title: Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay.
abstract: The proper orientation and lateral spacing of antibody molecules are a crucial element for an on-chip immunoassay in which the antibody or its antigen-binding fragments are immobilized on a solid surface. We covalently immobilized a modified protein G (Cys-protein G: protein G with only an N-terminal cysteine) on a dendron-coated surface to control its orientation and lateral spacing simultaneously. The cysteine-specific immobilization of Cys-protein G through the N-terminal cysteine resulted in 2.2-fold higher binding efficiency of Cys-protein G to IgG(2a) capture antibody than its random immobilization via lysine residues. The lateral spacing of 3.2 nm due to the surface modification with the 9-acid dendron molecule contributed to a 1.5-fold increase in the antibody-binding ability of Cys-protein G. Topographic images of atomic force microscopy exhibited a uniform coverage of Cys-protein G molecules immobilized on the thiol-reactive 9-acid dendron surface and homogeneous distribution of antibody bound to Cys-protein G. In the sandwich immunoassay, the control of the orientation of Cys-protein G led to 10-fold higher detection capability for rIL-2 compared with the randomly oriented protein G. The synergistic advantage of the unidirectional orientation and homogeneous lateral spacing of Cys-protein Gs on the dendron-coated surface can be applied to the development of more sensitive and reproducible antibody microarrays.
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, National Core Research Center for Systems Bio-Dynamics, Pohang University of Science and Technology, San 31 Hyoja-dong, Pohang, 790-784, South Korea.
authors.forename: Eung-Sam
authors.initials: ES
authors.lastname: Kim
authors.forename: Chang-Kyoon
authors.initials: CK
authors.lastname: Shim
authors.forename: Jae W
authors.initials: JW
authors.lastname: Lee
authors.forename: Joon Won
authors.initials: JW
authors.lastname: Park
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
mesh_terms: D000906:Antibodies / Q000276:immunology; D003545:Cysteine / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007118:Immunoassay; D007376:Interleukin-2 / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D000906:Antibodies; C029152:G-substrate; D055767:Immobilized Proteins; D007376:Interleukin-2; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D003545:Cysteine