title: Surface immobilized antibody orientation determined using ToF-SIMS and multivariate analysis.
abstract: Antibody orientation at solid phase interfaces plays a critical role in the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture as compared to their randomly oriented counterparts. Direct characterization of oriented proteins with surface analysis methods still remains a challenge however surface sensitive techniques such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody orientation. Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. Herein, ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr are used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) test against each of the models indicated preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab')2region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. Mass fragments were then compared to amino acid fragments and amino acid composition in F(ab')2and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab')2fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp. The systematic data analysis methodology employed herein offers a new approach for the investigation of antibody orientation applicable to a range of substrates.
  
  
  
  Controlled orientation of antibodies at solid phases is critical for maximizing antigen detection in biosensors and immunoassays. Surface-sensitive techniques (such as ToF-SIMS), capable of direct characterization of surface immobilized and oriented antibodies, are under-utilized in current practice. Selection of a small number of mass fragments for analysis, typically pertaining to amino acids, is commonplace in literature, leaving the majority of the information-rich spectra unanalyzed. The novelty of this work is the utilization of a comprehensive, unbiased mass fragment list and the employment of principal component analysis (PCA) and artificial neural network (ANN) models in a unique methodology to prove antibody orientation. This methodology is of significant and broad interest to the scientific community as it is applicable to a range of substrates and allows for direct, label-free characterization of surface bound proteins.
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, VIC 3086, Australia; CSIRO Manufacturing, VIC 3168, Australia.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Welch
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, VIC 3086, Australia; CSIRO Manufacturing, VIC 3168, Australia.
authors.forename: Robert M T
authors.initials: RMT
authors.lastname: Madiona
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, VIC 3086, Australia.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Payten
authors.affiliation: CSIRO Manufacturing, VIC 3168, Australia.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Easton
authors.affiliation: CSIRO Manufacturing, VIC 3168, Australia.
authors.forename: Luisa
authors.initials: L
authors.lastname: Pontes-Braz
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, VIC 3086, Australia.
authors.forename: Narelle
authors.initials: N
authors.lastname: Brack
authors.affiliation: CSIRO Manufacturing, VIC 3168, Australia.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Scoble
authors.affiliation: CSIRO Manufacturing, VIC 3168, Australia.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Muir
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, VIC 3086, Australia. Electronic address: p.pigram@latrobe.edu.au.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Pigram
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry*; D002857:Chromium / Q000737:chemistry; D005026:Ethylene Glycols / Q000737:chemistry*; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D055910:Antibodies, Immobilized; D005026:Ethylene Glycols; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D002857:Chromium; C013484:diethylene glycol
keywords: Antibody fragments; Antibody orientation; Artificial neural networks; Chromium (III) complex; Principal component analysis; ToF-SIMS