title: AFM imaging of ALYGNSA polymer-protein surfaces: evidence of antibody orientation.
abstract: Previous investigations found the combination of recombinant bacterial protein G (rProG) and poly(methyl methacrylate) (PMMA) to produce a greater proportion of oriented antibodies. PMMA-rProG yielded a sixfold greater availability of antibody Fab regions compared with other bacterial affinity linker protein and polymer pairings, including commercially available polystyrene (PS) high-binding 96-well microplates. Given the name ALYGNSA, the PMMA-rProG combination was developed into a fluorescence assay and evaluated in conjunction with commercially available cancer biomarker enzyme-linked immunosorbent assays (ELISAs). In each study, a lower limit of detection was seen with the ALYGNSA assay. The purpose of this investigation was to examine the ALYGNSA substrate in contrast with a commonly used ELISA substrate and analyze the affinity-immobilized antibodies for additional evidence of orientation. Non-contact atomic force microscopy is a logical method as it operates in ambient conditions, can be used directly on biological samples without modification, and offers the resolution necessary to identify the position of the antibody on the surface. Dynamic contact angle studies were employed to examine untreated PMMA and PS samples and revealed important differences in their surface characters. Comparative height threshold grain analysis of the prepared ALYGNSA surface, a similarly treated mica surface, and a gold colloid sizing standard evaluated and confirmed the antibody orientation of the ALYGNSA system.
authors: Department of Chemistry, College of Sciences, University of Massachusetts Lowell, Lowell, MA 01854, USA.
authors: Leslie R
authors: LR
authors: Farris
authors: Melisenda J
authors: MJ
authors: McDonald
mesh_terms: D055910:Antibodies, Immobilized / Q000032:analysis / Q000737:chemistry / Q000648:ultrastructure*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005453:Fluorescence; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry* / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000737:chemistry / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D009369:Neoplasms / Q000175:diagnosis / Q000276:immunology; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D011485:Protein Binding; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D001426:Bacterial Proteins; D014408:Biomarkers, Tumor; C034284:IgG Fc-binding protein, Streptococcus; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D011137:Polystyrenes; D019904:Polymethyl Methacrylate