Kinetic analysis of IgG antibodies to beta-amyloid oligomers with surface plasmon resonance

Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer’s disease.     

Advances in surface plasmon resonance biosensor analysis

The number and diversity of surface plasmon resonance (SPR) biosensor applications continue to increase. Evolutions in instrument and sensor chip technology, experimental methodology, and data analysis are making it possible to examine a wider variety of biomolecular interactions in greater mechanistic detail. SPR biosensors are poised to make a significant impact in basic research and pharmaceutical discovery.     

Studies of interactions with weak affinities and low-molecular-weight compounds using surface plasmon resonance technology

Interactions between the immobilized weak-affinity monoclonal IgG antibody 39.5, which is specific for the glucose-alpha 1,4-glucose motif, and various oligosaccharides were studied with surface plasmon resonance technology. The antibody was immobilized at high levels on the surface of the sensor chip and different concentrations of the analytes were injected at 25 and 40 degrees C. The 39.5 antibody exhibited specific binding to maltose, tetraglucose and maltotriose, with dissociation constants Kd in the range from 0.07 mM (25 degrees C) to 1.0 mM (40 degrees C). Association and dissociation rate constants (ka and kd) were rapid and baseline was obtained almost immediately after the end of each antigen injection. This excluded the need for a regeneration step but also made calculation of the kinetic values impossible. Owing to the weak affinity and the small size of the analytes (< 1000 Da), a careful design of control surfaces is demanded to exclude artefactual results.     

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.     

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.     

Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.     

Specificity analysis of human anti-DNA antibodies

Human hybrids producing anti-DNA antibodies were generated by the fusion of pokeweed mitogen-stimulated splenic lymphocytes from a child with sickle cell anemia to GM4672. Of 19 hybrids, three (15%) produced anti-DNA antibody as detected by an enzyme linked immunosorbent assay. One subclone from each of these three hybrids was then characterized. All produced IgM antibody in large amounts ranging from 22 to 266 micrograms/ml per million cells per 24 hr. All three antibodies bound both double- and single-stranded DNA. Competitive inhibition assays revealed the greatest inhibition of DNA binding with the ribohomopolymers polyinosinic and polyguanylic acid. A complex pattern of cross-reactivity with various other polynucleotides and with some phospholipids was observed. Subtle differences were found among the three antibodies in light chain class and some of the binding specificities. By using a modified Farr assay, all three monoclonals were found to be of low to intermediate affinity. These results confirm that anti-DNA antibodies apparently equivalent to those seen in patients with SLE can be derived from "normal" nonautoimmune individuals.     

Real time analysis of intact organelles using surface plasmon resonance

Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000 g brain supernatant (1-10 microg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, Kd), and protein profiling.     

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.     

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.     

Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates.     

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.     

Synthesis of polyrotaxane-biotin conjugates and surface plasmon resonance analysis of streptavidin recognition

A polyrotaxane-biotin conjugate was synthesized and its interaction with streptavidin measured using surface plasmon resonance (SPR) detection. A biodegradable polyrotaxane in whichca. 22 molecules of α-cyclodextrins (α-CDs) were threaded onto a poly(ethylene oxide) chain (M _n: 4,000) capped with benzyloxycarbonyl-L-phenylalanine was conjugated with a biotin hydorazide and 2-aminoethanol after activating the hydroxyl groups of α-CDs in the polyrotaxane usingN,N′-carbony diimidazole. The results of the high-resolution¹H-nuclear magnetic resonance (¹H-NMR) spectra and gel permeation chromatography of the conjugate showed thatca. 11 biotin molecules were actually introduced to the polyrotaxane scaffold. An SPR analysis showed that the binding curves of the biotin molecules in the conjugate on the streptavidin-deposited surface changed in a concentration dependent manner, indicating that the biotin in the conjugate was actually recognized by streptavidin. The association equilibrium constant (K _a) of the interaction between the conjugate and streptavidin tetramer was of the order 10⁷. These results suggest that polyrotaxane is useful for scaffolds as a polymeric ligand in biomedical fields.     

Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)–V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K_D = 0.93 nM to K_D = 348 nM). That decrease in the affinity of drug–ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug–ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.     

Monitoring the irradiation-induced conformational changes of ovalbumin by using monoclonal antibodies and surface plasmon resonance

Two types of conformationally specific anti-irradiated ovalbumin monoclonal antibodies were prepared in order to study and monitor irradiation-induced structural changes in the ovalbumin molecule. Surface plasmon resonance (SPR) detection was used to investigate the kinetic parameters of the reaction between antibodies and ovalbumin which had been administered with different doses of irradiation (0, 1.5, 2.0, 5.0, 10, 20, 50, and 100 kGy). The results demonstrate that the combination of monoclonal antibodies and the SPR method can be used to characterize the irradiation-induced conformational change with an unlabelled reagent.     

Biopharmaceutical production: Applications of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed.     

Experimental design for analysis of complex kinetics using surface plasmon resonance

Using BIAcore surface plasmon resonance technology, we found that the real-time association kinetics of Fabs specific for hen egg-white lysozyme did not conform to a 1:1 Langmuir association model. Heterogeneity of the components is not the source of the complex kinetics. Informed by independent structural data suggesting conformational flexibility differences among these antibodies, we chose global mathematical analysis based on a two-phase model, consistent with the encounter-docking view of protein-protein associations. Experimental association times (T(a)) from 2 to 250 min revealed that initial dissociation rates decreased with increasing T(a), confirming a multiphasic association. The relationship between observed dissociation rate and T(a) is characteristic of each antibody-antigen complex. We define a new parameter, T(50), the time at which the encounter and final complexes are of equimolar concentration. The observed T(50) is a function of analyte concentration and the encounter and docking rate constants. Simulations showed that when the ligand is saturated at high analyte concentrations, T(50) reaches a minimum value, T(50)(MIN), which can be used to compare antigen-antibody complexes. For high-affinity complexes with rapid rearrangement to a stable complex, T(50)(MIN) approaches T(1/2) of the rearrangement forward rate constant. We conclude that experiments with a range of T(a) are essential to assess the nature of the kinetics, regardless of whether a two-state or 1:1 model is applicable. We suggest this strategy because each T(a) potentially reveals a different distribution of molecular states; for two-step analysis, a range of T(a) that brackets T(50) is optimal.     

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCL_XL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.     

Mechanism for induction of anti-DNA antibodies by bacterial lipopolysaccharides in mice; II. Correlation between anti-DNA induction and polyclonal antibody formation by various polyclonal B lymphocyte activators.

The capacity of various polyclonal B lymphocyte activators (PBA) to induce, in mice, the formation of anti-DNA antibodies was compared with their ability to mediate the release of DNA in circulating blood and to stimulate polyclonal antibody synthesis in vivo. Anti-DNA antibodies or polyclonal antibody synthesis were induced in mice after the injection of at least 10 microgram lipopolysaccaride (LPS) from Salmonella typhimurium, 1 mg dextran sulfate (DS), or 2 mg purified protein derivative of tubercle bacteria RT32 (PPD). Smaller quantities of LPS (0.1 microgram) or DS (500 microgram) were sufficient to cause the release of DNA in circulating blood, whereas PPD was not able to provoke such a release at any concentration used. The association of anti-DNA antibodies with polyclonal antibody synthesis in mice injected with various PBA contrasts with the lack of correlation between the formation of anti-DNA antibodies and the release of measurable amounts of DNA in circulating blood. These results strongly suggest that the induction of anti-DNA antibodies by PBA is a consequence of the polyclonal B lymphocyte activation.