title: Selection of phage antibodies with GPX activity by combination of phage displayed antibody library with chemical modification and their characterization using a surface plasmon resonance biosensor.
abstract: Glutathione peroxidase (GPX) is an important antioxidant enzyme, which plays an important role in scavenging reactive oxygen species. To obtain humanized GPX catalytic antibodies, the phage displayed human antibody library on the surface of the filamentous bacteriophage was used to select novel antibodies by repetitive screening. Phage antibodies B8, H6 and C1 with the GSH-binding site were obtained from the library by enzyme-linked immunosorbent assay (ELISA) analysis with four rounds of selection against three haptens, S-2,4-dinitrophenyl t-butyl ester [GSH-S-DNP-Bu (B)], S-2,4-dinitrophenyl t-hexyl ester [GSH-S-DNP-He (H)] and S-2,4-dinitrophenyl cycle-hexyl ester [GSH-S-DNP-cHe (C)], and characterized using surface plasmon resonance (SPR) biosensor. The gold layer was modified by dithiodiglycolic acid (DDA) and three haptens were easily attached to DDA by self-assembling to form a biosensor membrane. The membrane bounds specifically corresponding antibodies. The kinetic process of the reaction between phage antibodies and their haptens was studied by SPR biosensor. In order to improve selectivity, chemical modification was used to incorporate directly catalytic group selenocysteine (Sec) into selected phage clone B8, H6 and C1 to form Se-B8, Se-H6 and Se-C1, respectively. The GPX activities of Se-B8, Se-H6 and Se-C1 were found to be 3000, 2000 and 700units/mumol, respectively. Compared with conventional ELISA analysis, the proposed method based on SPR biosensor is much more rapid and simpler.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, PR China; College of Chemistry, Jilin University, Changchun, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Mu
authors.forename: Daqian
authors.initials: D
authors.lastname: Song
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Han-Qi
authors.initials: HQ
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Gui-Min
authors.initials: GM
authors.lastname: Luo
authors.forename: Qin-Han
authors.initials: QH
authors.lastname: Jin