Perspectives for systematic in vitro antibody generation

Abstract

After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

“…When trying to match the available funding with the task of generating more than 100,000 reagents, it becomes clear that new solutions and technical optimizations must be found. High-throughput approaches must be adopted at all levels, from protein expression and binder production to multiplexed assays and multiparameter tests 35 , and will be an integral part of any design of a binder-generation pipeline 36 .…”

On the influence of vector design on antibody phage display

“…Second, antibody fragments can be displayed with increased efficiency on virions Rondot et al, 2001). Third, increased numbers of biopanned clones can be tested by robotic high throughput screening (de Wildt et al, 2000;Konthur et al, 2005).…”

Development of Human and Macaque Antibodies Using Antibody Phage Display for the Detection of Equine Encephalitis Viruses

“…Afterwards, these individual binders can be sequenced and further biochemically characterised (Hust et al, 2007a;Winter et al, 1994). This panning process can also be performed in a high-throughput manner (Buckler et al, 2008;Hallborn and Carlsson, 2002;Hust et al, 2011;Konthur et al, 2005). Because the gene sequence of the binder is available, the antibody -depending on the desired application -can be converted into different antibody formats (e.g.…”