Ivan Stojanovic

Analysis of cell surface antigens by Surface Plasmon Resonance imaging

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.

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

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.

Label-free cell profiling

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.

Capture of Tumor Cells on Anti-EpCAM-Functionalized Poly(acrylic acid)-Coated Surfaces

The presence of tumor cells in blood is predictive of short survival in several cancers and their isolation and characterization can guide toward the use of more effective treatments. These circulating tumor cells (CTC) are, however, extremely rare and require a technology that is sufficiently sensitive and specific to identify CTC against a background of billions of blood cells. Immuno-capture of cells expressing the epithelial cell adhesion molecule (EpCAM) are frequently used to enrich CTC from blood. The choice of bio conjugation strategy and antibody clone is crucial for adequate cell capture but is poorly understood. In this study, we determined the binding affinity constants and epitope binding of the EpCAM antibodies VU1D-9, HO-3, EpAb3-5, and MJ-37 by surface plasmon resonance imaging (SPRi). Glass surfaces were coated using a poly(acrylic acid) based coating and functionalized with anti-EpCAM antibodies. Binding of cells from the breast carcinoma cell line (SKBR-3) to the functionalized surfaces were compared. Although EpAb3-5 displayed the highest binding affinity HO-3 captured the highest amount of cells. Hence we report differences in the performance of the different antibodies and more importantly that the choice of antibody to capture CTC should be based on multiple assays.

Surface plasmon resonance is an analytically sensitive method for antigen profiling of extracellular vesicles

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 (RHS578T cells2 = 0.66, RMCF7 cells2 = 0.78, RSKBR3 cells2 = 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 (R2HS578T EVs = 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 (RHS578T2 = 0.77, RMCF72 = 0.49, RSKBR32 = 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.

Modeling single cell antibody excretion on a biosensor

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.

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

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.