Binding of Biotinylated Peptides to MHC Class II Proteins on Cell Surfaces

Jonathan B. Rothbard1, Robert Busch1

1 Stanford University Medical Center, Stanford, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.1
DOI:  10.1002/0471142735.im1801s25
Online Posting Date:  May, 2001
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Abstract

This unit describes a simple, reproducible, and semiquantitative assay for measuring ligand binding to cell surface receptors. This approach is useful as a quick screen for peptide binding to MHC class II proteins that avoids the need to purify the MHC class II molecules and that uses small numbers of cells. The basic protocol describes procedures for incubating cells expressing the MHC molecule of interest with a biotinylated peptide, washing off excess peptide, and detecting the bound peptides by staining with fluorescently labeled avidin. Bound fluorescence is then quantitated by flow cytometry. An alternate protocol provides a more sensitive method of measuring the peptide‐receptor complexes, and is useful when receptor density or binding site availability become limiting factors in the basic protocol. The alternate protocol uses an avidin/anti‐avidin/avidin sandwich, which has been found to increase sensitivity substantially without sacrificing specificity. Support protocols are provided for biotinylation of synthetic peptides in solution and for preparation of peptide stock solutions for use in the binding assays.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Simple Assay for Biotinylated Peptide Binding to MHC Class II Molecules on EBV‐Transformed B‐Lymphoblastoid Cells using Fluorescent Avidin
  • Alternate Protocol 1: Increasing the Sensitivity of Fluorescent Detection of Cell‐Bound Peptide using a Biotinylated Anti‐Avidin Antibody
  • Support Protocol 1: Biotinylation of Synthetic Peptides
  • Support Protocol 2: Preparation of Peptide Stocks for Cell‐Surface Binding Assays
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Simple Assay for Biotinylated Peptide Binding to MHC Class II Molecules on EBV‐Transformed B‐Lymphoblastoid Cells using Fluorescent Avidin

  Materials
  • Epstein‐Barr‐virus‐transformed B cells (EBV–B cells) expressing the histocompatibility leukocyte antigen DR (HLA‐DR) allele of interest ( appendix 1G)
  • Mutant EBV–B cell line lacking HLA‐DR expression
  • Complete RPMI‐10 ( appendix 2A penicillin/streptomycin optional)
  • recipeDulbecco's PBS (see recipe)
  • Mouse anti‐HLA‐DR monoclonal antibody in recipeDulbecco's PBS (L243, Becton Dickinson Immunocytometry; see recipes for Dulbecco's PBS and for recipestaining solutions)
  • 200 µM unbiotinylated peptide of interest in recipeDulbecco's PBS
  • Metabolic inhibitor in recipeDulbecco's PBS
  • 200 µM biotinylated peptide of interest (see protocol 3Support Protocols 1 and protocol 42)
  • recipeWash buffer (see recipe)
  • Saturated fluorescein isothiocyanate–conjugated avidin (FITC‐avidin) in recipewash buffer (see recipe for recipewash buffer and for recipestaining solutions): e.g., 10 µg/ml FITC–avidin D (Vector Labs) or 4.22 µg/ml FITC‐streptavidin (Calbiochem‐Novabiochem)
  • Saturated FITC‐conjugated goat anti‐mouse‐Ig antibody in recipewash buffer (Pharmingen; see reciperecipes for recipewash buffer and for recipestaining solutions)
  • 5 µg/ml propidium iodide in wash buffer
  • Tabletop centrifuge with inserts for tissue culture tubes and 96‐well plates
  • 96‐round‐bottom‐well plate (low protein binding)
  • Multichannel pipettor
  • Vacuum aspirator fitted with a syringe and needle or with an 8‐well aspirator (Drummond)
  • Additional reagents and equipment for sterile tissue culture ( appendix 2A), cell counting ( appendix 3A), and flow cytometry (see Chapter 5, particularly units 5.2 & 5.3)
CAUTION: Propidium iodide is a DNA intercalator and may be carcinogenic.

Alternate Protocol 1: Increasing the Sensitivity of Fluorescent Detection of Cell‐Bound Peptide using a Biotinylated Anti‐Avidin Antibody

  • recipeDulbecco's PBS (see recipe) containing 10 mM recipeEDTA
  • Trypsin/EDTA (Life Technologies)
  • 10 µg/ml biotinylated anti–avidin D antiserum (Vector Labs) in recipewash buffer

Support Protocol 1: Biotinylation of Synthetic Peptides

  • Peptide of interest
  • 6‐(biotinamido)hexanoyl N‐hydroxysuccinimide ester (long‐chain biotin NHS ester, or LCB‐NHS; store desiccated up to 2 years at −20°C)
  • Dimethylformamide (DMF)
  • 1 mg/ml dimethylaminocinnamaldehyde (DACA) solution (in ethanol containing 1% (v/v) sulfuric acid; store up to 1 year at room temperature)
  • Sonicating water bath
  • 50‐ and 100‐ml quick‐fit round‐bottomed flasks and appropriate lyophilizer adaptors
  • Rotary evaporator
  • Mass spectrometer (optional)
  • Amino acid analyzer (optional)
  • Silica TLC plate (optional)
  • Additional reagents and equipment for analytical and preparative reversed‐phase high‐performance liquid chromatography (reversed‐phase HPLC; unit 9.2).

Support Protocol 2: Preparation of Peptide Stocks for Cell‐Surface Binding Assays

  • Biotinylated peptide (see protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
   Busch, R. and Rothbard, J.B. 1990. Detection of peptide‐MHC class II complexes on the surface of intact cells. J. Immunol. Methods 134:1‐22.
   Busch, R., Strang, G., Howland, K., and Rothbard, J.B. 1990. Degenerate binding of immunogenic peptides to HLA‐DD proteins on B cell surfaces. Int. Immunol. 2:443‐451.
   Busch, R., Hill, C.M., Hayball, J.D., Lamb, J.R., and Rothbard, J.B. 1991. Effect of natural polymorphism at residue 86 of the HLA‐DR β chain on peptide binding. J. Immunol. 147:1292‐1298.
   Busch, R., Vturina, I.Y., Drexler, J., Momburg, F., and Hämmerling, G.J. 1995. Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain. Eur. J. Immunol. 25:48‐53.
   Busch, R., Cloutier, I., Sékaly, R.‐P., and Hämmerling, G.J. 1996. Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum. EMBO J. 15:418‐428.
   Creighton, T.E. 1993. Proteins: Structures and molecular properties. W.H. Freeman, New York.
   Hill, C.M., Hayball, J.D., Allison, A.A., and Rothbard, J.B. 1991. Conformational and structural characteristics of peptides binding to HLA‐DR molecules. J. Immunol. 147:189‐197.
   Jardetzky, T.S., Gorga, J.C., Busch, R., Rothbard, J., Strominger, J.L., and Wiley, D.C. 1990. Peptide binding to HLA‐DR1: A peptide with most residues substituted to alanine retains MHC binding. EMBO J. 9:1797‐1803.
   McCormick, D.B. and Roth, J.A. 1970. Specificity, stereochemistry, and mechanism of the color reaction between p‐dimethylaminocinnamaldehyde and biotin analogues. Anal. Biochem. 34:226‐236.
   Momburg, R., Fuchs, S., Drexler, J., Busch, R., Post, M., Hämmerling, G.J., and Adorini, L. 1993. Epitope‐specific enhancement of antigen presentation by invariant chain. J. Exp. Med. 178:1453‐1458.
   Mozes, E., Dayan, M., Zisman, E., Brocke, S., Licht, A., and Pecht, I. 1989. Direct binding of a myasthenia gravis–related epitope to MHC class II molecules on living murine antigen‐presenting cells. EMBO J. 8:4049‐4052.
   Rothbard, J.B., Busch, R., Bal, V., Trowsdale, J., Lechler, R., and Lamb, J. 1989a. Reversal of HLA restriction by a point mutation in an antigenic peptide. Int. Immunol. 1:487‐495.
   Rothbard, J.B., Busch, R., Howland, K., Bal, V., Fenton, C., Taylor, W.R., and Lamb, J.R. 1989b. Structural analysis of a peptide‐HLA class II complex: Identification of critical interactions for its formation and recognition by T cell receptor. Int. Immunol. 1:479‐486.
Key References
   Busch and Rothbard, 1990. See above.
  This review summarizes and compares cell‐surface peptide binding assays, discusses their advantages and drawbacks, and compares them with assays involving the use of purified proteins. Various uses of the protocols detailed here are illustrated.
   Busch et al., 1990. See above.
  This paper contains the original description of the Basic and Alternate Protocols outlined here for cell‐surface peptide binding assays. It describes specificity controls and dependence of peptide binding on physicochemical parameters.
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