Establishment of a phage display platform for the isolation of plasmodium falciparum specific monoclonal antibodies
Aachen / Publikationsserver der RWTH Aachen University (2015) [Dissertation / PhD Thesis]
Page(s): VI, 96 S. : Ill., graph. Darst.
Malaria still remains an important cause of morbidity and mortality in many parts of the world. Despite an enormous research effort, the goal of finding an effective vaccine still remains elusive. The antibody generation part of the Fraunhofer Foundation Project aims amongst other things to isolate human antibodies by antibody phage display from semi-immune individuals that might be used for short term protection or therapy. Human plasma samples were tested in an ELISA for their reactivity to MSP1-19, MSP3, AMA1 and crude plasmodium lysate and the responses scored. Purified IgG from these samples were then tested in vitro for direct invasion inhibition. From the highest responders, through a 2-step cloning strategy, an antibody Fab library of functional size (1.4x 107) was constructed and a subtractive panning strategy applied. The selected antibodies were expressed in Nicotiana benthamiana based on a protocol for rapid expression and purified through an affinity chromatography. A monoclonal Fab antibody that selectively binds to AMA1 was isolated and processed into full sized antibody. Functionality was assessed by surface plasmon resonance, reducing/non-reducing SDS PAGE, Western Blot, ELISA and growth inhibition assay. Analysis of the plasma immune responses revealed a significant correlation between responses to AMA1 and MSP3 and invasion inhibition. The recombinant antibody isolated from the Fab library specifically binds to the antigen AMA1 and also strongly inhibits the parasite invasion directly. The results also proved the applicability of the antibody phage display methodology for the isolation of functional recombinant human antibodies and thus a platform has been established that should allow for the isolation of antibodies against other Malaria blood stage antigens.