Mayer, Nikola, Beuvink, Iwan and Traggiai, Elisabetta (2014) EVALUATING SINGLE CELL PCR TO DISSECT THE HUMAN B CELL REPERTOIRE. master thesis.

Abstract

This thesis describes an improved single cell Polymerase Chain Reaction method able to amplify and express human monoclonal antibodies in a high-throughput manner. The technologies described so far in literature display critical experimental limits, namely low immunoglobulin gene coverage and more importantly the very restricted number of functional antibodies tested out of a numeric complex repertoire. Based on immunoglobulin and specific surface markers transitional, memory and naive B cells from peripheral blood of healthy donors were single cell sorted in 96 and 384 well plates using fluorescence-activated cell sorting and the performance of the single cell PCR assay was tested. Improvement in high-throughput cloning was achieved by implementing overlap extension PCR. Finally, the functionality of the single cell PCR method was tested by expressing cloned antibodies in eukaryotic cell lines and confirming their antigen binding by ELISA.

Item Type:	Article
Keywords:	single cell PCR B-cell Overlap Extension PCR antibody High-throughput cloning human
Date Deposited:	26 Apr 2016 23:46
Last Modified:	26 Apr 2016 23:46
URI:	https://oak.novartis.com/id/eprint/21212