title: Optimizing antibody immobilization strategies for the construction of protein microarrays.
abstract: Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.
authors.affiliation: Zyomyx, Inc., 26101 Research Road, Hayward, CA 94544, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Peluso
authors.forename: David S
authors.initials: DS
authors.lastname: Wilson
authors.forename: Duc
authors.initials: D
authors.lastname: Do
authors.forename: Huu
authors.initials: H
authors.lastname: Tran
authors.forename: Maanasa
authors.initials: M
authors.lastname: Venkatasubbaiah
authors.forename: David
authors.initials: D
authors.lastname: Quincy
authors.forename: Bettina
authors.initials: B
authors.lastname: Heidecker
authors.forename: Kelli
authors.initials: K
authors.lastname: Poindexter
authors.forename: Neil
authors.initials: N
authors.lastname: Tolani
authors.forename: Michael
authors.initials: M
authors.lastname: Phelan
authors.forename: Krista
authors.initials: K
authors.lastname: Witte
authors.forename: Linda S
authors.initials: LS
authors.lastname: Jung
authors.forename: Peter
authors.initials: P
authors.lastname: Wagner
authors.forename: Steffen
authors.initials: S
authors.lastname: Nock
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D019921:Biotinylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D019809:Streptavidin; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011506:Proteins; D019809:Streptavidin