Oriented Immobilization of Anti-Pneumolysin Tagged Recombinant Antibody Fragments |
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| Authors: | Maria del Mar Garcia-Suarez Roberto Villaverde Irene Gonzalez-Rodriguez Fernando Vazquez Francisco J Mendez |
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| Institution: | (1) Area de Microbiologia, Departamento de Biologia Funcional, Facultad de Medicina, Universidad de Oviedo, Julian Claveria 6, 33006 Oviedo, Asturias, Spain |
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| Abstract: | Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved
through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab
and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added
in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity
for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the
position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random
and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates.
The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels
of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve
the ELISA sensitivity. |
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