Multiplexed Analysis of Peptide Antigen‐Specific Antibodies

James E. Drummond1

1 Merck Research Laboratories, West Point, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.10
DOI:  10.1002/0471142956.cy1310s48
Online Posting Date:  April, 2009
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Many experiments require the simultaneous measurement of more than one parameter at a time. These protocols describe a multiplex method that can be used to detect two or more analytes simultaneously in the same sample preparation. Our example detects antibodies directed against peptide antigens but is amenable to most typical EIA format configurations to allow measurement of alternative analytes. We chose to use the LumAvidin bead‐based assay system due to the simplicity of coupling synthesized biotin‐peptides to the spectrally distinct bead types. The coupled beads were stored at 4°C. Using unlabeled beads as a negative control posed a significant risk of antigen migration from other labeled beads in the multiplex format. Therefore, labeled beads should be mixed immediately prior to performing the assay. Peptide‐specific units were assigned to a polyclonal standard control allowing for quantitation of sample responses. The assay has proven to be quite robust. Curr. Protocol. Cytom. 48:13.10.1‐13.10.7. © 2009 by John Wiley & Sons, Inc.

Keywords: multiplex; biotinylated peptides; LumAvidin; fluorescent microspheres

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Peptide‐Coated Microspheres
  • Basic Protocol 2: Analysis of Peptide Antigen–Specific Antibodies
  • Alternate Protocol 1: Antibody Specificity Confirmation (Competition)
  • Commentary
  • Literature Cited
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Basic Protocol 1: Preparation of Peptide‐Coated Microspheres

  • Appropriate solvent
  • Lyophilized biotinylated peptides
  • xMAP LumAvidin polystyrene fluorescent microsphere populations (5.6‐µm diameter; Luminex Corporation; store at 4°C in the dark)
  • Coupling buffer (CB): calcium‐ and magnesium‐free phosphate‐buffered saline (PBS; appendix 2A)/0.05% Tween‐20/0.05% sodium azide (make from 5% sodium azide solution, in calcium‐ and magnesium‐free PBS; 100× stock; store at room temperature); store buffer at 4°C
  • Isoton II diluent (Beckman Coulter)
  • Single‐use tubes (small volume to minimize dead air space)
  • Vortex
  • 1.5‐ml microcentrifuge tubes
  • Benchtop microcentrifuge
  • Hemacytometer or Z1 Series Coulter Counter (Beckman Coulter)

Basic Protocol 2: Analysis of Peptide Antigen–Specific Antibodies

  • Serum samples
  • Run buffer (RB): calcium‐ and magnesium‐free PBS/0.05% Tween‐20/10% normal goat serum (Sigma; store at −20°C)
  • Wash buffer (WB): calcium‐ and magnesium‐free PBS/0.05% Tween‐20 (store buffer at 4°C)
  • Peptide‐coupled LumAvidin bead (see protocol 1)
  • Anti‐human IgG‐phycoerythrin conjugate (store per manufacturer's instructions)
  • Standard control serum
  • 96‐well filter‐bottom plates
  • Filter‐well plate vacuum manifold
  • Thermomixer (Eppendorf)
  • Bio‐Plex instrument and software (Bio‐Rad Laboratories)

Alternate Protocol 1: Antibody Specificity Confirmation (Competition)

  • Outer membrane protein complex (peptide‐labeled OMPC‐A/M2 and OMPC‐B/HA 0; Marburg, ) or other inert support
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Literature Cited

Literature Cited
   Bianchi, E., Liang, X., Ingallinella, P., Finotto, M., Chastain, M.A., Fan, J., Fu, T.M., Song, H.C., Horton, M.S., Freed, D.C., Manger, W., Wen, E., Shi, L., Ionescu, R., Price, C., Wenger, M., Emini, E.A., Cortese, R., Ciliberto, G., Shiver, J.W., and Pessi, A. 2005. Universal influenza B vaccine based on the maturational cleavage site of the hemagglutinin precursor. J. Virol. 79:7380‐7388.
   Dias, D., Van Doren, J., Schlottmann, S., Kelly, S., Puchalski, D., Ruiz, W., Boerckel, P., Kessler, J., Antonello, J.M., Green, T., Brown, M., Smith, J., Chirmule, N., Barr, E., Jansen, K.U., and Esser, M.T. 2005. Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papilloma viruses 6, 11, 16, and 18. Clin. Diagn. Lab. Immunol. 12:959‐969.
   Drummond, J.E., Shaw, E.E., Antonello, J.M., Green, T., Page, G.J., Motley, C.O., Wilson, K.A., Finnefrock, A.C., Liang, X., and Casimiro, D.R. 2008. Design and optimization of a multiplex anti‐influenza peptide immunoassay. J. Immunol. Methods 334:11‐20.
   Fan, J., Liang, X., Horton, M.S., Perry, H.C., Citron, M.P., Heidecker, G.J., Fu, T.M., Joyce, J., Przysiecki, C.T., Keller, P.M., Garsky, V.M., Ionescu, R., Rippeon, Y., Shi, L., Chastain, M.A., Condra, J.H., Davies, M.E., Liao, J., Emini, E.A., and Shiver, J.W. 2004. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine 22:2993‐3003.
   Marburg, S.J.D., Tolman, R.L., Arison, B., McCauley, J., Kniskern, P.J., Hagoplan, A., and Vella, P.P. 1986. Bimolecular chemistry of macromolecules: Synthesis of bacterial polysaccharide conjugates with Neisseria meningitidis membrane protein. J. Am. Chem. Soc. 108:5282‐5287.
   O'Connell, M., Belanger, B.A., and Haaland, P.D. 1992. The four parameter logistic model for calibration and assay development. In Proceedings of the Biopharmaceutical Section pp. 180‐185. American Statistical Association, Washington D.C.
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