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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.


Schasfoort R.B.M.,University of Twente | Schasfoort R.B.M.,Ibis Technology | De Lau W.,University Utrecht | Van Der Kooi A.,Ibis Technology | And 2 more authors.
Analytical Biochemistry | Year: 2012

Affinity constants (k d, k a, and K D) can be determined by methods that apply immobilized ligands such as immunoassays and label-free biosensor technologies. This article outlines a new surface plasmon resonance (SPR) array imaging method that yields affinity constants that can be considered as the best estimate of the affinity constant for single biomolecular interactions. Calculated rate (k d and k a) and dissociation equilibrium (K D) constants for various ligand densities and analyte concentrations are extrapolated to the K D at the zero response level (K D R0). By applying this method to an LGR5-exo-Fc-RSPO1-FH interaction couple, the K D R0 was determined as 3.1 nM. © 2011 Elsevier Inc. All rights reserved.


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.


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.


Schasfoort R.B.M.,University of Twente | Schasfoort R.B.M.,Ibis Technology | Bentlage A.E.H.,University of Amsterdam | Stojanovic I.,University of Twente | And 4 more authors.
Analytical Biochemistry | Year: 2013

A surface plasmon resonance (SPR) array imaging method is outlined for label-free cell profiling. Red blood cells (RBCs) were injected into a flow chamber on top of a spotted sensor surface. Spots contained antibodies to various RBC membrane antigens. A typical sensorgram showed an initial response corresponding to cell sedimentation (S) followed by a specific upward response (T) corresponding to specific binding of cells during a critical wash step. The full analysis cycle for RBC profiling was less than 6 min. The sensor surface could be regenerated at least 100 times, allowing the determination of a cell surface antigen profile of RBCs. © 2013 Elsevier Inc. All rights reserved.


Stojanovic I.,University of Twente | Van Der Velden T.J.G.,University of Twente | Van Der Velden T.J.G.,Ibis Technology | Mulder H.W.,University of Twente | And 3 more authors.
Analytical Biochemistry | Year: 2015

Abstract Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10 h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95 pg (mean 2.96 pg per cell, SD 2.51) over a period of 10 h. Antibody excretion per cell per hour ranged from 0.02 to 1.19 pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells. © 2015 Elsevier Inc.


Patent
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.


PubMed | University of Twente and Ibis Technology
Type: | Journal: Analytical biochemistry | Year: 2016

The values of the affinity constants (kd, ka, and KD) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are exponentially interpolated to the KD at Rmax=100RU response level (KD(R100)).

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