Epitope Bin 96 mAbs in 24 hours
Determine epitope bins using state-of-the-art software
Fast and easy kinetics analysis


Handbook of Surface Plasmon Resonance, 2nd Edition - Richard Schasfoort, PhD

Surface Plasmon Resonance (SPR) plays a dominant role in real-time interaction sensing of biomolecular binding events and with the biosensor field expanding more applications are being found. In response to the market an update to the original title which was published in 2008 is now appropriate. With over fifty percent of the material being updated, this book provides a total system description including optics, fluidics and sensor surfaces.

Spanning theory, instrumentation and applications, it covers all the relevant issues for the practicing researcher. Unlocking the potential for SPR by showing highly exciting and unique opportunities for unraveling the functional relationships of complex biological processes, it is intended for a wide audience. A comprehensive and accessible source it contains expanded tutorial details to inspire students and guide them in this technology.

The second edition of the Handbook of Surface Plasmon Resonance will be launched at PEGS, Boston, 1-5th of May 2017


High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge.

ScienceDirect  DOI: 10.1016/j.drudis.2014.05.011

B. Brooks, A. Miles, Y. Abdiche
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.
Rinat Laboratories, Pfizer, South San Francisco, CA 94080, USA

Analytical tools are evolving to meet the need for the higher-Throughput characterization of therapeutic monoclonal antibodies. An antibody’s epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection.


The Importance of Epitope Binning for Biological Drug Discovery.

NCBI PMC  Curr Drug Discov Technol. Jun 2014; 11(2): 109-112

B. Brooks
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

The pharmaceutical industry is experiencing comeback sales growth due in large part to the industry’s R&D efforts that center on biologics drug development. To facilitate that effort, tools are being developed for more effective biologic drug development. At the forefront of this effort is epitope characterization, in particular epitope binning, primarily due to the role an epitope plays in drug function. Here we detail the financial advantages of epitope binning including (1) increased R&D productivity due to increased work in process, (2) reduced number of “dead-end”candidates, and (3) increased ability to re-engineer antibodies based on the epitope. With the arrival of high throughput biosensors, this manuscript serves as a call to push epitope binning earlier in the biological drug discovery process.


Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms.

mAbs, 8:2, 264-277, DOI: 10.1080/19420862.2015.1118596

Yasmina Noubia Abdiche, Rian Harriman, Xiaodi Deng, Yik Andy Yeung,
Adam Miles, Winse Morishige, Leila Boustany, Lei Zhu, Shelley Mettler
Izquierdo & William Harriman

The ability of monoclonal antibodies (mAbs) to target specific antigens with high precision has led to an
increasing demand to generate them for therapeutic use in many disease areas. Historically, the discovery
of therapeutic mAbs has relied upon the immunization of mammals and various in vitro display
technologies. While the routine immunization of rodents yields clones that are stable in serum and have
been selected against vast arrays of endogenous, non-target self-antigens, it is often difficult to obtain
species cross-reactive mAbs owing to the generally high sequence similarity shared across human
antigens and their mammalian orthologs. In vitro display technologies bypass this limitation, but lack an in
vivo screening mechanism, and thus may potentially generate mAbs with undesirable binding specificity
and stability issues. Chicken immunization is emerging as an attractive mAb discovery method because it
combines the benefits of both in vivo and in vitro display methods. Since chickens are phylogenetically
separated from mammals, their proteins share less sequence homology with those of humans, so human
proteins are often immunogenic and can readily elicit rodent cross-reactive clones, which are necessary
for in vivo proof of mechanism studies. Here, we compare the binding characteristics of mAbs isolated
from chicken immunization, mouse immunization, and phage display of human antibody libraries. Our
results show that chicken-derived mAbs not only recapitulate the kinetic diversity of mAbs sourced from
other methods, but appear to offer an expanded repertoire of epitopes. Further, chicken-derived mAbs
can bind their native serum antigen with very high affinity, highlighting their therapeutic potential.


High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with Functional Activity.

PLoS One. 2014 Mar 20;9(3):e92451. doi: 10.1371/journal.pone.0092451. eCollection 2014.

Abdiche YN, Miles A, Eckman J, Foletti D, Van Blarcom TJ, Yeung YA, Pons J, Rajpal A.
Pfizer Rinat, 230 E Grand Ave, South San Francisco, CA 94080, USA

Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb’s epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.


White Paper: Epitope Binning.

An important step in the engineering of biotherapeutics is to characterize and group a library of monoclonal
antibodies by the epitope binding regions generated against a specific antigen. This “epitope binning” enables the
maintenance of epitope diversity and provides important information to broaden intellectual property protection.
IBIS Technologies and Wasatch Microfluidics are pleased to announce a label free, array-based SPR package that
enables high throughput epitope binning of 96 antibodies directly from supernatant using only 150 μL of each
clone, overcoming the sample consumption, throughput limitations, and/or labeling restrictions of current
methods. IBIS and Wasatch have developed a powerful integrated hardware and software platform that delivers
the full potential of label-free, high throughput antibody binning, enabling you to…BIN THE FRIDGE.


White Paper: Combined ranking and binning.

In this white paper, the results are described of a combined kinetic ranking and binning study performed at UCB Pharma (Slough, UK). The ranking and binning was for 9 different antibodies using a single sensor, but up to 96 antibodies could be tested using a similar procedure as described here. Each antibody was spotted 5 times in a two-fold ligand density series for not only finding the affinity towards its target (Protein X) but also for identifying the bins (blockers and sandwichers). Due to the various ligand densities that were spotted the KDR0 method could be applied extrapolating the Rmax (a measure of ligand density) to zero. Also, a recognized global analysis evaluation using Scrubber2 software was carried out in order to compare with known “Biacored” values at UCB. The average, global and KDR0 values are given in this report for the clones directed toward Protein X. The experiment showed ranking results similar to previous tests as measured on different label free kinetic platforms at UCB.
The binning experiment showed a clear result of 3 bins and the best affinity antibody in these bins could be identified. Software for kinetic ranking and binning is available to analyze the data with enormous speed and accuracy. Hence the IBIS MX96 can become the high-performance work-horse for performing combined ranking and binning of antibodies to functional epitopes for finding the best therapeutic candidate in a limited period and at the lowest costs which is unrivalled in the market of high-end label-free interaction analysis equipment.


Poster: Smarter selection: building better reagents through Array SPRi-based high-throughput biophysical characterization and RabMAb® technologies.

The high-throughput Array SPRi system can be used to provide comprehensive epitope binning and kinetic screening information directly at the primary screening stage. In combination with RabMAb® hybridoma and B cell fusion technologies, this approach can quickly identify and develop high quality monoclonal antibodies (MAbs) for research and diagnostic applications. To demonstrate the value of this approach, we characterized a panel of MAbs corresponding to a subset of highly abundant B cells that circulate after immunization with a particular antigen. Crude and purified preparations of the panel were included in the assay to test assay performance at the very earliest stagesofproduct development. High-throughput epitope binning and kinetic screening on the Array SPRi platform allowed us to profile the binding character of each MAb, and assess the breadth of epitope recognition represented in the panel using only 20 μL of each supernatant and a few micrograms of the target analyte.

As presented at the Antibody Engineering and Therapeutics Congress, December 7-10 2015, San Diego, CA, USA.


White Paper: Epitope Mapping on digested proteins.

In this white paper, a new method is presented for discovery and mapping of the immunogenic epitopes of auto-antigens. Antigens containing undetermined antigenic epitopes were subjected to tryptic digestion and then separated using strong cation exchange liquid chromatography. The resulting fractions were then used to create arrays on the surface of surface plasmon resonance (SPR) sensors dedicated for use as part of IBIS’ MX96 System. The arrayed fractions were then exposed to a series of patient serum samples containing auto-antibodies or case controls; fractions containing the dominant immunogenic epitopes derived from the auto-antigen digests were then revealed by the resultant response signals.
In the example given, auto-antibodies contained within the sera of Rheumatoid Arthritis (RA) patients were used to identify dominant immunogenic epitopes. Rheumatoid Arthritis is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. In this white paper, the hypothesis that specific post-translational modifications in proteins may result in auto-immunity was tested. To investigate this possibility, proteins were synthetically citrullinated, digested and then artificially modified peptide-containing fractions were used to create arrays. The auto-antibody binding response generated using just minute quantities of finite patient sera samples showed that LysN- and trypsin-digested peptide fractions independently showed presence of auto-antigens. In particular, the Cit1 and Cit2 peptides and the protein could be a potential new biomarker for RA. Interestingly, there was significant variability between patients in terms of the epitope identity of the protein auto-antigen investigated.
In depth investigations of clinical features including the profiling anti-citrullinated protein autoantibodies of RA patients are now feasible and under further examination. This approach could be extended to investigate the role of citrullination or other post-translational modifications in auto-immune diseases.


White Paper: Epitope mapping at the amino acid level.

In this white paper, a new method is presented for the antibody epitope discovery at the amino acid level. Antigens contain many undetermined epitopes which all can be a target for antibodies. If the antigen amino acid sequence is known, linear epitopes can be defined through epitope mapping. Here we studied a total of 40 different 12-mer peptides, all biotinylated at the N-terminus, comprising amino acids 149 -201 of the RSV-G protein. This region of the protein contains the epitopes of two previously described antibodies (3D3 and 131-2G). The resultant 12-mer overlapping peptides were then used to create arrays on a streptavidin-coated (3D) SensEye® sensor which is inserted in the IBIS MX96 imager. The arrayed peptides are then exposed to the RSV-G protein specific antibodies 3D3, 131-2G and antibody AIMM-1 of AIMM therapeutics [1]. Each antibody only binds a specific group of peptides, from which the antibody epitopes can be defined. 3D3 and 131-2G bind a very similar epitope, with 131-2G requiring more amino acid contacts to bind. The unique AIMM-1 antibody bound to a new epitope, which is not recognized by 3D3 and 131-2G. The experiments were conducted on the IBIS MX96 imager in the labs of AIMM Therapeutics, Amsterdam, The Netherlands.


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

Arthritis Res Ther. 2010;12(6):R219. doi: 10.1186/ar3205. Epub 2010 Dec 23.

van Beers JJ, Raijmakers R, Alexander LE, Stammen-Vogelzangs J, Lokate AM, Heck AJ, Schasfoort RB, Pruijn GJ.

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.


Label-Free Glycoprofiling with Multiplex Surface Plasmon Resonance: A Tool To Quantify Sialylation of Erythropoietin

Anal. Chem., 2015, 87 (16), pp 8115–8122 DOI: 10.1021/acs.analchem.5b00870

Karin P. M. Geuijen, Liem A. Halim, Huub Schellekens, Richard B. Schasfoort, René H. Wijffels, and Michel H. Eppink

Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.


White Paper: First Pass Kinetics™ Screening of Captured Antibodies.

The following white paper shows an example of a binding kinetics screening strategy using capture of IL-6R antibody on immobilized anti-IgG Fc surface followed by probing with a serial titration of IL-6R. Varied antibody densities were generated across the chip surface to demonstrate both measures of reproducibility as well as to highlight the dynamic range of this system for looking at kinetics of ligands with broad differences in concentration. This approach to rapid profiling and ranking, termed First-Pass Kinetics™, presents a robust means of screening high numbers of candidates while still enabling the benefits of detailed antigen-antibody binding kinetics.


White Paper: Affinity of Fc -VH interactions.

This white paper describes the determination of the kinetic rate and equilibrium affinity constants of Fc – VH interactions as performed in the labs of Crescendo Biologics, Cambridge, UK. For determining affinity constants it is well known to apply low ligand densities to reduce rebinding effects and factors that influence kinetics. Two approaches were applied for determining the affinity constants of VH antibodies for a Fc fusion protein: (a) A capture approach using Protein G and (b) direct immobilization of the Fc fusion protein (Mw ~ 60 kD) on the sensor surface; both followed by the VH (MW~ 12 kD) injection. Both experiments were performed in a single run and the data obtained was comparable with Biacore T200 data. The capture of the Fc fusion protein on the protein G spots lead to a decaying baseline for the VH binding. The decaying baseline correction was performed by a double referencing principle using blank subtraction in both Scrubber and SPRintX software. The equilibrium dissociation constants obtained with the IBIS MX96 were similar for VH binding to directly coupled Fc fusion protein, or to Fc fusion protein captured with Protein-G. Earlier blind control measurements using a Biacore T200 in a kinetic titration experiment showed similar values.


High-throughput and multiplexed regeneration buffer scouting for affinity-based interactions.

Anal Biochem. 2014 Jun 1;454:38-40. doi: 10.1016/j.ab.2014.03.011. Epub 2014 Mar 21.

Geuijen KP, Schasfoort RB, Wijffels RH, Eppink MH. 
Synthon, Microweg 22, 6545 CM Nijmegen, The Netherlands.

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.


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

Nature. 2011 Jul 4;476(7360):293-7. doi: 10.1038/nature10337.

de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, Stange DE, van Es JE, Guardavaccaro D, Schasfoort RB, Mohri Y, Nishimori K, Mohammed S, Heck AJ, Clevers H.

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.


Structures of Wnt-antagonist ZNRF3 and its complex with R-spondin 1 and implications for signaling.

PLoS One. 2013 Dec 12;8(12):e83110. doi: 10.1371/journal.pone.0083110. eCollection 2013.

Peng WC, de Lau W, Madoori PK, Forneris F, Granneman JC, Clevers H, Gros P.

Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 – R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.


Analyzing a kinetic titration series using affinity biosensors.

Anal Biochem. 2006 Feb 1;349(1):136-47. Epub 2005 Oct 13.

Karlsson R, Katsamba PS, Nordin H, Pol E, Myszka DG.

The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call “kinetic titration.” This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate.


White Paper: Specifications of the IBIS MX96.

In this white paper, the specifications of the IBIS MX96 are described. The large dynamic and linear range of the scanning angle optics allows measurements ranging from less than 1 Resonance Unit (RU) up to 30 000 RU with noise levels < 0.5 RU RMSD, making the MX96 the most sensitive SPR imaging instrument in the market. The imaging platform facilitates real-time sensing of analyte interactions on a 6×8, 4×12 or an 8×12 ligand array. Strong feature of the IBIS MX96 is the possibility to apply unlimited interaction time per sample for sub-picomolar limits of detection. Valve-less injection of samples and patented “back-and-forth” flow-based fluidics requires a sample volume of only 100 microliter to simultaneously measure the interactions on 96 ligand spots. The unsurpassed multiplex capacity of this platform provides scope to examine multiple interactions, simultaneously, whilst offering multiple referencing possibilities (up to 48 for 96 spots). An analysis cycle of 96 sample injections of 100 microliters in combination with a 96 spot microarray, generates 9216 referenced sensorgrams during unattended operation in a single run. MX96-SPRi software has been developed to analyze biomolecular interactions in such an organized way that kinetic evaluation of the measurement is straightforward. It enables the user to analyze high content screening assays with convenient data processing. The software is structured in an unique way enabling the highest performance in biomolecular interaction sensing.


White Paper: X-scan TCR HLA-peptide interactions.

Immunocore prepares therapeutics that can re-direct the immune response to recognise and kill diseased cells. Immunocore’s “ImmTAC” technology requires production of high affinity monoclonal T-Cell receptors (mTCR) that bind to cancer-specific peptides presented on the surface of diseased cells as a peptide-human leukocyte antigen (pHLA) complex. Since these mTCRs have high (picomolar) affinities and long binding half-lives it is imperative that they retain antigen specificity. The mTCR specificity is currently investigated in a cellular X-scan experiment whereby cells are pulsed with peptides similar in sequence to the wild-type peptide. Using this method a specificity motif can be elucidated when membrane-bound mTCR binds to target pHLA containing a single amino acid exchange at each position along the cognate 9-mer peptide antigen.
In this white paper Immunocore utilises surface plasmon resonance (SPR) (IBIS MX96, IBIS Technologies) to compare the specificity of two soluble mTCRs binding to 1-substituted peptides. A key feature of the IBIS MX96 instrument is that the affinity of up to 96 HLA-peptide interactions can be determined for each TCR with only a single injection of only 100 μl of) highly concentrated TCR in serial diluted concentrations. Issues due to HLA-peptide precipitates were observed and definitely it is an advantage that the precipitates can be visually inspected by SPR imaging after the spotting process of the biotinylated HLA peptide complexes.
It is concluded that the multiplexing nature of the IBIS MX96 and CFM printer (IBIS Technologies) would allow full X-scan motifs to be elucidated for several low affinity mTCRs on a single pHLA-loaded SensEye® sensor.


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

Anal Biochem. 2016 Feb 13. pii: S0003-2697(16)00047-6. doi: 10.1016/j.ab.2016.01.023. [Epub ahead of print]

Schasfoort RB, Andree KC, van der Velde N, van der Kooi A, Stojanovic I, Terstappen LW.

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 (KDR100).


Method for estimating the single molecular affinity.

Anal Biochem. 2012 Feb 15;421(2):794-6. doi: 10.1016/j.ab.2011.12.011. Epub 2011 Dec 13.

Schasfoort RB, Lau Wd, Kooi Av, Clevers H, Engbers GH.

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.


Selection of LNA-containing DNA aptamers against recombinant human CD73.

Mol Biosyst. 2015 May;11(5):1260-70. doi: 10.1039/c5mb00045a.

Elle IC, Karlsen KK, Terp MG, Larsen N, Nielsen R, Derbyshire N, Mandrup S, Ditzel HJ, Wengel J.

LNA-containing DNA aptamers against CD73 (human ecto-5′-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.


Optimal conditions for protein array deposition using continuous flow.

Anal Chem. 2008 Nov 15;80(22):8561-7. doi: 10.1021/ac8014609. Epub 2008 Oct 22.

Natarajan S, Hatch A, Myszka DG, Gale BK.

Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.


Identification Of Autoantibody Profiles By Monitoring Autoantibody Biomarkers In Rheumatoid Arthritis With Microarray Surface Plasmon Resonance Imaging.

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.

Van Beers, Joyce J.B.C., Segbers-Lokate, Angelique M.C., Egberts, Wilma T.M. Vree, Schasfoort, Richard B.M., Pruijn, Ger J.M.

Autoantibodies against citrullinated proteins (ACPA) are specifically found in approximately 75% of rheumatoid arthritis (RA) patients. Citrullination is the post-translational conversion of peptidylarginine into peptidylcitrulline, which is catalyzed by peptidylarginine deiminase (PAD) in a calcium-dependent manner. Several citrullinated antigens have been identified in the inflamed joints of RA patients. These include fibrinogen, alpha-enolase, vimentin and collagen type II. Accumulating evidence suggests a role of citrullinated proteins and ACPA in the pathophysiology of RA. The results of many studies indicate that the ACPA response is highly heterogeneous with diverse patterns of reactivity to distinct citrullinated epitopes. This study aimed to identify clinically meaningful ACPA profiles in RA patients using a microarray containing different citrullinated peptides in combination with surface plasmon resonance imaging (iSPR).


Imaging surface plasmon resonance for multiplex microassay sensing of mycotoxins.

Anal Bioanal Chem. 2011 Jul;400(9):3005-11. doi: 10.1007/s00216-011-4973-8. Epub 2011 Apr 12.

Dorokhin D, Haasnoot W, Franssen MC, Zuilhof H, Nielen MW.

A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin-protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 μg/kg for DON and 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed samples.


Continuous scaling 3d micro flow printing for improved spot morphology in protein microarrays - biomed 2013.

Biomed Sci Instrum. 2013;49:25-31.

Romanov V, Gale B, Eckman J, Miles A, Brooks B.

The protein microarray platform while innovative still poses a number of challenges which can only be met through creative and sophisticated system design. Pin printing while allowing for flexibility as to the type of medium printed does not offer the kind of spot reproducibility that a very sensitive application may require. The Continuous Flow Microspotter (CFM) was designed to not only allow for flexibility and reproducibility but to also achieve solution stability through flow scaling. This study uses the emerging CFM for printing protein and antibodies three dimensionally for general protein microarray applications. Consistent spot morphology, a continual and persistent problem in traditional pin printed microarrays, was compared under variable printed flow rates. The final assessment was performed using a rudimentary shear model. Force effects discussion and statistical data was used to demonstrate the versatility of the system.


Sensitivity of protein array deposition using continuous flow printing for fluorescent microarray applications - biomed 2013.

Biomed Sci Instrum. 2013;49:117-23.

Romanov V, Miles A, Gale B, Eckman J, Brooks B.

The promise of antibody and protein microarrays to revolutionize disease diagnostics has failed to live up to the hype primarily due to the problems associated with the printing of the antibodies and/or proteins onto the detection surface. The current standard in printing proteins is pin printing. An alternative to the pin printer is the continuous-flow microspotter (CFM), a protein printer that uses microfluidic flow to print down the proteins. The advantages of the CFM include consistent spot morphology, spot-to-spot uniformity and enhanced surface concentration. Further, the CFM is effective at capturing proteins and antibodies from either dilute or complex (e.g. blood or tissue) samples. In this study, the sensitivity of CFM printing Cy3 and Cy5 fluorescently labeled proteins was determined. Values were obtained at low concentrations tens of ng/mL with low coefficients of variation. Thus, the CFM can effectively print and quantify proteins and antibodies from low concentration and complex buffered samples.


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

Blood. 2015 Mar 12;125(11):1793-802. doi: 10.1182/blood-2014-05-579110. Epub 2014 Dec 29.

Kapur R, Heitink-Pollé KM, Porcelijn L, Bentlage AE, Bruin MC, Visser R, Roos D, Schasfoort RB, de Haas M, van der Schoot CE, Vidarsson G.

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.


Label free cell profiling.

Anal Biochem. 2013 Aug 1;439(1):4-6. doi: 10.1016/j.ab.2013.04.001. Epub 2013 Apr 11.

Schasfoort RB, Bentlage AE, Stojanovic I, van der Kooi A, van der Schoot E, Terstappen LW, Vidarsson G.
Medical Cell Biophysics Group, Faculty of Science and Technology, University of Twente, PO Box 217, 7500AE 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.


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

Anal Biochem. 2015 Sep 15;485:112-8. doi: 10.1016/j.ab.2015.06.018. Epub 2015 Jun 18.

Stojanović I, van der Velden TJ, Mulder HW, Schasfoort RB, Terstappen LW.

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.


Analysis of cell surface antigens by Surface Plasmon Resonance imaging.

Biosens Bioelectron. 2014 Feb 15;52:36-43. doi: 10.1016/j.bios.2013.08.027. Epub 2013 Aug 27.

Stojanović I, Schasfoort RB, Terstappen LW.

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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