Recombinant Antibodies – Tools for building proteomic platforms

Sammanfattning: This thesis, which is based on four original papers, covers different aspects of the evolvement and development of proteomic platforms and the construction of human synthetic antibody libraries for the isolation of antibody reagents has been very central. There is an accelerating need for technologies capable of providing proteomic analysis of high sensitivity with good accuracy and an overall broad coverage as well as for biomarker validation. Paper I, focused on the design and construction of human synthetic antibody libraries for the isolation of scFv antibodies suitable for the use in different affinity proteomic platforms. High-throughput procedures for the isolation as well as screening of antibodies was established and provides a pipeline capable of selecting antibodies against a large number of target antigens in parallel. Furthermore, scFv antibodies generated from these antibody libraries were validated for the use in antibody microarrays and were also utilized in various affinity-based proteomic platforms. In paper II and III, two different approaches are taken to address problems associated with proteomic analysis. Technologies capable of biomarker validation are highly required for increasing the implementation of protein biomarkers in clinical use. A method combining affinity enrichment with the use of scFv antibodies and mass spectrometric read-out were developed in paper II. The platform allows multiplexed detection of low abundant target proteins in human serum samples. Paper III focused on establishing a platform to facilitate the detection and investigation of bacterial virulence factors by using Streptococcus pyogenes as a model system. Bacterial infections are still a serious health problem around the world and analysis exploring bacterial strategies promoting survival and proliferation within the human host are of great importance. The performance of an affinity-based platform is dependent on the functionality of the used affinity reagents. In paper IV, a semi-targeted platform for biomarker discovery were enhanced by taking advantage of an oriented antibody immobilization strategy. In conclusion, this thesis presents a pipeline for development of human scFv antibodies suitable for proteomic analysis as well as the establishment and evolvement of various proteomic platforms.