TY  - JOUR
A1  - Schmidt, Carsten
A1  - Roediger, Stefan
A1  - Gruner, Melanie
A1  - Moncsek, Anja
A1  - Stohwasser, Ralf
A1  - Hanack, Katja
A1  - Schierack, Peter
A1  - Schroeder, Christian
T1  - Multiplex localization of sequential peptide epitopes by use of a planar microbead chip
JF  - Analytica chimica acta : an international journal devoted to all branches of analytical chemistry
N2  - Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28 gamma (proteasome activator 28 gamma) polyclonal serum, for a murine monoclonal antibody against PA28 gamma, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks. (C) 2016 Elsevier B.V. All rights reserved.
KW  - Epitope mapping
KW  - In vivo biotinylation
KW  - Multiplexed assays
KW  - Microbeads
KW  - VideoScan technology
Y1  - 2016
U6  - https://doi.org/10.1016/j.aca.2015.12.030
SN  - 0003-2670
SN  - 1873-4324
VL  - 908
SP  - 150
EP  - 160
PB  - Elsevier
CY  - Amsterdam
ER  -