title: Atomic force microscopy analysis of IgG films at hydrophobic surfaces: a promising method to probe IgG orientations and optimize ELISA tests performance.
abstract: IgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions-the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance.
authors.affiliation: Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, UMR 7564, Villers-lès-Nancy, F-54600, France; CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, UMR 7564, 405 rue de Vandoeuvre, Villers-lès-Nancy, F-54600, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: de Thier
authors.affiliation: Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, UMR 7564, Villers-lès-Nancy, F-54600, France; CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, UMR 7564, 405 rue de Vandoeuvre, Villers-lès-Nancy, F-54600, France.
authors.forename: Jalal
authors.initials: J
authors.lastname: Bacharouche
authors.affiliation: Université de Lorraine, Laboratoire Interdisciplinaire des Environnements Continentaux, UMR 7360, Vandœuvre-lès-Nancy, F-54501, France; CNRS, Laboratoire Interdisciplinaire des Environnements Continentaux, UMR 7360, Vandœuvre-lès-Nancy, F-54501, France.
authors.forename: Jérôme F L
authors.initials: JF
authors.lastname: Duval
authors.affiliation: Université de Lorraine, Laboratoire d'Energétique et de Mécanique Théorique et Appliquée, UMR 7563, Vandœuvre-lès-Nancy, F-54504, France; CNRS, Laboratoire d'Energétique et de Mécanique Théorique et Appliquée, UMR 7563, 2 avenue de la Forêt de Haye, BP 160, Vandœuvre-lès-Nancy, F-54504, France.
authors.forename: Salaheddine
authors.initials: S
authors.lastname: Skali-Lami
authors.affiliation: Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, UMR 7564, Villers-lès-Nancy, F-54600, France; CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, UMR 7564, 405 rue de Vandoeuvre, Villers-lès-Nancy, F-54600, France. Electronic address: gregory.francius@univ-lorraine.fr.
authors.forename: Grégory
authors.initials: G
authors.lastname: Francius
mesh_terms: D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D015850:Interleukin-6 / Q000276:immunology; D051379:Mice; D018625:Microscopy, Atomic Force; D013499:Surface Properties
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; D015850:Interleukin-6
keywords: Atomic force microscopy; ELISA; Molecular conformation; Protein adsorption; Thin films