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Stojanovic I.,University of Twente | Schasfoort R.B.M.,University of Twente | Schasfoort R.B.M.,Ibis Technology | Terstappen L.W.M.M.,University of Twente
Biosensors and Bioelectronics | Year: 2014

Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells. © 2013 Elsevier B.V. Source

Geuijen K.P.M.,Downstream Processing | Geuijen K.P.M.,Wageningen University | Schasfoort R.B.,University of Twente | Schasfoort R.B.,Ibis Technology | And 3 more authors.
Analytical Biochemistry | Year: 2014

Affinity-based analyses on biosensors depend partly on regeneration between measurements. Regeneration is performed with a buffer that efficiently breaks all interactions between ligand and analyte while maintaining the active binding site of the ligand. We demonstrated a regeneration buffer scouting using the combination of a continuous flow microspotter with a surface plasmon resonance imaging platform to simultaneously test 48 different regeneration buffers on a single biosensor. Optimal regeneration conditions are found within hours and consume little amounts of buffers, analyte, and ligand. This workflow can be applied to any ligand that is coupled through amine, thiol, or streptavidin immobilization. © 2014 Elsevier Inc. All rights reserved. Source

van Beers J.J.B.C.,Radboud University Nijmegen | Raijmakers R.,University Utrecht | Alexander L.-E.,Radboud University Nijmegen | Stammen-Vogelzangs J.,Radboud University Nijmegen | And 5 more authors.
Arthritis Research and Therapy | Year: 2010

Introduction: Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.Methods: Human fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays.Results: In total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the β-chain.Conclusions: A comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides. © 2010 van Beers et al.; licensee BioMed Central Ltd. Source

Stichting Sanquin Bloedvoorziening and Ibis Technology | Date: 2013-12-19

The invention relates to means and methods for detecting a cell surface molecule in a cell sample. The invention further relates to a method for blood group typing and screening and to SPR sensors and SPR measuring systems suitable for use in such methods. The method includes steps of allowing a liquid cell sample to flow to and along the sensor surface, temporarily reducing the shear rate of the liquid sample to allow cells in the sample to sediment by binding to a binding compound immobilised on a metal film on the sensor surface, and removing unbound cells or non-specifically bound cells or fragments thereof. Moreover, the ratio of the magnitude of the specific total signal response to the signal response during sedimentation is determined.

The present invention relates to a method for the measurement of at least one sample by the interaction with the surface in the field of at least one sensor surface, such as surface plasmon resonance measurement, comprising the steps of: i) sampling the sample and a buffer; ii) transporting the sample and the buffer to at least one flow cell which is in liquid contact with the sensor surface of at least one sensor for measuring a parameter of a sample by interaction of the sample at the sensor surface in the field of the sensor surface; iii) transporting the sample into the flow cell into contact with the sensor surface; iv) handling a separation fluidim by inserting and/or removing the separation fluidim between the sample and the buffer upstream and/or downstream of the sensor surface; v) measuring the interaction of the sample at the sensor surface; and vi) dispensing the sample from the flow cell, and to a measuring system for such method.

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