Extracting kinetic rate constants from surface plasmon resonance array systems

Surface plasmon resonance imaging systems, such as Flexchip from Biacore, are capable of monitoring hundreds of reaction spots simultaneously within a single flow cell. Interpreting the binding kinetics in a large-format flow cell presents a number of potential challenges, including accounting for mass transport effects and spot-to-spot sample depletion. We employed a combination of computer simulations and experimentation to characterize these effects across the spotted array and established that a simple two-compartment model may be used to accurately extract intrinsic rate constants from the array under mass transport-limited conditions. Using antibody systems, we demonstrate that the spot-to-spot variability in the binding kinetics was <9%. We also illustrate the advantage of globally fitting binding data from multiple spots within an array for a system that is mass transport limited.     

Real time kinetic studies of the interaction between folded antisense and target RNAs using surface plasmon resonance

Antisense RNAs interact with their complementary target RNAs as folded structures. The formation of early binding intermediates is the most important step in determining the overall rates of stable complex formation in vitro and the efficiency of control in vivo. In the case of CopA and CopT (antisense/target RNA pair of plasmid R1), recent studies have identified a four-way junction structure as the major binding intermediate. Previously, the kinetics of antisense/target RNA interaction was studied by indirect methods. Here we have used surface plasmon resonance to follow the binding of CopI (a truncated variant of CopA) to CopT in real time. A protocol was developed that permitted the determination of association and dissociation rate constants for wild-type and mutant CopI-CopT pairs. The K(D)-values calculated from these rate constants were in good agreement with the results obtained by indirect methods. In comparison to earlier model studies of interactions between simple complementary nucleic acids, we observe a different temperature dependence for dissociation rate constants. This may be indicative of the complexity of the steps required for interacting folded RNAs; intramolecular structure competes with intermolecular helix progression during complex formation. The association rate constants were not significantly dependent on temperature. The analysis presented shows that the stability of a kissing complex is not the primary determinant of the rate of stable CopA/CopT complex formation.     

Exploring computational lead optimisation with affinity constants obtained by surface plasmon resonance for the interaction of PorA epitope peptides with antibody against Neisseria meningitidis.

LUDI is a program used for de novo structure-based design of ligands and can predict binding of ligands quantitatively using a scoring function. Here we evaluate LUDI in a lead optimisation study with ligands for the antibody MN12H2, that has been raised against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. The ligands were synthetic peptides that are derived from the smallest binding epitope (182)DTNNN(186). LUDI's fragment building rules are used for the proposal of new peptide-ligands for MN12H2 and were focused on replacements of Asp(186) in the epitope. Accordingly, a series of peptides was synthesised with isosteric mutations. The interaction of the peptides with MN12H2 was analysed with a surface plasmon resonance competition assay yielding equilibrium binding constants in solution (K(S)). The binding affinity seems to be largely determined by entropy, and the side chain of Asn(186) is sensitive for charge, inversion, hydrophobicity and size. Head-to-tail cyclisation of the peptide in a nine-amino-acid ring gives little reduction in affinity. It is concluded that the scoring function of LUDI does not help in optimisation of the peptide lead for MN12H2 binding. Other more elaborate molecular mechanics calculations show similar results. This implies that our current knowledge of molecular recognition is insufficient for explaining a case of peptide-protein binding, where the design process requires subtle changes in structure (from lead finding to lead optimisation).     

Exploring computational lead optimisation with affinity constants obtained by surface plasmon resonance for the interaction of PorA epitope peptides with antibody against Neisseria meningitidis

LUDI is a program used for de novo structure-based design of ligands and can predict binding of ligands quantitatively using a scoring function. Here we evaluate LUDI in a lead optimisation study with ligands for the antibody MN12H2, that has been raised against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. The ligands were synthetic peptides that are derived from the smallest binding epitope ¹⁸²DTNNN¹⁸⁶. LUDI’s fragment building rules are used for the proposal of new peptide-ligands for MN12H2 and were focused on replacements of Asp¹⁸⁶ in the epitope. Accordingly, a series of peptides was synthesised with isosteric mutations. The interaction of the peptides with MN12H2 was analysed with a surface plasmon resonance competition assay yielding equilibrium binding constants in solution (K_S). The binding affinity seems to be largely determined by entropy, and the side chain of Asn¹⁸⁶ is sensitive for charge, inversion, hydrophobicity and size. Head-to-tail cyclisation of the peptide in a nine-amino-acid ring gives little reduction in affinity. It is concluded that the scoring function of LUDI does not help in optimisation of the peptide lead for MN12H2 binding. Other more elaborate molecular mechanics calculations show similar results. This implies that our current knowledge of molecular recognition is insufficient for explaining a case of peptide-protein binding, where the design process requires subtle changes in structure (from lead finding to lead optimisation).     

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.     

Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.

We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.     

Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance

Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins’ respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.     

Binding of anti-human-interleukin-6 monoclonal antibodies to synthetic peptides of human interleukin-6 studied using surface plasmon resonance.

Biosensor technology employing surface plasmon resonance (SPR) detection provides a highly-sensitive (sub ng), non-extrinsic labelling approach for monitoring protein interactions in real-time. We have used this approach to map the binding sites on human interleukin-6 (hIL-6) for a series of anti-hIL-6 monoclonal antibodies (mAbs). Epitopes were localised by monitoring the ability of ten synthetic peptides, spanning the sequence of hIL-6, to inhibit the binding of anti-hIL-6 mAbs to immobilised hIL-6. Peptide P8 (Pro139-Gln153) inhibited binding of anti-IL-6-mAbs 1, 2 and 7. To increase the sensitivity of detection of antibody-synthetic peptide interactions, a procedure was developed for immobilising the synthetic peptides directly to the sensor surface of the SPR instrument. From this study, association equilibrium constants of 2.1 x 10(6)M-1 and 3.6 x 10(4)M-1 were calculated for the mAb7-immobilised P8 and mAb7-free P8 interactions, respectively.     

Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides

Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.     

Development and validation of a kinetic assay for analysis of anti-human interleukin-5 monoclonal antibody (SCH 55700) and human interleukin-5 interactions using surface plasmon resonance

A method to assess the kinetic interactions of a humanized anti-human interleukin-5 (IL-5) monoclonal antibody (SCH 55700) with native human IL-5 using surface plasmon resonance (SPR) has been developed and validated. Since there are no clearly defined validation requirements for a SPR-based binding kinetic assay, the validation strategy was based on the guidelines stipulated by the International Conference on Harmonization for Analytical Method Validation. Due to the uniqueness of the method, however, proper interpretation of the guidance was critical for establishing a validation plan. Validation was designed to assess repeatability, intermediate precision, specificity, linearity, and robustness which included analysis of baseline stability and reproducibility of ligand immobilization. Additionally, system suitability criteria were established to assure that the assay consistently performs as it was intended. The experimental artifacts that can complicate kinetic analysis using biosensor technology, such as heterogeneity of the ligand, mass transport, and nonspecific binding, were considered during the development of this assay. For each run, replicate concentrations of SCH 55700 were injected randomly over the immobilized surfaces to acquire association- and dissociation-phase data. The data were transformed and double referenced to remove systematic deviations seen in the binding responses. Association and dissociation rates were determined using a bivalent analyte model for curve fitting.     

The influence of signal noise on the accuracy of kinetic constants measured by surface plasmon resonance experiments.

An analysis is carried out to investigate the accuracy of kinetic parameters obtained using surface plasmon resonance methodology with a BIAcore instrument. The Cramer Rao lower bound for the least possible variance of an estimator of the kinetic parameters is determined. Using simulations it is shown that the standard least-squares estimation technique provides estimates that achieve this bound. The theoretical and simulation results are compared with experimental data obtained from an analysis of the interaction of the myc peptide with the anti-myc antibody, 9E10. This investigation indicates that the accuracy of the results depends on the signal level which has particular relevance to the design of experiments with low signal levels. It is shown how the accuracy of the estimates of the kinetic constants depends on the kinetic constants themselves and how the accuracy of the association constants depends on the concentration of the analyte that is used in the experiment. In addition, the effects of increasing the number of data points in the analysis of dissociation data on the accuracy of the estimates are quantitated. It is also demonstrated that signal averaging of data derived from repeat sensorgrams can result in a significant decrease in the standard deviation of the estimates.     

Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy.

Hsp70 chaperones assist protein folding through ATP-regulated transient association with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze ATP and, consequently, to close its substrate binding cavity allowing trapping of substrates. We analyzed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with DnaJ using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a sensor chip with coupled DnaJ. This interaction was specific as it was not detected with a functionally defective DnaJ mutant protein, DnaJ259, that carries a mutation in the HPD signature motif of the conserved J-domain. Detectable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was competitively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. These findings indicate that the detected response signals resulted from DnaJ and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. Although not considered as physiologically relevant, this association allowed us to experimentally unravel the mechanism of DnaJ action. Accordingly, DnaJ stimulates ATP hydrolysis only after association of a substrate with the substrate binding cavity of DnaK. Further analysis revealed that this coupling mechanism required the J-domain of DnaJ and was also functional for natural DnaK substrates, and thus is central to the mechanism of action of the DnaK chaperone system.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Purification of recombinant human B-domain-deleted factor VIII using anti-factor VIII monoclonal antibody selected by the surface plasmon resonance biosensor.

The surface plasmon resonance (SPR) biosensor measures the real-time kinetics of noncovalent interaction between a receptor and its ligand. Monoclonal antibodies (mAbs) against recombinant factor VIII (rFVIII) were screened from 127 mAb candidates using the SPR biosensor for the purpose of affinity purification of rFVIII. Each mAb showed a different association and dissociation capacity for rFVIII at each buffer condition. One mAb, F8-38, was selected for immunopurification of rFVIII. To characterize the selected mAb F8-38, the immunopurification results on the anti-FVIII mAb F8-38 affinity gel and the anti-von Willebrand factor (vWF) mAb affinity gel were studied. Immunopurification by the anti-vWF affinity gel showed a lower binding capacity of rFVIII and resulted in low purification efficiency. On the other hand, immunopurification by the anti-FVIII affinity gel exhibited a 3.5-fold binding capacity and a 2-fold purification efficiency compared to those of the anti-vWF affinity gel. The amounts of proteins and DNAs derived from host cells and mouse IgGs derived from the affinity matrix in the affinity eluate were similar to those of the anti-vWF affinity gel. In conclusion, the SPR method of immunopurification is a useful technology in the screening of mAbs aimed at the development of an affinity purification procedure, and the mAb F8-38 was selected using this technology on the basis of the purification procedure of rFVIII.     

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.     

The investigation of recognition interaction between phenylboronate monolayer and glycated hemoglobin using surface plasmon resonance

Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.     

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.