Citation
Liu, Fang; Dubey, Manish; Takahashi, Hironobu; Castner, David G.; & Grainger, David W. (2010).
Immobilized Antibody Orientation Analysis Using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-Generated Patterns.
Anal Chem, 82(7), 2947-2958.
Abstract
This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein A- and fluorescein- copatterned or control surfaces using antibody?ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten- and protein A-patterned regions through antigen?antibody recognition and natural protein A?Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copatterned surfaces generated side-by-side full antibody ?heads-up? and ?tails-up? oriented surface patterns. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in ?heads-up? regions, and ions from amino acids enriched in Fc domains for antibodies oriented in ?tails-up? regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each ?heads-up? versus ?tails-up? patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain- and structure-specific compositional differences by TOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays.
Reference Type
Journal Article
Secondary Title
Anal Chem
Author(s)
Liu, FangDubey, ManishTakahashi, HironobuCastner, David G.Grainger, David W.
Year Published
2010
Date Published
1270080000
Volume Number
82
Issue Number
7
Pages
2947-2958
DOI
10.1021/ac902964q
