Development of Escherichia coli Asparaginase II for Immunosensing: A Trade-Off between Receptor Density and Sensing Efficiency

摘要

The clinical success of Escherichia colil-asparaginase II (EcAII) as a front line chemotherapeutic agent for acute lymphoblastic leukemia (ALL) is often compromised because of its silent inactivation by neutralizing antibodies. Timely detection of silent immune response can rely on immobilizing EcAII, to capture and detect anti-EcAII antibodies. Having recently reported the use of a portable surface plasmon resonance (SPR) sensing device to detect anti-EcAII antibodies in undiluted serum from children undergoing therapy for ALL (Aubé et al., ACS Sensors>2016, 1 (11), 1358–1365), here we investigate the impact of the quaternary structure and the mode of immobilization of EcAII onto low-fouling SPR sensor chips on the sensitivity and reproducibility of immunosensing. We show that the native tetrameric structure of EcAII, while being essential for activity, is not required for antibody recognition because monomeric EcAII is equally antigenic. By modulating the mode of immobilization, we observed that low-density surface coverage obtained upon covalent immobilization allowed eachtetrameric EcAII to bind up to two antibody molecules, whereas high-densitysurface coverage arising from metal chelation by N- or C-terminalhistidine-tag reduced the sensing efficiency to less than one antibodymolecule per tetramer. Nonetheless, immobilization of EcAII by metalchelation procured up to 10-fold greater surface coverage, thus resultingin increased SPR sensitivity and allowing reliable detection of loweranalyte concentrations. Importantly, only metal chelation achievedhighly reproducible immobilization of EcAII, providing the sensingreproducibility that is required for plasmonic sensing in clinicalsamples. This report sheds light on the impact of multiple factorsthat need to be considered to optimize the practical applicationsof plasmonic sensors.