Variable wavelength surface plasmon resonance (SPR) in biosensing

In this study, we fabricated a novel variable wavelength surface plasmon resonance (SPR) sensor, which detects resonance conditions such as a maximum attenuation wavelength, measuring change of microscopic refractive index. Such a change was measured to detect a salmonella antigen–antibody reaction and a penicillinase–penicillin reaction. Our experiments were performed after immobilizing a salmonella antibody on the sensor chip. We measured the shift in resonant wavelength during the antigen–antibody reaction for 30 min by injecting 5 × 10⁷ cells/ml concentration of salmonella antigen solution into the sample chamber. Also, after immobilizing penicillinase on the sensor chip, we measured the shift in resonant wavelength during the reaction. Penicillin solution at 10 mM was injected into the sample chamber. The shift of resonant wavelength for each experiment was measured using a white light source, multimode optical fiber, a part of sensor chip and an optical spectrum analyzer.As a result, the resonant wavelength shifted about 0.26 nm/min owing to the salmonella antibody–antigen reaction. Thus, we could detect the change in wavelength (0.8 nm/min) through the interaction of penicillin and penicillinase for 15 min using variable wavelength SPR sensor.     

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.     

Synthesis of interphotoreceptor retinoid-binding protein (IRBP) by monkey retina in organ culture: effect of monensin

Whole monkey retinas were incubated in short-term organ culture with either radiolabeled amino acids or glucosamine. Soluble retinal proteins and proteins in the culture medium were analyzed by SDS-poly-acrylamide gel electrophoresis. Fluorography showed that the interphotoreceptor retinoid-binding protein (IRBP), a 146,000 Mr glycoprotein localized in the extracellular matrix, is synthesized by the neural retina and rapidly secreted into the medium. Secretion is blocked by 10-5M monensin. No significant IRBP synthesis was observed in the pigment-epithelium-choroid complex. IRBP is thus the major component synthesized and secreted by the neural retina into the interphotoreceptor space. This, and its affinity for retinoid makes it a prime candidate for an extracellular retinoid transport vehicle.     

Reduced level of interphotoreceptor retinoid-binding protein (IRBP), a possible cause for retinal degeneration in the Abyssinian cat

Summary Retinae of Abyssinian cats homozygous for a retinal degeneration gene, and normal controls, have been investigated using antibodies directed against opsin, transducin (TD-), S-antigen (48K protein), interphotoreceptor retinoid-binding protein (IRBP), and cone outer segments. IRBP-immunoreactivity (IR) is much reduced at stage 2 of the disease in affected retinae; later massive photoreceptor cell death occurs. In cats, at a late stage of the disease, the retina exhibits few S-antigen-IR cells in the peripheral part of the retina whereas, in the central part, some patches of cells exhibiting opsin-IR, TD--IR, and S-antigen-IR are present in remnants of the outer nuclear layer (ONL). No IRBP-IR is detectable at this stage. The form and size of the majority of these remaining cells, however, does not resemble that of normal photoreceptors. No, or only rudimentary, inner and outer segments are present; long bifurcating basal protrusions often occur. These cells, which could be remains of cone elements, are S-antigen immunoreactive. Double labelling for different retina-specific proteins reveals a co-localization of opsin, TD- and S-antigen in some, but not all, remaining photoreceptor elements. Cells exhibiting opsin-IR also show TD--IR and S-antigen-IR located within the entire cell and its protrusions. In control retinae and retinae at early stages of the disease, immunoreactions are comparable with all antibodies used. However, TD--IR is less intensive in the photoreceptor terminals. S-antigen-IR cones are most frequently present in the peripheral retina. Reduction of IRBP at an early stage of the disease could be one of the factors leading to photoreceptor cell death at later stages.     

A fusion protein expression analysis using surface plasmon resonance imaging

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.     

Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands

Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z′ factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.     

High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates

We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR)

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.     

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.     

Comparison between the surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) method in a structural analysis of human endothelin-1

In this study, an automated surface plasmon resonance (SPR)-based biosensor was compared with a quartz crystal microbalance (QCM) biosensor. The two biosensor systems were used for characterizing a site-directed monoclonal antibody (mAb), raised against the C-terminal heptapeptide ET-1_15–21 of the human endothelin (ET-1). The mAb was characterized by its capacity for binding to ET-1, ET-3, Big.ET-1_22–38, the C-terminal (ET-1_15–21, ET-1_16–21, ET-1_17–21), and six derivates of ET-1_16–21, each containing a substitution with alanine (Ala) of a single aminoacid from position 16–21, respectively. The mAb reacted well with ET-1 and its fragments ET-1_15–21, ET-1_16–21, ET-1_17–21, but showed only a partial cross-reaction with ET-3, and did not bind human Big.ET-1_22–38. The Ala substitution on position 16,17, or 19 of ET-1_16–21 did not affect the antibody binding capacity of the hexapaptide ET-1_16–21. On the contrary, Ala substitution or Asp¹⁸, Ile²⁰ and particularly Trp²¹, inhibited its immunoreactivity. Thus the C-terminal represents an immunodominant epitope in ET-1 and is important for antibody binding. The SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, we found a good correlation between results obtained using the BIA technology and the QCM.     

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10⁻¹³ M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.     

A method for microbial cell surface fingerprinting based on surface plasmon resonance

A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).     

NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance

Nitrilotriacetate (NTA)-mediated capture of a histidine-tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA-capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine-tagged SH3 domain of the human phosphatidylinositol 3-kinase p85alpha subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%-deviation against the theoretical binding capacity.     

Enhancement of the sensitivity of surface plasmon resonance (SPR) immunosensor for the detection of anti-GAD antibody by changing the pH for streptavidin immobilization

Surface plasmon resonance (SPR) immunobiosensor was developed for the detection of anti-glutamic acid decarboxylase (GAD) antibody. In this study, carboxylic terminated self-assembled monolayer, which was prepared by mixing of 3-mercaptopropionic acid (3-MPA) and 11-mercaptoundecanoic acid (11-MUA) (10:1 ratio), was used to evaluate the effect of external pH on the affinity between streptavidin and sensor surface. At pH values ranging from 4.0 to 5.5, it was found that streptavidin could more easily access onto the sensor surface at higher pH, and the enhanced binding of streptavidin at high pH allowed more extensive immobilization of biotin-GAD, which serves as the epitope for anti-GAD antibody. Consequently, the increase of RU caused by immuno-response between GAD and anti-GAD antibody was remarkably higher when streptavidin was bound on to the sensor surface at pH 5.5 than at pH 4.5. Therefore, we could conclude that the pH of coupling buffer greatly influences the sensitivity of immunosensor.     

A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates,casein and calmodulin

Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, -subunits, and two regulatory, -subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is down regulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit, and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30°C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2 subunit and protein substrates share a common, or at least an overlapping site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine.     

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

Combinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(β1-O)Thr, Gal(β1-S)Cys/Gal(β1-N)Asn, and Lac(β1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR)

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody–antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (<1.25 μg/ml) in a physiologically relevant range (1–40 μg/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.