A novel approach for generation of fully human monoclonal antibodies using single cell PCR and in vitro translation

2554 Since the establishment of hybridoma technology, monoclonal antibodies have been playing a pivotal role in basic research, diagnostics and human healthcare. Hybridoma technology and phage display, represent two common approaches for the generation of monoclonal antibodies. Both of these methods comprise some drawbacks. In the hybridoma technology, obtaining of stable clones is a major hurdle, thus diminishing diversity of the antibodies, since only a limited number of B-cells are successfully fused. In the phage display approach, there is the issue of mis- and/or dis-optimal pairings of heavy and light chains, resulting in cross reacting antibodies. A possibility to overcome these drawbacks consists of a method based on the amplification of cognate IgH and IgL chains of an IgG isotype from a single B-cell in combination with in vitro translation. The high sensitivity of this method can be exploited to amplify even minute amounts of mRNA from single cells. This method permits the investigation of the expressed antibody from each B-cell, thus providing higher diversity as opposed to the hybridoma technology. Furthermore, since the cognate antibody chains are obtained from mature B-cells after antigen contact, selectively high specific and accurately paired antibodies can be generated. This confers a high advantage over the generation of unspecific antibodies via phage display.
 In this study, a multiplex semi nested PCR for amplification of cognate IgH and IgL chains from a single CD19 positive human B-cell isolated from human PBMCs was established. The Fab PCR product was subsequently translated in vitro. The expression was examined using ELISA and western blot. The genes encoding the IgH and IgL were amplified by single cell RT-PCR and the combined with regulatory elements for in vitro transcription and translation by overlapping PCR technique. The in vitro translation of such a linear mRNA template has previously been described by Nakano et al. The IgG-specific PCR amplification established in this study, was optimized and modified resulting in an 10 fold increase in measured OD-values in comparison to values reported by Nakano et al.
 To accomplish this, three different PCR-based approaches were established: a two step PCR approach, a nested and a semi nested PCR approach. The nested PCR approach proved to be highly sensitive and resulted in high recovery ratios of the amplified heavy and light chains. However, the obtained Fab products could not be translated in vitro. The two step approach lead to the generation of functional Fab fragments with a plasmid template positive control, but was insensitive with single B-cell templates. On the other hand, the expression of functional and stable Fab fragments was observed after in vitro translation of PCR products obtained via the semi nested approach. With single B-cell templates, production of Fab protein was in the range of 180 to 310 ng/mL.