Unit

UNIT 13.10 Multiplexed Analysis of Peptide Antigen-Specific Antibodies

  1. James E. Drummond

Published Online: 1 APR 2009

DOI: 10.1002/0471142956.cy1310s48

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Drummond, J. E. 2009. Multiplexed Analysis of Peptide Antigen-Specific Antibodies. Current Protocols in Cytometry. 48:13.10:13.10.1–13.10.7.

Author Information

  1. Merck Research Laboratories, West Point, Pennsylvania

Publication History

  1. Published Online: 1 APR 2009
  2. Published Print: APR 2009

Abstract

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