Competition‐Based Cellular Peptide Binding Assay for HLA Class I

Jan H. Kessler1, Willemien E. Benckhuijsen1, Tuna Mutis1, Cornelis J.M. Melief1, Sjoerd H. van der Burg1, Jan W. Drijfhout1

1 Leiden University Medical Center, Leiden
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.12
DOI:  10.1002/0471142735.im1812s61
Online Posting Date:  September, 2004
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This unit describes a competition assay to determine binding of unlabeled test peptides to thirteen of the most prevalent HLA class I molecules. It uses cells expressing the HLA class I molecule of interest on their surface, fluorescently labeled reference peptides, and unlabeled test peptides. Cells of interest are stripped from their natural HLA‐bound peptides using acid treatment and subsequently incubated with a mixture of labeled reference peptide and titrating concentrations of test peptide. Subsequently, FACS analysis is performed to determine the amount of bound reference peptide, which is a measure of the ability of test peptide to compete for binding to HLA. The assay provides IC50 values for binding of test peptides to HLA molecules. It can be performed in a normally equipped cellular laboratory, requires no additional equipment besides a flow cytometer (FACS), and is relatively easy to perform. Assay‐specific parameters for several HLA alleles are provided.

Keywords: HLA; peptide binding; synthetic peptide; FACS analysis; fluorescence; cellular assay

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Competition‐Based Cellular Peptide Binding Assay for HLA Class I
  • Support Protocol 1: Synthesis, Analysis, and Quantification of Fluorescent Reference Peptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Competition‐Based Cellular Peptide Binding Assay for HLA Class I

  • Test peptide in PBS ( appendix 2A)
  • Positive‐control peptides in PBS (Table 18.12.2)
  • PBS ( appendix 2A)
  • Fluorescent reference (Fl) peptide in PBS (see protocol 2 and Strategic Planning)
  • Cells (see Strategic Planning and Table 18.12.1) in flasks
  • Elution buffer (see recipe)
  • IMDM‐2 ( appendix 2A) with and without 2 µg/ml β 2M (see recipe)
  • Ice‐cold PBS ( appendix 2A) containing 0.5% (w/v) BSA
  • PBS ( appendix 2A) containing 1% (w/v) paraformaldehyde (PFA)
  • 96‐well V‐bottom microtiter plates
  • Multichannel pipettor
  • 15‐ml centrifuge tubes
  • Centrifuge with rotors for tubes and microtiter plates
  • Reagent reservoir
  • FACScan flow cytometer (or equivalent instrument) and appropriate tubes
  • Software for nonlinear regression analysis (e.g., CurveExpert 1.3, SPSS Science Software)
  • Additional reagents and equipment for counting cells ( appendix 3A)
    Table 8.2.2   Materials   Positive‐Control Peptides c and Their IC 50 Values   Positive‐Control Peptides and Their IC 50 Values

    HLA class I allele d Peptide sequence Origin IC 50 (µM) Reference
    A1 (A*0101) YLEPAIAKY Consensus sequence 0.2 Sette et al. ( )
    A2 (A*0201) FLPSDFFPSV HBV cAg 18‐27 0.5 Bertoletti et al. ( )
    YIGEVLVSV mHag HA‐2 3.5 den Haan et al. ( )
    A3 (A*0301) KVFPCALINK Consensus sequence 0.7 Sette et al. ( )
    QVPLRPMTYK HIV‐1nef 73‐82 0.2 Koenig et al. ( )
    KQSSKALQR BCR‐ABL b3a2 2.2 Bocchia et al. ( )
    A11 (A*1101) QVPLRPMTYK HIV‐1nef 73‐82 2.0 Koenig et al. ( )
    KQSSKALQR BCR‐ABL b3a2 6.7 Bocchia et al. ( )
    A24 (A*2402) RYLKDQQLL HIV‐1env gp41 583‐591 1.8 Dai et al. ( )
    AYIDNYNKF Consensus sequence 0.6 Kast et al. ( )
    A68 (A*6801) KTGGPIYKR Influenza A NP 91‐99 1.3 Guo et al. ( )
    B7 (B*0702) APAPAPSWPL Human p53 84‐93 0.5 Not published
    SPSVDKARAEL Human SMCY 950‐960 0.7 Wang et al. ( )
    B8 (B*0801) FLRGRAYGL EBNA‐3 339‐347 0.2 Burrows et al. ( )
    GFKQSSKAL BCR‐ABL b3a2 fusion region 1.5 Bocchia et al. ( )
    B14 (B*1402) ERYLKDQQL HIV‐1env gp41 584‐592 7.5 Johnson et al. ( )
    B35 (B*3501) NPDIVIYQY HIV‐1 RT 330‐338 1.2 Sipsas et al. ( )
    B60 (B*4001) KESTLHLVL Ubiquitin 63‐71 1.9 Falk et al. ( )
    B61 (B*4002) GEFGGFGSV Histone acetyltransferase 127‐135 0.2 Falk et al. ( )
    GEFVDLYV 40S ribosomal protein S21 6‐13 0.3 Falk et al. ( )
    B62 (B*1501) YLGEFSITY 40S ribosomal protein S15 114‐122 0.6 Falk et al. ( )

     cA positive‐control peptide is defined here as one which exhibits good binding for the particular HLA molecule being investigated.
     dRefer to appendix 1D for nomenclature.

Support Protocol 1: Synthesis, Analysis, and Quantification of Fluorescent Reference Peptides

  • Sephadex G‐10
  • 3:7 and 9:1 acetic acid/water
  • 1.0 M Tris·Cl, pH 9.5 ( appendix 2A; store at 4°C)
  • Acetonitrile
  • Synthetic reference peptide containing Cys at the position where a Cys(Fl) is required (Table 18.12.1 and Table 18.12.4)
  • 5‐(iodoacetamido)fluorescein (Fluka Chemie AG or Molecular Probes)
  • Acetic acid
  • Mobile phase for reversed‐phase HPLC
  • 200 mM sodium phosphate buffer, pH 7.5 ( appendix 2A)
  • ∼1.7 × 12–cm disposable column (e.g., Bio‐Rad Econo‐Pac)
  • Additional reagents and equipment for reversed‐phase HPLC (Henzel and Stults, ) and mass spectroscopy (Coligan et al., )
    Table 8.2.4   MaterialsMolecular Masses of Fluorescent Reference Peptides

    HLA class I allele f Fl peptide g Expected mass (monoisotopic) Expected mass (average)
    A1 (A*0101) YLEPAXAKY 1443.569 1444.57
    A2 (A*0201) FLPSDXFPSV 1497.579 1498.62
    A3 (A*0301) KVFPXALINK 1518.721 1519.77
    A11 (A*1101) KVFPXALINK 1518.721 1519.77
    A24 (A*2402) RYLKXQQLL 1550.722 1551.78
    A68 (A*6801) KTGGPIXKR 1345.612 1346.52
    B7 (B*0702) APAPAPXWPL 1408.579 1409.58
    B8 (B*0801) FLRGRAXGL 1378.612 1379.55
    B14 (B*1402) DRYIHAXLL 1489.633 1490.65
    B35 (B*3501) NPDIVXYQY 1500.554 1501.58
    B60 (B*4001) KESTXHLVL 1415.606 1416.56
    B61 (B*4002) GEFGGXGSV 1198.391 1199.21
    B62 (B*1501) YLGEFSXTY 1468.516 1469.54

     fRefer to appendix 1S for nomenclature.
     gX is the Cys(Fl) moiety.
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Literature Cited

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Internet Resources
  Information on the availability and ordering of the B cell lines can be obtained via the home page of the International Histocompatibility Workshop.
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