Determination of affinities and antigenic epitopes of bovine cardiac troponin I (cTnI) with monoclonal antibodies by surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor based on wavelength modulation was used for real-time detection of the interaction of three monoclonal antibodies and antigens of bovine cardiac troponin I (cTnI). In order to recognize antigenic epitopes of bovine cTnI, two experimental modes were applied. In the first experimental mode, three monoclonal antibodies were divided into three groups and three experiments were performed on biosensor surfaces prepared with protein A. In the second experimental mode, antigen was immobilized on the biosensor surface prepared by the amine-coupling method and three monoclonal antibodies were detected in turn. The results obtained by the two modes are consistent. In addition, the affinities of the monoclonal antibodies for the antigen were also determined by the association rate and the disassociation rate in real-time. These results validate the biosensor technology and illustrate how biosensors based on wavelength modulation can be used to study the interaction of monoclonal antibodies and antigens in real time.     

Biotinylated steroid derivatives as ligands for biospecific interaction analysis with monoclonal antibodies using immunosensor devices

Systematic ligand-binding studies of the biospecific interaction between steroids and antisteroid antibodies can be performed in real time using biosensor techniques. In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensor systems were applied. Different biotinylated testosterone (T) and 17beta-estradiol (E2) derivatives were preincubated with streptavidin and immobilized on the sensor surfaces. We obtained low matrix densities of antigen enabling the investigation of the binding kinetics and position specificities of various anti-E2 and anti-T monoclonal antibodies (mAbs) to these steroidal compounds. The highest immunoreactivity of anti-E2 and anti-T mAbs is not necessarily for the specific modified steroid that was used as a protein-coupled hapten for immunization. The kinetic data confirm that both 3- and 19-specific anti-T mAbs do not discriminate between the 3- and 19-biotinylated T derivatives, whereas the 7alpha-biotinylated T probe showed no affinity to these two anti-T mAbs. In the case of the 3-specific anti-E2 mAb, comparable interaction data were found for 3- and 6alpha-biotinylated E2 compounds. The 6-specific anti-E2 mAb showed comparable ligand binding, but a significant higher dissociation rate to the position-specific antigen. The QCM and SPR results correspond well to the data from cross-reactivity studies in solution as well as to enzyme immunoassay equilibrium measurements.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.     

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.     

Surface plasmon resonance detection of interactions between peptide fragments of N-telopeptide and its monoclonal antibodies.

As populations age, osteoporosis is becoming an important public health care problem. Urinary level of the cross-linked N-telopeptide of type I collagen has been reported to be a sensitive marker of bone resorption. Recently, we synthesized and characterized 10 overlapping peptides covering the N-telopeptide of alpha-2 type I collagen and reported their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibodies determined by a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). In this study, we design an assay based on the surface plasmon resonance (SPR) technology to detect binding interaction of each peptide fragment of NTX with the anti-NTX monoclonal antibodies. Anti-NTX monoclonal antibodies were immobilized on the surface of sensor chip by amine-coupling procedure. Serial dilutions of each peptide were prepared and injected separately onto the antibodies-immobilized sensor chip. The real-time association and dissociation interactions of each peptide were detected and reported as sensorgrams. Binding response of each peptide to the monoclonal antibodies was determined, and the SPR results were compared with the ELISA results. We demonstrate that the trends of binding potency of peptide fragments detected by SPR are in good correlation to the results obtained by ELISA, indicating that our developed SPR-based method can be further applied to detect the NTX fragments in urine and to monitor the bone loss in humans. The potent peptide fragments identified by both assays are promising for further preparation of specific monoclonal antibodies in order to develop bioassays for bone loss in humans.     

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed ''ligand'') is immobilized onto a sensor chip surface, while the other (the ''analyte'') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method''s high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter''s cognate substrate binding protein).     

Capture and reconstitution of G protein-coupled receptors on a biosensor surface

Surface plasmon resonance (SPR) biosensors offer a unique opportunity to study the binding activity of G protein-coupled receptors (GPCRs) in real time with minimal sample preparation. Using two chemokine receptors (CXCR4 and CCR5) as model systems, we captured the proteins from crude cell preparations onto the biosensor surface and reconstituted a lipid environment to maintain receptor activity. The conformational states of the receptors were probed using conformationally dependent antibodies, and by characterizing the binding properties of a native chemokine ligand (stromal cell-derived factor 1alpha). The results suggest that the detergent-solubilized receptors are active for ligand binding in the presence and absence of a reconstituted bilayer. There are three advantages to using this receptor-capturing approach: (1) there is no need to purify the receptor prior to immobilization on the biosensor surface, (2) the receptors are homogeneously immobilized through the capturing step, and (3) the receptors can be captured at high enough densities to allow the study of relatively low-molecular-mass ligands (2000-4000Da). We also demonstrated that the receptors are sensitive to the solubilizing conditions, which illustrates the potential for using SPR biosensors to rapidly screen solublization conditions for GPCRs.     

Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry

BACKGROUND: The kinetics of protein-protein interactions can be monitored with optical biosensors based on the principles of either surface plasmon resonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane. METHODS: We monitored with an IASys biosensor system, based on a resonant mirror: (1) the binding of cells to an immobilized ligand, (2) the binding of a soluble ligand to immobilized cells, and (3) the binding of a soluble ligand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic flow cytometry. RESULTS: With an optical biosensor, the useful configuration is the one based on immobilized plasma membrane vesicles. However, signals can be detected only for very abundant binding sites (>10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor. CONCLUSIONS: Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode.     

Capture molecules preconditioned for kinetic analysis of high-affinity antigen-antibody complex in Biacore A100

Surface plasmon resonance (SPR) is routinely applied on determining association or dissociation constant rates of antigen-antibody complexes. In a SPR system such as Biacore, the capture method is a widely accepted procedure in kinetic analysis for association or dissociation of soluble antigen analytes with antibody ligands initially captured by anti-Fc molecules immobilized on the sensor chip. Appropriate preparations of anti-immunoglobulin G (IgG)-Fc molecules on sensor chips have not been examined yet for stable kinetic analysis of antibodies with several affinities to soluble antigens. Here, we constructed murine monoclonal antibodies (MoAbs) with various affinities to hen egg lysozyme (HEL) and performed kinetic analysis of these MoAbs captured by rat MoAbs against mouse IgG-Fc immobilized on the sensor chip. When capture molecules maximally immobilized on the sensor chip, we observed no apparent dissociation of MoAbs with extremely high affinity to soluble HEL antigens. In contrast, on the limited amount (1000-2000 response units) of capture molecule immobilized on the sensor chip, we could perform stable kinetic analysis of MoAbs with highest affinities to the antigen as well as those with lower or moderate binding affinities. Thus, in some cases, accurate kinetic analysis of high-affinity antibodies can be performed by minimization of capture molecule densities on the sensor chip in SPR.     

Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology

The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.     

Surface plasmon resonance study of the molecular recognition between polymerase and DNA containing various mismatches and conformational changes of DNA-protein complexes

Although surface plasmon resonance (SPR) biosensor technique has been used to study protein-protein interactions and to detect conformational changes of proteins, it has not been shown whether the SPR biosensor can be used to study a complex kinetic system such as the protein-DNA binding, which sometimes involves several binding steps as well as dynamic conformational changes of the complexes. In this study, we have used SPR biosensor and T7 polymerase as the model system to study the interactions of the polymerase with a series of DNA template-primer duplexes containing different number of mismatches and GC contents at various positions near the primer 3'-end. In general, the binding constants measured by the SPR are several magnitudes smaller than those determined in solution, indicating the limitation of the surface-based technique for measuring solution-based interactions. However, the distinct polymerase binding profiles obtained for DNA duplexes differed by as low as a single mismatch suggest that the SPR data can be used for relative comparison purpose among a set of experiments carried out under identical conditions. The successful fitting of the binding profiles using the established translocation model also demonstrated that SPR can be used to monitor conformational changes, as well as to derive relative kinetic values, within a complicated DNA-protein interaction system. The results also demonstrated that SPR biosensor may be used as a sensitive technique for studying molecular recognition events, such as single-base discrimination involved in protein-DNA interactions.     

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 10³ cells/ml or 50 cells were detected by the SPR biosensor.     

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.     

Immunosensor with a controlled orientation of antibodies by using NeutrAvidin-protein A complex at immunoaffinity layer

The orientation of antibody was controlled by using NeutrAvidin-protein A complex on the gold surface of SPR biosensor. The surface density of receptor antibody (anti-hIgG) was compared by treatment of receptor antibody to the layer of avidin, NeutrAvidin, protein A, NeutrAvidin-protein A complex and bare gold surface of SPR biosensor. The ligand antibody (hIgG) was injected to each IA layer and the binding ratio of ligand antibody per unit receptor was estimated as a parameter of orientation control. The NeutrAvidin-protein A complex on gold surface of SPR biosensor showed the highest surface density of receptor antibody as well as the binding ratio of ligand antibody per receptor antibody. The NeutrAvidin-protein A complex was also prepared on biotin-labelled SAM, and the binding ratio of ligand per receptor was found to be significantly improved in comparison to the IA layer prepared by chemical coupling of receptor antibody to the SAM layer. The NeutrAvidin-protein A complex which showed the highest efficiency for the binding of ligand antibodies, was applied for the detection of a cancer marker called CEA. By using NeutrAvidin-protein A complex and sandwich assay for signal amplification, sensitivity was improved to be 1.5-fold higher than bare gold surface and the detection of CEA with the detection limit of 30 ng/ml was achieved.     

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.     

Optical surface plasmon resonance biosensors in molecular fishing

An optical biosensor employing surface plasmon resonance (SPR; SPR-biosensor) is a highly efficient instrument applicable for direct real time registration of molecular interactions without additional use of any labels or coupled processes. As an independent approach it is especially effective in analysis of various ligand receptor interactions. SPR-biosensors are used for validation of studies on intermolecular interactions in complex biological systems (affinity profiling of various groups of proteins, etc.). Recently, potential application of the SPR-biosensor for molecular fishing (direct affinity binding of target molecules from complex biological mixtures on the optical biosensor surface followed by their elution for identification by LCMS/MS) has been demonstrated. Using SPR-biosensors in such studies it is possible to solve the following tasks: (a) SPR-based selection of immobilization conditions required for the most effective affinity separation of a particular biological sample; (b) SPR-based molecular fishing for subsequent protein identification by mass spectrometry; (c) SPR-based validation of the interaction of identified proteins with immobilized ligand. This review considers practical application of the SPR technology in the context of recent studies performed in the Institute of Biomedical Chemistry on molecular fishing of real biological objects.     

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG₁), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1–1297) and in the middle region (mAb 6B: aa 1480–1693; mAbs 2A, 3B: aa 2152–2377; mAbs 9A, L2 and L4: aa 2657–3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG₁-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).     

Measurement of the affinity of melittin for zwitterionic and anionic membranes using immobilized lipid biosensors.

The binding of melittin to zwitterionic dimyristyphosphatidylcholine (DMPC) and anionic dimyristylphosphatidylglycerol (DMPG) was analysed using two different immobilized model membrane systems. The first system used surface plasmon resonance (SPR), which monitors the real-time binding of peptides to an immobilized hybrid bilayer. SPR experiments reflected a stronger binding of melittin for DMPG than for DMPC, while kinetic analysis suggested the existence of at least two distinct binding steps. The second lipid biosensor system involved an immobilized phospholipid monolayer covalently attached to a microporous silica surface. The binding of melittin to the immobilized monolayer was then monitored using dynamic elution chromatography with varied methanol concentrations to analyse the binding of melittin to DMPC and DMPG. The nonlinear binding behaviour observed for melittin with the phosphatidylcholine (PC) and phosphatidylglycerol (PG) monolayers compared with the linear retention plots and Gaussian peak shapes observed for the control molecule demonstrated that melittin undergoes significant conformational and orientational changes upon binding to the immobilized PC and PG ligands. The dependence of log k' on per cent methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while other forces, presumably electrostatic in nature, also made a contribution to the affinity of the peptides for the lipid monolayer, particularly at lower temperatures. The complementary use of these two lipid biosensors thus allows the role of hydrophobic and electrostatic forces in peptide-membrane interactions to be studied.     

HX531, a retinoid X receptor antagonist, inhibited the 9-cis retinoic acid-induced binding with steroid receptor coactivator-1 as detected by surface plasmon resonance

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.