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Monoclonal Antibodies

Anti-MR1 [8G3] monoclonal antibody

Invented by Jose Villadangos , Hamish McWilliam
Invented at University of Melbourne
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A novel antibody against MR1 reveals a β2m-deficient HC conformer exists in the ER. (A) Schematic representation of the conformational states of steady-state MR1 as distinguished by each antibody. MR1-HC (blue) shown bound or not to β2m (gray), and empty or loaded with VitBAg. The charged lysine-43 side chain in empty MR1-HC is indicated by a plus symbol (+). (B) Structural alignment of MR1–β2m-6-FP (PDB ID code 4GUP; gray) and HLA-B27 (PDB ID code 1HSA; green) with their bound antigens. Epitopes for mAbs 8G3 and HC10 are highlighted in purple and red, respectively. (C) C1R cells transfected with MR1 were treated without ligand (lanes 1–5) or with Ac-6-FP (lanes 6–9) for 6 h, then lysed and sequential IPs performed using antibodies against the cytosolic tail (αMR1-CT) or conformational-specific mAbs 8G3 or 8F2.F9. After the primary IP (1°) was carried out (lanes 1, 2, 6) the remainder was then subjected to a secondary IP (2°) with each antibody (lanes 3–5, 7–9). Precipitates were treated with endo H or not, then separated by SDS/PAGE and immunoblotted with αMR1-CT or anti-β2m. Endo H-resistant (R) or -sensitive (S) MR1 are indicated. Taken from McWilliam HEG, Mak JYW, Awad W, Zorkau M, Cruz-Gomez S, Lim HJ, Yan Y, Wormald S, Dagley LF, Eckle SBG, Corbett AJ, Liu H, Li S, Reddiex SJJ, Mintern JD, Liu L, McCluskey J, Rossjohn J, Fairlie DP, Villadangos JA. Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens. Proc Natl Acad Sci U S A. 2020 Oct 6;117(40):24974-24985. doi: 10.1073/pnas.2011260117. Epub 2020 Sep 21. PMID: 32958637; PMCID: PMC7547156.
Endogenous levels of MR1 from C1R cells are largely endo H-sensitive.a. MR1 was immunoprecipitated (IP) from 2 x 107 WT C1R cells, treated with or without endoglycosidase H (endo H), then detected by immunoblot (IB) with mAb 8G3. MR1 is largely endo H sensitive (S) with a minor component being resistant (R). b. Mouse M12 or M12 overexpressing mouse MR1 (mMR1) were treated with or without 2 µM Ac-6-FP for 8 h, then cell lysates were immunoblotted using mAb 8G3 or against actin. Data are representative of two independent experiments (a-b). Taken from McWilliam HEG, Mak JYW, Awad W, Zorkau M, Cruz-Gomez S, Lim HJ, Yan Y, Wormald S, Dagley LF, Eckle SBG, Corbett AJ, Liu H, Li S, Reddiex SJJ, Mintern JD, Liu L, McCluskey J, Rossjohn J, Fairlie DP, Villadangos JA. Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens. Proc Natl Acad Sci U S A. 2020 Oct 6;117(40):24974-24985. doi: 10.1073/pnas.2011260117. Epub 2020 Sep 21. PMID: 32958637; PMCID: PMC7547156.
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Catalogue Number 160705
Applications IP WB
Antigen/Gene or Protein Targets MR1
Synonyms Major histocompatability complex class I-related gene protein
Reactivity Human and Mouse
Relevance MR1 presents small metabolite antigens from diverse microbes to activate an abundant T cell population called mucosal associated invariant T cells (MAIT cells). In this way MR1 activates MAIT cells which results in anti-microbial immunity and tissue repair.
Host Mouse
Immunogen Peptide sequence from MR1: pwmaenlapdhwer
Immunogen UniProt ID Q95460
Positive Control The peptide or a lysate of cells expressing MR1
Subclass IgG2a kappa
Molecular Weight (kDa) 42
Myeloma Used Sp2/0
Recommended Growing Conditions Gibco HSFM supplemented with 10% FBS and IL6
Strain C57BL/6
Notes Please note, for immunoprecipitations of MR1, only one conformational form is precipitated – this is the form not bound to beta-2-microglobulin (see PMID:32958637 for details).
Research Area Immunology, Cancer

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McWilliam et al. 2020. Proc Natl Acad Sci U S A. 117(40):24974-24985. PMID: 32958637.

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References: 1 entry

McWilliam et al. 2020. Proc Natl Acad Sci U S A. 117(40):24974-24985. PMID: 32958637.

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Inventor Information

Inventors

Jose Villadangos

Jose Villadangos

Hamish McWilliam

Hamish McWilliam

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Invented at

University of Melbourne
University of Melbourne

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