Publications by authors named "Richard B M Schasfoort"

35 Publications

Presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Biosens Bioelectron 2021 Mar 16;183:113165. Epub 2021 Mar 16.

Department of Clinical Chemistry, Medlon, BV, 7512KZ, Enschede, the Netherlands; Department of Clinical Chemistry and Laboratory Medicine, Ziekenhuis Groep Twente, PO BOX 7600, 7600, SZ, Almelo, the Netherlands.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 119 CoViD-19 patients. The SPRi assay measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19 and this new method can furthermore be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 in vaccination programs.
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http://dx.doi.org/10.1016/j.bios.2021.113165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962981PMC
March 2021

IgG Subclass Determines Suppression Versus Enhancement of Humoral Alloimmunity to Kell RBC Antigens in Mice.

Front Immunol 2020 16;11:1516. Epub 2020 Jul 16.

Bloodworks Northwest Research Institute, Seattle, WA, United States.

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.
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http://dx.doi.org/10.3389/fimmu.2020.01516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378678PMC
July 2020

Multiplex Label Free Characterization of Cancer Cell Lines Using Surface Plasmon Resonance Imaging.

Biosensors (Basel) 2019 May 27;9(2). Epub 2019 May 27.

Medical Cell BioPhysics Group, MIRA institute, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500AE Enschede, The Netherlands.

Rapid multiplex cell surface marker analysis can expedite investigations in which large number of antigens need to be analyzed. Simultaneous analysis of multiple surface antigens at the same level of sensitivity is however limited in the current golden standard analysis method, flow cytometry. In this paper we introduce a surface plasmon resonance imaging (SPRi)-based technique for 44-plex parameter analysis using a single sample, in less than 20 min. We analyzed the expression on cells from five different cancer cell lines by SPRi on a 44-plex antibody array including 4 negative controls and compared the output with flow cytometry. The combined correlation of the markers that showed expression by flow cytometry was 0.76. The results demonstrate as a proof of principle that SPRi can be applied for rapid semi-quantitative multiplex cell surface marker analysis.
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http://dx.doi.org/10.3390/bios9020070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628007PMC
May 2019

A microwell array platform to print and measure biomolecules produced by single cells.

Lab Chip 2019 05;19(10):1850-1859

Department of Medical Cell BioPhysics, University of Twente, Hallenweg 23, Enschede, 7522 NH, The Netherlands.

Here we describe a combined method to monitor the secretion of molecules produced by single cells, followed by a method to isolate the individual cells that produced these molecules. The method is based on a self-sorting microwell chip that is connected to an activated membrane that collects the produced molecules. The produced molecules are printed by diffusion in small spots onto the membrane. The location of the printed spots can be correlated to the microwell number and the cell that produced these molecules. To demonstrate the method, we used the EpCAM antibody producing hybridoma cell line VU1D9 and a genetically engineered CHO cell-line producing Her2. VU1D9 cells produced 4.6 ± 5.6 pg (mean ± SD) of EpCAM antibody per 24 h and CHO cells 6.5 ± 8.2 pg per 24 h of Herceptin antibody.
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http://dx.doi.org/10.1039/c9lc00100jDOI Listing
May 2019

Trends in SPR Cytometry: Advances in Label-Free Detection of Cell Parameters.

Biosensors (Basel) 2018 Oct 30;8(4). Epub 2018 Oct 30.

Medical Cell BioPhysics Group (MCBP), University of Twente, 7500 AE Enschede, The Netherlands.

SPR cytometry entails the measurement of parameters from intact cells using the surface plasmon resonance (SPR) phenomenon. Specific real-time and label-free binding of living cells to sensor surfaces has been made possible through the availability of SPR imaging (SPRi) instruments and researchers have started to explore its potential in the last decade. Here we will discuss the mechanisms of detection and additionally describe the problems and issues of mammalian cells in SPR biosensing, both from our own experience and with information from the literature. Finally, we build on the knowledge and applications that has already materialized in this field to give a forecast of some exciting applications for SPRi cytometry.
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http://dx.doi.org/10.3390/bios8040102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315638PMC
October 2018

Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging.

Anal Chem 2018 06 15;90(11):6563-6571. Epub 2018 May 15.

Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine , University of Twente , Enschede , 7522 NB , The Netherlands.

There is a large unmet need for reliable biomarker measurement systems for clinical application. Such systems should meet challenging requirements for large scale use, including a large dynamic detection range, multiplexing capacity, and both high specificity and sensitivity. More importantly, these requirements need to apply to complex biological samples, which require extensive quality control. In this paper, we present the development of an enhancement detection cascade for surface plasmon resonance imaging (SPRi). The cascade applies an antibody sandwich assay, followed by neutravidin and a gold nanoparticle enhancement for quantitative biomarker measurements in small volumes of complex fluids. We present a feasibility study both in simple buffers and in spiked equine synovial fluid with four cytokines, IL-1β, IL-6, IFN-γ, and TNF-α. Our enhancement cascade leads to an antibody dependent improvement in sensitivity up to 40 000 times, resulting in a limit of detection as low as 50 fg/mL and a dynamic detection range of more than 7 logs. Additionally, measurements at these low concentrations are highly reliable with intra- and interassay CVs between 2% and 20%. We subsequently showed this assay is suitable for multiplex measurements with good specificity and limited cross-reactivity. Moreover, we demonstrated robust detection of IL-6 and IL-1β in spiked undiluted equine synovial fluid with small variation compared to buffer controls. In addition, the availability of real time measurements provides extensive quality control opportunities, essential for clinical applications. Therefore, we consider this method is suitable for broad application in SPRi for multiplex biomarker detection in both research and clinical settings.
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http://dx.doi.org/10.1021/acs.analchem.8b00260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990928PMC
June 2018

Rapid screening of IgG quality attributes - effects on Fc receptor binding.

FEBS Open Bio 2017 10 5;7(10):1557-1574. Epub 2017 Sep 5.

Downstream processing Synthon Biopharmaceuticals BV Nijmegen the Netherlands.

The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High-throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress-induced modifications to identify potential impact . Interestingly, we found that asparagine deamidation (D-N) reduced the binding of IgG to the low-affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short-term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in-process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.
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http://dx.doi.org/10.1002/2211-5463.12283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623700PMC
October 2017

Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles.

Clin Chem 2017 Oct 7;63(10):1633-1641. Epub 2017 Aug 7.

Department of Biomedical Engineering and Physics.

Background: Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface.

Methods: We investigated the potential of a 48-multiplex surface plasmon resonance imaging (SPRi) system to perform EV phenotyping. Antigen surface density of 11 antigens was measured on the human breast cancer cell lines HS578T, MCF7, and SKBR3 and their EVs by use of both SPRi and the widely used flow cytometry (FCM).

Results: For cells, the SPRi and FCM signals for antigen exposure correlated (R = 0.66, R = 0.78, R = 0.60). With regard to EVs, SPRi detected 31 out of 33 tested antibody-EV pairs, whereas our flow cytometer detected 5 antibody-EV pairs because of high blank and isotype control signals. For HS578T-derived EVs, the SPRi and FCM signals correlated (R = 0.98). However, on MCF7- and SKBR3-derived EVs, insufficient antigens were detected by our flow cytometer. To confirm that the SPRi responses correlated with mean antigen density on EVs, the SPRi responses of EVs were correlated with antigen density on parental cells as measured by FCM (R = 0.77, R = 0.49, R = 0.52).

Conclusions: SPRi responses correlate with mean antigen density. Moreover, SPRi detects lower antigen-exposure levels than FCM because SPRi measures an ensemble of EVs binding to the sensor surface, whereas FCM detects antigens of single EV.
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http://dx.doi.org/10.1373/clinchem.2016.271049DOI Listing
October 2017

Rapid Buffer and Ligand Screening for Affinity Chromatography by Multiplexed Surface Plasmon Resonance Imaging.

Biotechnol J 2017 Sep 10;12(9). Epub 2017 Aug 10.

Downstream Processing, Synthon Biopharmaceuticals BV, PO Box 7071, 6503 GN, Nijmegen, The Netherlands.

Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the purification process is applied to speed up the development (e.g., microtiterplates, robocolumns). The application of surface plasmon resonance imaging (SPRi) as a tool to simultaneously screen many buffer conditions for wash and elution steps in an affinity-based purification process is studied. Additionally, the protein A ligand stability after exposure to harsh cleaning conditions often limits the reuse of resins and is determined at lab scale. The SPRi technology to screen ligand life-time with respect to alkali stability is used. It is also demonstrated that SPRi can successfully be applied in screening experiments for process developments in a miniaturized approach. The amount of resin, protein and buffer in these studies is reduced 30-300-fold compared to 1 mL column scale, and approximately 10-1000-fold compared to filter plate experiments. The overall development time can be decreased from several months towards days. The multiplexed SPRi can be applied in screening affinity chromatography conditions in early stage development for ligand development and recombinant protein production.
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http://dx.doi.org/10.1002/biot.201700154DOI Listing
September 2017

Modeling single cell antibody excretion on a biosensor.

Anal Biochem 2016 07 31;504:1-3. Epub 2016 Mar 31.

Medical Cell Biophysics Group, MIRA Institute, Faculty of Science and Technology, University of Twente, 7500 AE Enschede, The Netherlands; IBIS Technologies, 7521 PR Enschede, The Netherlands.

We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.
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http://dx.doi.org/10.1016/j.ab.2016.03.018DOI Listing
July 2016

Interpolation method for accurate affinity ranking of arrayed ligand-analyte interactions.

Anal Biochem 2016 May 13;500:21-3. Epub 2016 Feb 13.

Medical Cell Biophysics Group, MIRA Institute, University of Twente, 7500 AB Enschede, The Netherlands.

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 = 100 RU response level (KD(R100)).
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http://dx.doi.org/10.1016/j.ab.2016.01.023DOI Listing
May 2016

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging.

Anal Biochem 2015 Sep 18;485:112-8. Epub 2015 Jun 18.

Medical Cell BioPhysics Group, MIRA Institute, Faculty of Science and Technology, University of Twente, 7500 AE Enschede, The Netherlands.

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 10h. 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 2.96p g 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.
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http://dx.doi.org/10.1016/j.ab.2015.06.018DOI Listing
September 2015

Platinum(II) as bifunctional linker in antibody-drug conjugate formation: coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model.

ChemMedChem 2015 May 25;10(5):797-803. Epub 2015 Mar 25.

Department of Radiology and Nuclear Medicine, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam (The Netherlands).

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48 h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.
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http://dx.doi.org/10.1002/cmdc.201402496DOI Listing
May 2015

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.

Blood 2015 Mar 29;125(11):1793-802. Epub 2014 Dec 29.

Department of Experimental Immunohematology, Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;

Immune-mediated platelet destruction is most frequently caused by allo- or autoantibodies via Fcγ receptor-dependent phagocytosis. Disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors play a role. Here we show that the acute phase protein C-reactive protein (CRP), a ligand for Fc receptors on phagocytes, enhances antibody-mediated platelet destruction by human phagocytes in vitro and in vivo in mice. Without antiplatelet antibodies, CRP was found to be inert toward platelets, but it bound to phosphorylcholine exposed after oxidation triggered by antiplatelet antibodies, thereby enhancing platelet phagocytosis. CRP levels were significantly elevated in patients with allo- and autoantibody-mediated thrombocytopenias compared with healthy controls. Within a week, intravenous immunoglobulin treatment in children with newly diagnosed immune thrombocytopenia led to significant decrease of CRP levels, increased platelet numbers, and clinically decreased bleeding severity. Furthermore, the higher the level of CRP at diagnosis, the longer it took before stable platelet counts were reached. These data suggest that CRP amplifies antibody-mediated platelet destruction and may in part explain the aggravation of thrombocytopenia on infections. Hence, targeting CRP could offer new therapeutic opportunities for these patients.
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http://dx.doi.org/10.1182/blood-2014-05-579110DOI Listing
March 2015

Analysis of cell surface antigens by Surface Plasmon Resonance imaging.

Biosens Bioelectron 2014 Feb 27;52:36-43. Epub 2013 Aug 27.

Medical Cell Biophysics Group, MIRA institute, Faculty of Science and Technology, University of Twente, PO Box 217, 7500AE Enschede, The Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bios.2013.08.027DOI Listing
February 2014

Label-free cell profiling.

Anal Biochem 2013 Aug 11;439(1):4-6. Epub 2013 Apr 11.

Medical Cell Biophysics Group, MIRA Institute, University of Twente, Enschede, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.ab.2013.04.001DOI Listing
August 2013

Method for estimating the single molecular affinity.

Anal Biochem 2012 Feb 13;421(2):794-6. Epub 2011 Dec 13.

Medical Cell Biophysics Group, MIRA Institute, University of Twente, 7500 AE Enschede, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.ab.2011.12.011DOI Listing
February 2012

Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.

Nature 2011 Jul 4;476(7360):293-7. Epub 2011 Jul 4.

Hubrecht Institute and University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.
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http://dx.doi.org/10.1038/nature10337DOI Listing
July 2011

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance.

Arthritis Res Ther 2010 23;12(6):R219. Epub 2010 Dec 23.

Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands.

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.
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http://dx.doi.org/10.1186/ar3205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046532PMC
June 2011

Electrokinetic lab-on-a-biochip for multi-ligand/multi-analyte biosensing.

Anal Chem 2010 May;82(10):4145-50

BIOS Lab-on-a-Chip Group, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.

We present a simple electrokinetic lab-on-a-biochip (EKLB) with four microchannels integrated with a surface plasmon resonance imaging (iSPR) label-free biosensor that is operated using a single electrical voltage for the simultaneous transport of reagents in all microchannels without conventional fluidic plumbing. We demonstrate the utility of the simple approach with various biosensing experiments, including single injection kinetics (multiple varied ligand densities and single analyte concentration), one shot kinetics (single ligand densities and multiple varied analyte concentrations), and multi-ligand/multianalyte detection. In all cases, the binding kinetics and affinity were extracted using a conventional 1:1 interaction model. Since the reagent transport is done with a single electrical voltage source, scaling up to hundreds to thousands of simultaneous experiments is straightforward.
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http://dx.doi.org/10.1021/ac1003163DOI Listing
May 2010

Electrokinetic label-free screening chip: a marriage of multiplexing and high throughput analysis using surface plasmon resonance imaging.

Lab Chip 2010 Apr 9;10(8):986-90. Epub 2010 Mar 9.

BIOS Lab-on-a-Chip Group, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.

We present an electrokinetic label-free biomolecular screening chip (Glass/PDMS) to screen up to 10 samples simultaneously using surface plasmon resonance imaging (iSPR). This approach reduces the duration of an experiment when compared to conventional experimental methods. This new device offers a high degree of parallelization not only for analyte samples, but also for multiplex analyte interactions where up to 90 ligands are immobilized on the sensing surface. The proof of concept has been demonstrated with well-known biomolecular interactant pairs. The new chip can be used for high throughput screening applications and kinetics parameter extraction, simultaneously, of interactant-protein complex formation.
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http://dx.doi.org/10.1039/c000705fDOI Listing
April 2010

Single injection microarray-based biosensor kinetics.

Anal Methods 2009 Dec;1(3):162-169

BIOS Lab-on-a-Chip Group, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500, AE Enschede, The Netherlands.

Binding affinity of biomolecular interactions can be directly extracted from measured surface plasmon resonance biosensor sensorgrams by fitting the data to the appropriate model equations. The conventional method for affinity estimation uses a series of analytes and buffers that are injected serially to a single immobilized ligand on the sensing surface, including a regeneration step between each injection, to generate information about the binding behavior. We present an alternative method to estimate the affinity using a single analyte concentration injected to multiple ligand densities in a microarray format. This parameter estimation method eliminates the need for multiple analyte injections and surface regeneration steps, which can be important for applications where there is limited analyte serum, fragile ligand-surface attachment, or the detection of multiple biomolecule interactions. The single analyte injection approach for binding affinity estimation has been demonstrated for two different interactant pairs, β2 microglobulin/anti-β2 microglobulin (β2M) and human IgG/Fab fragments of anti-human IgG (hIgG), where the ligands are printed in a microarray format. Quantitative comparisons between the estimated binding affinities measured with the conventional method are β2M: KD = 1.48 ± 0.28 nM and hIgG: KD = 12.6 ± 0.2 nM and for the single injection method are β2M: KD = 1.52 ± 0.22 nM and hIgG: KD = 12.5 ± 0.6 nM, which are in good agreement in both cases.
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http://dx.doi.org/10.1039/b9ay00176jDOI Listing
December 2009

Integrated electrokinetic sample focusing and surface plasmon resonance imaging system for measuring biomolecular interactions.

Anal Chem 2009 Mar;81(5):1957-63

BIOS Lab-On-A-Chip Group, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.

Label-free biomolecular binding measurement methods, such as surface plasmon resonance (SPR), are becoming increasingly more important for the estimation of real-time binding kinetics. Recent advances in surface plasmon resonance imaging (iSPR) are emerging for label-free microarray-based assay applications, where multiple biomolecular interactions can be measured simultaneously. However, conventional iSPR microarray systems rely on protein printing techniques for ligand immobilization to the gold imaging surface and external pumps for analyte transport. In this article, we present an integrated microfluidics and iSPR platform that uses only electrokinetic transport and guiding of ligands and analytes and, therefore, requires only electrical inputs for sample transport. An important advantage of this new approach, compared to conventional systems, is the ability to direct a single analyte to a specific ligand location in the microarray, which can facilitate analysis parallelization. Additionally, this simple approach does not require complicated microfluidic channel arrangements, external pumps, or valves. As a demonstration, kinetics and affinity have been extracted from measured binding responses of human IgG and goat antihuman IgG using a simple 1:1 model and compared to responses measured with conventional pressure driven analyte transport. The measured results indicate similar binding kinetics and affinity between the electrokinetic and pressure-driven sample manipulation methods and no cross contamination to adjacent measurement locations has been observed.
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http://dx.doi.org/10.1021/ac802668zDOI Listing
March 2009

Bubble-free operation of a microfluidic free-flow electrophoresis chip with integrated Pt electrodes.

Anal Chem 2008 Jun 25;80(11):4111-8. Epub 2008 Apr 25.

MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500AE Enschede, The Netherlands.

In order to ensure a stable and efficient separation in microfluidic free-flow electrophoresis (FFE) devices, various methods and chips have been presented until now. A major concern hereby is the generation of gas bubbles caused by electrolysis and the resulting disturbances in the position of the separated analyte lanes. Instable lane positions would lead to a decreased resolution in sample collection over time which certainly would be problematic when incorporating a stationary detector system. In contrast to our previous publications, in which we implemented laborious semipermeable membranes to keep bubbles outside the separation region, here we describe an electrochemical approach to suppress the electrolysis of water molecules and therefore bubble formation. This approach allowed a simpler and additionally a closed chip device with integrated platinum electrodes. With the use of this chip, the successful separation of three fluorescent compounds was demonstrated. Quinhydrone, which is a complex of hydroquinone and p-benzoquinone, was added only to the local flow streams along the electrodes, preventing mixing with the separation media and sample. The electrical current was generated via the oxidization and reduction of hydroquinone and p-benzoquinone up to a certain limit of the electrical current without gas formation. The separation stability was investigated for the chip with and without quinhydrone, and the results clearly indicated the improvement. In contrast to the device operating without quinhydrone, a 2.5-fold increase in resolution was achieved. Furthermore, separation was demonstrated within tens of milliseconds. This chemical approach with its high miniaturization possibilities offers an interesting alternative, in particular for low-current miniaturized FFE systems, in which large and open electrode reservoirs are not tolerable.
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http://dx.doi.org/10.1021/ac800275cDOI Listing
June 2008

Electrokinetic sorting and collection of fractions for preparative capillary electrophoresis on a chip.

Lab Chip 2008 May 18;8(5):801-9. Epub 2008 Mar 18.

MESA+ Institute for Nanotechnology, Faculty of Science and Technology, University of Twente, P.O Box 217, 7500AE, Enschede, The Netherlands.

A microfabricated device capable of selecting and collecting multiple components from a mixture separated by capillary electrophoresis (CE) is described. This collection is automated and can be easily controlled by a set of rules defined by an operator, enabling fast and consistent operation. The device consists of an electrokinetically steered fluidic network that can be divided into three sections: a CE part, a fractions distribution region and a set of storage channels. Sample fractions leave the CE channel and are detected in the interfacial region by fluorescence intensity measurements. If an upcoming peak is detected, separation is withheld and the potentials are reconfigured to force the fraction into one of the collection channels, where they become available for further processing or analysis. The sequence of separation and collection is repeated until all the bands of interest are captured. A mixture of three fluorescent dyes (Rhodamine 6G, Rhodamine B and Fluorescein) was used to demonstrate the principle. The components were repeatedly separated by means of CE and pooled in their respective storage channels. In comparison to previous developments, the system presented in this paper offers automatic collection of all fractions in a single run. Furthermore, it is possible to run the system in a repetitive mode for accumulative pooling if more fractionated sample is required.
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http://dx.doi.org/10.1039/b717785bDOI Listing
May 2008

Miniaturizing free-flow electrophoresis - a critical review.

Electrophoresis 2008 Mar;29(5):977-93

Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.

Free-flow electrophoresis (FFE) separation methods have been developed and investigated for around 50 years and have been applied not only to many types of analytes for various biomedical applications, but also for the separation of inorganic and organic substances. Its continuous sample preparation and mild separation conditions make it also interesting for online monitoring and detection applications. Since 1994 several microfluidic, miniaturized FFE devices were developed and experimentally characterized. In contrast to their large-scale counterparts microfluidic FFE (mu-FFE) devices offer new possibilities due to the very rapid separations within several seconds or below and the requirement for sample volumes in the microliter range. Eventually, these mu-FFE systems might find application in so-called lab-on-a-chip devices for real-time monitoring and separation applications. This review gives detailed information on the results so far published on mu-FFE chips, comprising its four main modes, namely free-flow zone electrophoresis (FFZE), free-flow IEF (FFIEF), free-flow ITP (FFITP), and free-flow field-step electrophoresis (FFFSE). The principles of the different FFE modes and the basic underlying theory are given and discussed with special emphasis on miniaturization. Different designs as well as fabrication methods and applied materials are discussed and evaluated. Furthermore, the separation results shown indicate that similar separation quality with respect to conventional FFE systems, as defined by the resolution and peak capacity, can be achieved with mu-FFE separations when applying much lower electrical voltages. Furthermore, innovations still occur and several approaches for hyphenated, more integrated systems have been proposed so far, some of which are discussed here. This review is intended as an introduction and early compendium for research and development within this field.
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http://dx.doi.org/10.1002/elps.200700725DOI Listing
March 2008

Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays.

Biosens Bioelectron 2008 Jan 11;23(6):839-44. Epub 2007 Sep 11.

BIOS, Lab-on-a-Chip Group, MESA+ Research Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.

In this paper we describe the use of a commercial surface plasmon resonance (SPR) imaging instrument for monitoring the binding of biomolecules on user-defined regions of interest of a microarray. By monitoring the angle shift of the SPR-dip using a continuous angle-scanning mode instead of monitoring the change in reflectivity at a fixed angle, a linear relationship with respect to the mass density change on the surface will remain over a wide dynamic angle range of 8 degrees. Peptides (2.4 kDa) and proteins (150 kDa) were both spotted on the same sensor chip to illustrate that both, low and high molecular weight ligands with initial large differences in off-set SPR angles, can be applied within the same experiment. By using a fluorescently labeled antibody, SPR results can be confirmed by means of fluorescence microscopy after completion of a SPR experiment. SPR imaging in angle-scanning operation provides sensitive, accurate, and label-free detection of analyte binding on microarrays containing different molecular weight ligands.
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http://dx.doi.org/10.1016/j.bios.2007.08.025DOI Listing
January 2008

Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging.

J Am Chem Soc 2007 Nov 17;129(45):14013-8. Epub 2007 Oct 17.

Department of Biomolecular Chemistry, Nijmegen Centre for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands.

A new commercial surface plasmon resonance (SPR) imaging analysis system with a novel SPR dip angle scanning principle allows the measurement, without the need for labeling, of the exact SPR dip angle. With this system hundreds of biomolecular interactions can be monitored on microarrays simultaneously and with great precision. The potency of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. The smallest antibody concentration that could be measured in this experimental setup was 0.5 pM.
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http://dx.doi.org/10.1021/ja075103xDOI Listing
November 2007

Microfluidic high-resolution free-flow isoelectric focusing.

Anal Chem 2007 Nov 29;79(21):8190-8. Epub 2007 Sep 29.

MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500AE Enschede, The Netherlands.

A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of Delta(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is Delta(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.
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http://dx.doi.org/10.1021/ac071419bDOI Listing
November 2007

Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes.

Lab Chip 2006 Mar 26;6(3):374-80. Epub 2006 Jan 26.

Biochip Group, MESA+ Research Institute, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500AE Enschede, The Netherlands.

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.
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http://dx.doi.org/10.1039/b514731jDOI Listing
March 2006