Surface Plasmon Resonance Approach Research Articles

Establishment of platform for screening insulin-like growth factor-1 receptor inhibitors and evaluation of novel inhibitors

The insulin-like growth factor-1 receptor (IGF1R) is over-expressed in a wide variety of tumors and contributes to tumor cell proliferation, metastasis and drug resistance. The aim of this study was to establish a sensitive screening platform to identify novel IGF1R inhibitors. The catalytic domain of IGF1R was expressed using the Bac-to-Bac baculovirus expression system. The screening platform for IGF1R inhibitors was established based on ELISA. The binding profile of IGF1R with the inhibitors was predicted with molecular docking and then subjected to the surface plasmon resonance (SPR) approach. The growth inhibition of cancer cells by the inhibitors was assessed with MTT assay. Apoptosis was analyzed using flow cytometry and Western blotting. A naturally occurring small molecule compound hematoxylin was identified as the most potent inhibitor (IC₅₀ value=1.8±0.1 μmol/L) within a library of more than 200 compounds tested. Molecular simulation predicted the possible binding mode of hematoxylin with IGF1R. An SPR assay further confirmed that hematoxylin bound directly to IGF1R with high binding affinity (Kd=4.2 × 10⁻⁶ mol/L). In HL-60 cancer cells, hematoxylin inactivated the phosphorylation of IGF1R and downstream signaling and therefore suppressed cell proliferation. Mechanistic studies revealed that hematoxylin induced apoptosis in HL-60 cells via both extrinsic and intrinsic pathways. A simple, sensitive ELISA-based screening platform for identifying IGF1R inhibitors was established. Hematoxylin was identified as a promising IGF1R inhibitor with effective antitumor activity that deserves further investigation.

Evaluation of Surface Plasmon Resonance Biosensors for Detection of Tetrodotoxin in Food Matrices and Comparison to Analytical Methods

Tetrodotoxin (TTX) is a low molecular weight neurotoxin found in a number of animal species, including pufferfish. One emerging method for TTX detection employs surface plasmon resonance (SPR) immunosensors. SPR, an optical technique that allows for label-free, real-time, multiplexed analysis, can have detection limits that rival many of the conventional transduction methods. Preliminary SPR approaches for TTX were successful, yet suffered from low throughput and used noncommercial instrumentation. To advance this method for broader use, the immunoassay was transferred to a commercial instrument and optimized for improved detection. This manuscript outlines the assay development and results for complex matrices relevant to seafood safety (pufferfish) and food adulteration (milk, apple juice). In addition, results are compared to those obtained using receptor binding assay, ELISA, HPLC-FD, and LC/MS/MS detection techniques. Results highlight the advantages of SPR assays, including rapid screening capability with low reagent consumption and low- to subppb detection limits.

PH006, a novel and selective Src kinase inhibitor, suppresses human breast cancer growth and metastasis in vitro and in vivo

The central role of Src in tumor progression and metastasis has validated it as an attractive therapeutic target for the treatment of human breast cancer. The aim of this study was to identify potential Src kinase inhibitor, explore its activity, and mechanism of action in human breast cancer. A strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and high-throughput screening was adopted and a novel 6-hydrazinopurine-based inhibitor of c-Src kinase PH006 was obtained. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay. The binding mode between PH006 and Src was profiled by surface plasmon resonance approach and molecular simulation. The anti-proliferative activity was evaluated by Sulforhodamin B (SRB) and Colony formation. The anti-invasion and anti-migration activities were assessed by trans-well and wound healing assay. Results indicated that PH006 was an ATP-competitive Src inhibitor, which selectively inhibited c-Src with an IC₅₀ of 0.38 μM among a panel of 14 diverse tyrosine kinases. PH006 potently inhibited c-Src phosphorylation and c-Src-dependent signal transduction, resulting in inhibition of cell proliferation, migration, and invasion in human breast cancer MDA-MB-231 cells. Further study demonstrated that the anti-proliferative activity of PH006 was ascribed to its capability to arrest cells in G1 phase, while its anti-motility activity was related to suppression of MMP2/9 and HGF secretion. Moreover, PH006 exhibited potent activity against tumor growth as well as metastasis of human breast cancer MDA-MB-435 xenograft beard in nude mice, which was accompanied with reduced Src/FAK signaling in tumor tissue. Taken together, PH006 is a novel selective inhibitor of c-Src and possesses potent activity against breast cancer growth and metastasis, which could be potentially developed as a lead candidate against breast cancers with elevated Src tyrosine kinase activity.

Investigation of the effects of design parameters on sensitivity of surface plasmon resonance biosensors

Surface based analytical tools have gained more importance for rapid, sensitive and label-free monitoring of molecular recognition events. The surface plasmon resonance (SPR) approach has played a prominent role in real time monitoring of surface binding events. This paper first provides a brief description of the existing biosensing methods. Next, an investigation of the role of thin films of gold, silver, and aluminum for protein detection in SPR biosensors is presented. It is shown that the sensitivity, which is indicated by the shift of plasmon dip, is not linearly related to the thickness of protein but quadratic over a specific range. The approach involves plotting the reflectivity curve as a function of the angle of incidence.

Surface plasmon resonance instrument as a refractometer for liquids and ultrathin films

A surface plasmon resonance (SPR) setup in Kretschmann configuration is being utilized as a refractometer for both liquids as well as ultrathin films. The SPR signal detection technology used is based on a goniometer approach providing a wide angular scan range which facilitates highly accurate liquid and gas phase measurements. Attention was paid to improve sample handling and preparation. In order to avoid cross-contamination between measurements an easily removable and exchangeable molded PDMS flow cell was used during the measurements. By careful choice of components for liquid handling the dead volume of the system could be reduced down to some microliters. The angular change and thus the refractive index for sucrose, ethylene glycol (EG) and ethanol solutions with different concentrations, the thickness and refractive index of deposited Langmuir-Blodgett (LB) films, and the interaction kinetics between a biotin containing self-assembled monolayer (SAM) and streptavidin were determined. The measured refractive indices of sucrose, EG and ethanol solutions corresponded well with literature values. LB films were characterized by measuring the complete SPR curve in an angular scan range from 40 to 78°. A two-color SPR approach combined with two-media measurements was successfully employed for simultaneous and unambiguous determination of both refractive index and thickness of stearic acid monolayers. The thickness obtained for the stearic acid monolayer was 2.66 nm, and the refractive indices at 635 and 670 nm were 1.5800 and 1.4138, respectively. The developed sensor-plate holder enabled functionalization of the SPR gold chip outside the instrument, therefore making the sample handling more flexible. The affinity constant obtained for the streptavidin–biotin interaction was 1.01 × 10 −8 M. The total angle SPR method used in this study clearly shows its potential to be used as a refractometer for both liquids and ultrathin films, as well as for traditional liquid phase biomolecular kinetic studies.

The Marine-Derived Oligosaccharide Sulfate (MdOS), a Novel Multiple Tyrosine Kinase Inhibitor, Combats Tumor Angiogenesis both In Vitro and In Vivo

Despite the emerging success of multi-targeted protein tyrosine kinase (PTK) inhibitors in cancer therapy, significant side effects and resistance concerns seems to be avoided unlikely. The aim of the present study was to identify novel multi-targeting PTK inhibitors. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay (ELISA). The antiproliferative activities in human microvascular endothelial cells (HMECs) were evaluated by sulforhodamine (SRB) assay. The phosphorylation of kinases and their downstream molecules was probed by western blot analysis. The binding mode between MdOS and PTKs was profiled by surface plasmon resonance (SPR) approach and molecular simulation. Tube formation assay, rat aortic ring method and chicken chorioallantoic membrane assay were combined to illustrate the in vitro and in vivo anti-angiogenic effects. Results indicated that MdOS, a novel marine-derived oligosaccharide sulfate, exhibited a broad-spectrum PTK inhibitory action. At an enzymatic level, MdOS inhibited HER2, EGFR, VEGFR, PDGFR, c-Kit, FGFR1 and c-Src, with little impact on FGFR2. In cellular settings, MdOS inhibited phosphorylation of PTKs, exemplified by HER2, EGFR and VEGFR2, and downstream molecules of Erk1/2 and AKT. Further studies demonstrated that MdOS acted as an ATP-competitive inhibitor via directly binding to the residues of entrance rather than those of the ATP-binding pocket. Furthermore, MdOS inhibited proliferation and tube formation of HMECs, arrested microvessel outgrowth of rat aortic rings and hindered the neovascularization of chick allantoic membrane. Taken together, results presented here indicated that MdOS exhibited anti-angiogenic activity in a PTK-dependent manner and make it a promising agent for further evaluation in PTK-associated cancer therapy.

Tissue transglutaminase modulates α‐synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated alpha-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant alpha-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective alpha-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/alpha-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all alpha-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal alpha-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal beta-sheet-containing oligomers, the tTG/alpha-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant alpha-synuclein variants. We propose that tTG cross-linking imposes structural constraints on alpha-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

Real-time protein biosensor arrays based on surface plasmon resonance differential phase imaging

This paper reports the application of differential phase surface plasmon resonance (SPR) imaging in two-dimensional (2D) protein biosensor arrays. Our phase imaging approach offers a distinct advantage over the conventional angular SPR technique in terms of utilization efficiency of optical sensor elements in the imaging device. In the angular approach, each biosensor site in the biosensor array requires a linear array of optical detector elements to locate the SPR angular dip. The maximum biosensor density that a two-dimensional imaging device can offer is a one-dimensional SPR biosensor array. On the other hand, the phase-sensitive SPR approach captures data in the time domain instead of the spatial domain. It is possible that each pixel in the captured interferogram represents one sensor site, thus offering high-density two-dimensional biosensor arrays. In addition, our differential phase approach improves detection resolution through removing common-mode disturbances. Experimental results demonstrate a system resolution of 8.8 × 10 −7 RIU (refractive index unit). Real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels was achieved using a biosensor array. The detection limit was 0.77 μg/ml. The reported two-dimensional SPR biosensor array offers a real-time and non-labeling detection tool for high-throughput protein array analysis. It may find promising applications in protein therapeutics, drug screening and clinical diagnostics.

Real-time detection of Cu 2+ sequestration and release by immobilized apo-metallothioneins using SECM combined with SPR

Scanning electrochemical microscopy (SECM) combined with surface plasmon resonance (SPR), SECM–SPR, was applied for real-time detection of the incorporation of Cu 2+ by apo-metallothionein (apo-MT) immobilized on the SPR substrate and release of Cu 2+ from surface-confined metallothionein (MT). Cu 2+ anodically stripped from a Cu-coated SECM Au tip was sequestered by apo-MT upon its diffusion to the SPR substrate, and release of Cu 2+ by MT was accomplished by generating protons via oxidation of hydroquinone at the tip. The high sensitivity of the SPR instrument is capable of following the structural and compositional changes of MT molecules during the metal sequestration and release processes. Due to the enhanced mass transfer rate at the SECM tip, the complication of mass transfer limitation on kinetic measurements, commonly encountered in flow injection SPR, is circumvented. The time-resolved SPR response reveals stepwise changes among three stable MT structures and allows the number of copper ions coordinated in each structure to be determined. The numbers of copper ions incorporated by each MT molecule in the three structures were determined to be 5, 9, and 12. This work expands the SECM–SPR approach to assessments of the dynamics and affinity of binding of small ions to surface-confined proteins and to studies of proteins that do not undergo facile electron transfer reactions.

Rapid Characterization of Drug-drug Interaction in Plasma Protein Binding using a Surface Plasmon Resonance Biosensor

High-throughput characterization of drug-drug interactions in plasma protein binding was demonstrated by using a surface plasmon resonance (SPR) biosensor. The method used in this study enabled the discrimination between the two modes of binding inhibition, direct competition and negative allosteric effect, which was difficult in conventional SPR approaches. Two theoretical equations were used representing SPR binding response for directly competitive binding or for independent binding. The experimental binding data for human serum albumin was processed by non-linear least squared regression of the equations. By this approach, drug-drug interactions were classified into three modes, direct competition, independent binding, and allosteric interaction, which were almost consistent with previous reports. In addition, dissociation constants were also estimated roughly for direct competition and for independent binding. The analytical throughput was almost as high as in the previous reports; three minutes per injection. This method is a powerful tool for the characterization of drug-drug interaction at an early stage of new drug development.

Chemical sensors based on molecularly modified metallic nanoparticles

This paper presents a concise, although admittedly non-exhaustive, didactic review of some of the main concepts and approaches related to the use of molecularly modified metal nanoparticles in or as chemical sensors. This paper attempts to pull together different views and terminologies used in sensors based on molecularly modified metal nanoparticles, including those established upon electrochemical, optical, surface Plasmon resonance, piezoelectric and electrical transduction approaches. Finally, this paper discusses briefly the main advantages and disadvantages of each of the presented class of sensors.

Kinetics of cholesterol extraction from lipid membranes by methyl-β-cyclodextrin—A surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-β-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-β-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl- sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.

Comparative expression of different antibody formats in mammalian cells and Pichia pastoris

Generation of recombinant antibody fragments has been advanced by phage display technology but their broad use in biochemical or analytical applications is often hindered by their univalence. For enhancement of functional affinity and overall applicability, the fusion of scFvs (single-chain variable fragments) to IgG constant domains has become an attractive approach. In order to evaluate characteristics and expression behaviour of different IgG-analogous antibody formats, we fused an scFv to different portions of the heavy chain constant region of human IgG1. Two types of antibodies, an scFv-C(H)2-3 antibody and an scFv-C(H)1-3 antibody, a new intermediate with retained C(H)1 domain, were generated with or without an affinity tag for purification. Additionally, the scFv was reconverted into the heterotetrameric IgG molecule. To allow a reliable comparison of expression behaviours of different antibodies, we established vector systems that allow isogenetic and efficient expression of the recombinant antibodies based on site-specific recombination. Upon recombinant expression in mammalian cells and the methylotrophic yeast Pichia pastoris, disulfide-linked and glycosylated oligomers were obtained. Establishment of isogenetic cell lines revealed that the presence of the C(H)1 domain is not critical for secretion efficiency. Reactivity of the different constructs with antigen and Fc receptors was verified by ELISA, surface plasmon resonance approaches, as well as FACS analysis of HEK-293 cells (human embryonic kidney cells) stably transfected with human FcgammaRI (high-affinity IgG receptor) (CD64). In summary, the results obtained provide evidence for comparable behaviour of the different antibody formats and the vectors for isogenetic expression will contribute to a broader application of phage display-derived antibodies.

Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

A new approach for analyzing lipid–lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids, and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in Escherichia coli, purified to homogeneity by mutated streptavidin (SoftLink) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [ 14C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on- and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD–cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thereby validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) the ability to study lipids in their natural states (i.e., without relatively large reporter groups) and (ii) the ability to measure on- and off- rate constants as well as equilibrium constants.

Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process

Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen–oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

Characterization of Human Complement Receptor Type 2 (CR2/CD21) as a Receptor for IFN-α: A Potential Role in Systemic Lupus Erythematosus

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha, a multifunctional cytokine important in the innate immune system, has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus, IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction.

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.