Multiplexed analysis of peptide antigen-specific antibodies.

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