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. 2017 Feb 8;8:96.
doi: 10.3389/fimmu.2017.00096. eCollection 2017.

A Simple and Rapid Method for Quality Control of Major Histocompatibility Complex-Peptide Monomers by Flow Cytometry

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Free PMC article

A Simple and Rapid Method for Quality Control of Major Histocompatibility Complex-Peptide Monomers by Flow Cytometry

P Anoop Chandran et al. Front Immunol. .
Free PMC article

Abstract

Major histocompatibility complex (MHC) multimers are essential tools in T cell immunomonitoring, which are employed both in basic and clinical research, as well as for assessing clinical samples during therapy. The generation of MHC monomers loaded with synthetic peptides is an elaborate and time-consuming process. It would be beneficial to assess the quality of these monomers prior to downstream applications. In this technical note, we describe a novel flow cytometry-based, cell-free, quick, and robust assay to check the quality of MHC monomers directly after refolding or after long-term storage.

Keywords: MHC–peptide monomers; UV-peptide exchange; antigen-specific T cells; multimers; quality control.

Figures

Figure 1
Figure 1
Workflow of the bead-based assay for controlling the quality of major histocompatibility complex-monomers: the five steps and the three assay conditions tested (i)–(iii) are shown. Details are given in the Section “Materials and Methods.”
Figure 2
Figure 2
Assessment of major histocompatibility complex (MHC) monomer biotinylation. (A) We titrated the biotinylated and non-biotinylated monomers (0.00005–5 µg/mL) on the beads and stained them with anti-MHC and relevant isotype control monoclonal antibodies. The complexes were subsequently detected using a PE-labeled secondary antibody. Specific fluorescence indices of the PE signal are plotted. (B) Histograms showing the binding of the respective anti-MHC antibodies to 0.05 µg/mL of the indicated biotinylated and non-biotinylated monomers. Representative data from one of two experiments are shown in (A,B).
Figure 3
Figure 3
Detection of denatured major histocompatibility complex (MHC) monomers. Beads were loaded with monomers, treated with an acidic buffer or with PBS (control), and then stained with the indicated anti-MHC monoclonal antibodies and relevant isotype controls, followed by detection with a PE-labeled secondary antibody. Representative histograms of PE fluorescences from one of two experiments are shown.
Figure 4
Figure 4
Quality control of UV-exchanged major histocompatibility complex (MHC) monomers. Monomers containing UV-labile peptides were left untreated or were exposed to UV-light in the presence or absence of a replacement peptide. Binding of the indicated anti-MHC monoclonal antibodies to these monomers was detected using a PE-labeled secondary antibody. Median fluorescence of the PE signal from two representative experiments out of three is plotted. Histograms of one of the experiments are shown in Figure S3A in Supplementary Material.
Figure 5
Figure 5
Application of the bead assay to test in-house produced major histocompatibility complex (MHC) monomers. Two batches of conventional HLA-A*02 monomers (Batch 1 and Batch 2), each generated with the Melan A peptide (ELAGIGLTV) were tested. (A) PBMCs from two HLA-A02(+) donors (indicated) were stained with APC-labeled MHC multimers generated from Batch 1 and PE-labeled MHC multimers generated from Batch 2. Multimer-APC/PE vs. CD8 PE-Cy7 dot plots are shown. (B) Fluorescence histograms obtained in the bead test.

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