A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin-mature bovine prion fusion protein (TrxA-bPrP c ). After five rounds of panning against recombinant bovine prion protein (rb-PrP c ) and ELISA test, two positive clones with high affinity to rb-PrP c , named Z163 and Z186, were obtained. They were conjugated with a linker-streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186-L-SBP/Z163-Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27-30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb-PrP c were 3.24 Â 10 28 M, 8.82 Â 10 28 M, and 8.10 Â 10 29 M for Z186-L-SBP, Z163, and Z163-Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186-L-SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163-Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb-PrP c with detection limit of 1 pg/ml.
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