Measurement of MHC/Peptide Interactions by Gel Filtration or Monoclonal Antibody Capture

John Sidney1, Scott Southwood1, Carrie Moore1, Carla Oseroff1, Clemencia Pinilla1, Howard M. Grey1, Alessandro Sette1

1 La Jolla Institute for Allergy and Immunology, La Jolla, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.3
DOI:  10.1002/0471142735.im1803s100
Online Posting Date:  February, 2013
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This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high‐affinity, assay‐specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size‐exclusion gel‐filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained. Curr. Protoc. Immunol. 100:18.3.1‐18.3.36. © 2013 by John Wiley & Sons, Inc.

Keywords: MHC class I; MHC class II; T cell epitope; peptide ligand; binding affinity; CTL; epitope recognition

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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Peptide Binding to Affinity‐Purified Class I and Class II MHC Molecules
  • Support Protocol 1: MHC Purification
  • Support Protocol 2: Radiolabeling of Peptides by the Chloramine T Method
  • Alternate Protocol 1: Direct Binding Assays to Identify Appropriate High‐Affinity Ligands
  • Support Protocol 3: Separation of MHC‐Peptide Complexes by Size‐Exclusion Gel‐Filtration Chromatography
  • Support Protocol 4: Separation of MHC‐Peptide Complexes by Antibody‐Based MHC Capture
  • Support Protocol 5: Preparation of Immunoaffinity Columns
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Determination of Peptide Binding to Affinity‐Purified Class I and Class II MHC Molecules

  • Inhibitor peptides
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.2 (Invitrogen)
  • Dimethylsulfoxide (DMSO)
  • 0.05% (v/v) Nonidet P‐40 (NP‐40; Fluka)/PBS, pH 7.2
  • Citrate/phosphate buffer (optional; see recipe)
  • MHC (see protocol 2 and protocol 4 for preparation and titration, respectively)
  • Protease inhibitor cocktail (prepare at step indicated, not in advance; see recipe)
  • 1 to 3 µM human β 2‐microglobulin (Class I only; Scripps Laboratories, cat. no. M0114)
  • 1.6% (v/v) NP‐40/PBS: PBS, pH 7.2 (Class II only)
  • 0.82% Pluronic in PBS, pH 7.2
  • 10% digitonin in water
  • Radiolabeled peptide (see protocol 3)
  • Reaction vessels (e.g., 96‐well polypropylene round‐bottom plates from Costar, or 12 × 75–mm culture tubes)
  • Mylar film plate sealer with adhesive backing (ICN Biomedicals) or Costar storage mat III (Corning)
  • Additional reagents and equipment for gel filtration or MHC capture and analysis (see Support Protocols protocol 53 and protocol 64)

Support Protocol 1: MHC Purification

  • Cell line(s): examples include Epstein‐Barr virus (EBV)–transformed human B cell lines; mouse B cell lymphomas or mastocytomas; singly transfected fibroblast, C1R, or 721.221 lines; or Drosophila cells (see Tables 18.3.1, 18.3.2, and 18.3.3 for specific lines that have been used). Cells should be checked for MHC expression prior to purification (or at harvest when freezing for later use).
  • Complete RPMI‐10 ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Lysis buffer (see recipe), ice cold
  • Washing buffer: 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A) with 1% Nonidet P‐40 (store up to 6 months at 4°C)
  • 0.4% (w/v) octylglucoside in PBS
  • Elution buffer (see recipe)
  • 2 M Tris⋅Cl, pH 6.8 ( appendix 2A)
  • 2.5 M glycine, pH 2.5
  • 225‐cm2 tissue culture flasks or roller bottle apparatus
  • Refrigerated centrifuge
  • 0.8‐µm filter
  • Columns (50‐ml borosilicate glass containing one each of the following): inactivated Sepharose CL‐4B (10‐ml bed volume), protein A–Sepharose CL‐4B (5‐ml bed volume), and protein A‐Sepharose CL‐4B conjugated to the appropriate anti‐MHC antibody (Table 18.3.5; 10‐ml bed volume; see protocol 7 for conjugation)
  • Centriprep‐30 concentrator (Amicon)
  • Additional reagents and equipment for counting cells ( appendix 3A)
    Table 8.3.5   MaterialsMonoclonal Antibodies Used in MHC Purification or Capture

    Monoclonal antibody Specificity Source d
    M1/42 H‐2 Class I ATCC
    28‐14‐8S H‐2 Db and Ld ATCC
    34‐5‐8S H‐2 Dd ATCC
    Y3JP H‐2 IAb, IAs, IAu Janeway et al. ( )
    MKD6 H‐2 IAd ATCC
    10.3.6 H‐2 IAk ATCC
    14.4.4 H‐2 IEd, IEk ATCC
    B8‐24‐3 H‐2 Kb ATCC
    Y‐3 H‐2 Kb, Kk ATCC
    SF1‐1.1.1 H‐2 Kd ATCC
    B123.2 HLA B and C e Rebai and Malissen ( )
    W6/32 HLA Class I ATCC
    HB180 HLA Class II ATCC
    B7/21 HLA DP ATCC
    SPVL3 HLA DQ Nepom et al. ( )

     dATCC, American Type Culture Collection.
     eThe B123.2 antibody will also bind some HLA A molecules. To date, we have identified HLA A*2301, A*2601, A*2902, A*3001, A*3002, A*3101, A*3201 and A*3301 as B123.2 reactive. We would presume that corresponding subtypes are also reactive.

Support Protocol 2: Radiolabeling of Peptides by the Chloramine T Method

  • Tyrosinated peptide (10 to 20 mg/ml)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A) with and without 0.05% Tween 20 (Sigma)
  • ∼40 µM [Na125]I (∼100 µCi/µl; NEN Life Sciences, Perkin Elmer)
  • 0.1 mg/ml chloramine T (Sigma) in PBS/0.05% Tween 20
  • 0.1 mg/ml sodium metabisulfite (Fisher) in PBS/Tween
  • 10% (w/v) sodium azide
  • Ethanol
  • 0.82% NP‐40
  • Sephadex G‐10 column: multispin separation kits (Genesee Scientific) with a 0.8 ml bed volume suspended in PBS, pH 7.2
  • Microcentrifuge: Labnet International Spectrafuge 16M (cat. no. C0160‐R;
  • Polyethylene storage vessel (e.g., 1.5‐ml microcentrifuge tubes)

Alternate Protocol 1: Direct Binding Assays to Identify Appropriate High‐Affinity Ligands

  • Anti‐MHC monoclonal antibody (see first annotation to step 9): 30 µg/ml in 0.1 M Tris⋅Cl, pH 8.0 ( appendix 2A; the same antibodies used for the affinity columns for MHC purification are used in the capture assay)
  • Blocking solution: 0.3% (v/v) Tween 20 in PBS or 1% (w/v) BSA in PBS
  • 96‐well round‐bottom polystyrene microtiter plate (Greiner‐bio‐one, cat. no. 650201,
  • Mylar plate sealer with adhesive back (MP Biomedicals, LLC, cat. no. 76‐402‐05)
  • View Seal (Greiner‐bio‐one, cat. no. 676070,
  • Costar Sealing Mat (Corning, cat. no. 3080)
  • 96‐well flat‐bottom white polystyrene Optiplate (Greiner‐bio‐one, cat. no. 655074,
  • TopSeal‐A for 96‐well microplates (PerkinElmer, cat. no. 6005185)
  • Microscint‐20 (PerkinElmer; Cat. #6013621)
  • Topcount microscintillation counter (Perkin‐Elmer Instruments)

Support Protocol 3: Separation of MHC‐Peptide Complexes by Size‐Exclusion Gel‐Filtration Chromatography

  • Protein A–Sepharose CL4 B (Sigma, cat. no. P‐3391)
  • Sepharose CL4 B (Sigma, cat. no. CL4B‐200; for use in uncoupled pre‐columns)
  • 100 mM borate buffer, pH 8.2: dissolve 6.18 g boric acid (Sigma, cat no. B‐7660)/9.54 g borax (Sigma, cat. no. B‐0127)/4.38 g NaCl in H 2O
  • Monoclonal antibody (approximately 20 to 30 mg; see annotation to step 3) in PBS, pH 7.2 (see below) at a concentration of about 2 mg/ml or higher
  • PBS, pH 7.2: 20 mM Na 2HPO 4/150 mM NaCl/0.05% NaN 3
  • 200 mM triethanolamine, pH 8.2 (Sigma, cat. no. T‐1377)
  • 20 mM dimethyl pimelimidate (DMP; Pierce, cat. no. 21667) in 200 mM triethanolamine, pH 8.2
  • Phosphate‐buffered saline (PBS, Invitrogen, cat. no. 10010‐023) containing 0.02% sodium azide (NaN 3;Fisher Scientific, cat. no. S227‐500)
  • 20 mM ethanolamine pH 8.2 (Sigma, cat. no. E‐9508)
  • 0.02% sodium azide (NaN 3) (Fisher Scientific, cat. no. S227‐500) in PBS, pH 7.2 (Invitrogen, cat. no. 10010‐023)
  • Elution buffer (see recipe)
  • 2 M glycine, pH 2.5
  • Washing buffer: 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A) with 1% Nonidet P‐40 (store up to 6 months at 4°C)
  • 50‐ml borosilicate glass column with stopcock
  • Rotator
  • Spectrophotometer
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Key References
   Buchli et al., 2004, 2005. See above.
  Two papers from William Hildebrand's group demonstrating the use of fluorescence polarization techniques and soluble MHC for developing sensitive binding assays. Willie's group has done pioneering work in developing expression systems for the production of soluble MHC molecules.
   Harndahl et al. 2009, 2011. See above
  Soren Buus has been a leader for over two decades in the MHC binding field, and the steady stream of excellent work from his laboratory is always creative, interesting and definitely relevant to the present context. His work covers a number of novel assay approaches, as well as related bioinformatics‐based tools.
   Justesen et al. 2009. See above.
  A well‐written review of antigen processing, which gives an appropriate backdrop for understanding MHC‐peptide binding.
   Germain and Margulies, 1993. See above.
  A recent effort towards the classification of HLA Class II binding specificities into supertypes, defining sets of molecules with shared or largely overlapping repertoires.
   Greenbaum et al., 2011. See above.
  Very detailed and clearly presented review of the structural aspects of MHC function.
   Madden, 1995. See above.
  Very detailed review of peptide binding to MHC Class II.
   McFarland and Beeson, 2002. See above.
  A large listing of MHC‐peptide binding motifs, mostly as defined using MHC‐peptide elution methodology, is available at the web site described in these references.
   Rammensee et al., 1995, 1999. See above.
  A recent analysis and compilation of HLA Class I binding specificities to define supertypes describing sets of molecules with shared or largely overlapping repertoires.
   Sidney et al., 2008b. See above.
Internet Resources
  The ImMunoGeneTics Database: The IMGT/HLA Database, initiated by Marie‐Paule Lefranc, provides a specialist database for sequences of the human major histocompatibility complex (HLA) and includes the official sequences for the WHO Nomenclature Committee For Factors of the HLA System. The IMGT/HLA Database is part of the international ImMunoGeneTics project (IMGT), and is hosted by the European Bioinformatics Institute (EBI).
  Kenneth Parker provides predictions for peptide binding to a number of different HLA Class I alleles based on pioneering matrices published in 1994.
  SYFPEITHI is a pioneering database developed under the direction of Hans‐Georg Rammensee. It comprises more than 7000 peptide sequences known to bind Class I and Class II MHC molecules. The entries are compiled from published reports only.
  This is the Web site for the American Society of Histocompatibility and Immunogenetics.
  The Immune Epitope Database and Analysis Resource (IEDB) contains data related to antibody and T cell epitopes for humans, nonhuman primates, rodents, and other animal species. Curation of peptidic and nonpeptidic epitope data relating to all infectious diseases (including NIAID Category A, B, and C priority pathogens and NIAID Emerging and Re‐emerging infectious diseases), allergens, autoimmune diseases, and transplant/alloantigens is current and constantly being updated. The database also contains MHC binding data from a variety of different antigenic sources and immune epitope data from the FIMM (Brusic), HLA Ligand (Hildebrand), TopBank (Sette), and MHC binding (Buus) databases.
  The NetMHC 3.2 server, developed by Morten Nielsen, Soren Buus, and co‐workers, and hosted by the Center for Biological Sequence Analysis at the Technical University of Denmark, predicts binding of peptides to a number of different HLA alleles using artificial neural networks (ANNs) and weight matrices. Also available is a graphic MHC‐peptide motif viewer.
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