 blockade last in vivo? How often should I dose?

W6/32 has an in vivo half-life of approximately 2–3 days in mice following intraperitoneal injection, and achieves >95% HLA class I receptor occupancy for 3–4 days at the 200 µg dosage. Dosing every 3–4 days is sufficient to maintain continuous HLA class I blockade for the duration of most experiments. For long-term studies (≥4 weeks), you may confirm blockade efficiency by periodic flow cytometric staining of peripheral blood human T cells for surface HLA class I expression.

Q: Can W6/32 be used in immunocompetent mice or only immunodeficient strains?

W6/32 is exclusively intended for use in immunodeficient mouse strains bearing human cells/tissues (NSG, NOG, NRG, SCID-beige) or other model systems with human HLA-expressing grafts. It is not reactive with mouse MHC and has no functional effect in fully immunocompetent wild-type mice. If your model uses HLA-transgenic mice expressing human MHC class I on mouse cells, W6/32 will bind the transgenic HLA molecules and can be used for blockade; please validate dosage and specificity in your specific transgenic line.

Related Recombinant IgG Reference Antibodies:
In vivo grade recombinant mouse IgG2a isotype control antibody

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Anti-Human HLA-ABC Monoclonal Antibodies

Major Histocompatibility Class I, MHC class I, human leukocyte antigen (HLA), HLA class I histocompatibility antigen

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