title: Molecular interference in antibody-antigen interaction studied with magnetic force immunoassay.
abstract: Molecular interferences are an important challenge in biotechnologies based on antibody-antigen interactions, such as sandwich immunoassays. We report how a sandwich immunoassay with magnetic particles as label can be used to probe interference by surfactants. Surfactants are often used to improve the performance of immunoassays, however the surfactants can affect the involved proteins and the mechanism of action of surfactant molecules on the antibody-antigen system is mostly unknown. As an example, we investigated molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay with two monoclonal antibodies. The influence of the surfactant below the critical micelle concentration (0.00-0.04%) on dissociation properties was quantified in a magnetic tweezers setup, where a force is applied to the molecules via magnetic particle labels. The force-dependent dissociation curves revealed the existence of two distinct cTn-dependent bond types, namely a weak bond attributable to non-specific binding of cTn, and a strong bond attributable to the specific binding of cTn. The dissociation rate constant of the strong bonds increased with the surfactant concentration by about a factor of two. Circular dichroism spectroscopy data showed that the nonionic surfactant influences the conformation of cTn while not noticeably affecting the two monoclonal antibodies. This suggests that the surfactant-induced increase of the dissociation rate of the specific sandwich-type cTn binding may be related to a conformational change of the antigen molecule. The described methodology is an effective tool to study the influence of surfactants and other interferences on assays based on protein interactions.
authors: Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors: D
authors: D
authors: Dorokhin
authors: Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors: L J
authors: LJ
authors: van IJzendoorn
authors: Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors: A M
authors: AM
authors: de Jong
authors: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors: L
authors: L
authors: Nieto
authors: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors: L
authors: L
authors: Brunsveld
authors: Philips Research, High Tech Campus 11, Eindhoven, The Netherlands.
authors: J G
authors: JG
authors: Orsel
authors: Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands. Electronic address: m.w.j.prins@tue.nl.
authors: M W J
authors: MW
authors: Prins
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D002942:Circular Dichroism; D007118:Immunoassay / Q000379:methods*; D008280:Magnetics; D020442:Poloxamer / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D014336:Troponin / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D013501:Surface-Active Agents; D014336:Troponin; D020442:Poloxamer