scholarly journals High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A

2001 ◽  
Vol 69 (1) ◽  
pp. 570-574 ◽  
Author(s):  
Dorothy D. Pless ◽  
Edna R. Torres ◽  
Emily K. Reinke ◽  
Sina Bavari
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Botulinum Neurotoxin ◽  
Epitope Mapping ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Domain ◽  
Protective Antibodies ◽  
Fusion Products

ABSTRACT Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A HC. Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A HC was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A HC fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.

scholarly journals Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 22 ◽  
Author(s):  
Devendra Bhandari ◽  
Fur-Chi Chen ◽  
Shreya Hamal ◽  
Roger Bridgman
Keyword(s):  
Monoclonal Antibodies ◽  
Surface Plasmon Resonance ◽  
Salmonella Typhimurium ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Epitope Mapping ◽  
Dissociation Constants ◽  
Interaction Model ◽  
Dissociation Rate ◽  
Surface Plasmon Resonance Biosensor

Salmonella Typhimurium is one of the leading causes of foodborne diseases worldwide. Biosensors and immunoassays utilizing monoclonal antibodies are widely used for the detection and subtyping of S. Typhimurium. However, due to insufficient information on the nature of binding with S. Typhimurium flagellin, the selection of appropriate antibodies for assay development is a cumbersome task. Hence, we aimed to compare the binding kinetics of a panel of monoclonal antibodies and their relative binding sites to flagellin antigen using a surface plasmon resonance biosensor. Initially, the flagellin was captured on the sensor surface through an immobilized anti-flagellin antibody. The interactions of different concentrations of monoclonal antibodies to flagellin were determined, and binding curves were fitted using 1:1 bio-interaction model to calculate the kinetic parameters. For epitope mapping, pairwise comparisons were completed to determine the binding inhibition of each paired combination of monoclonal antibodies. It was found that these monoclonal antibodies differed significantly (p < 0.05) in association rate, dissociation rate, and equilibrium dissociation constants. Of the five monoclonal antibodies, only two interfered with the binding of each other. Four distinct epitopes located within a 23 kDa domain of flagellin were identified. Findings from this study provide crucial information needed for the further development and optimization of biosensors and other immunoassays for the detection and subtyping of Salmonella.

Localized and Propagating Surface Plasmon Resonance Sensors: A Study Using Carbohydrate Binding Protein

2005 ◽  
Vol 876 ◽  
Author(s):  
Chanda Yonzon ◽  
Richard P. Van Duyne
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Specific Binding ◽  
Gold Surface ◽  
Optical Biosensor ◽  
Localized Surface Plasmon ◽  
Carbohydrate Binding ◽  
Binding Studies ◽  
Assembled Monolayers

AbstractThis work encompasses a comparative analysis of the properties of two optical biosensor platforms: (1) the propagating surface plasmon resonance (SPR) sensor based on a planar, thin film gold surface and (2) the localized surface plasmon resonance (LSPR) sensor based on surface confined Ag nanoparticles fabricated by nanosphere lithography. The binding of Concanavalin A (ConA) to mannose-functionalized self-assembled monolayers (SAMs) is chosen to illustrate the similarities and the differences of these sensors. A comprehensive set of non-specific binding studies demonstrate that the single transduction mechanism is due to the specific binding of ConA to the mannose-functionalized surface. Finally, an elementary (2x1) multiplexed version of a LSPR carbohydrate sensing chip to probe the simultaneous binding of ConA to mannose and galactose-functionalized SAMs is also demonstrated.

scholarly journals Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor

2005 ◽  
Vol 390 (2) ◽  
pp. 475-484 ◽  
Author(s):  
L. Niv-Spector ◽  
N. Raver ◽  
M. Friedman-Einat ◽  
J. Grosclaude ◽  
E. E. Gussakovsky ◽  
...  
Keyword(s):  
Amino Acids ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Leptin Receptor ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Surface Plasmon Resonance Analysis

The binding domain of the chicken leptin receptor [chLBD (chicken leptin-binding domain)], subcloned from the full-size chicken leptin receptor and prepared in an Escherichia coli system, was subjected to site-directed mutagenesis to identify the amino acids involved in leptin binding. A total of 22 electrophoretically pure, >90% monomer-containing mutants were expressed, refolded and purified. The effects of the mutations were tested by the ability to form complexes with ovine leptin, and the kinetic parameters of interaction were determined by surface plasmon resonance. Six mutants were used to determine whether mutations of several amino acids that differ between chLBD and mammalian LBDs will affect affinity: none showed any such effect, except the mutant A105D (Ala105→Asp), which exhibited some decrease in affinity. Surface plasmon resonance analysis identified six mutants in which binding activity was totally abolished (F73A, Y14A/F73A, V76A/F77A, L78A/L79A, V76A/F77A/L78A/L79A and A105D/D106V) and six mutants (Y14A, R41A, R41A/S42A/K43A, V103A, V135A/F136A and F136A) in which affinity for the hormone was reduced, mainly by increased dissociation rates. Gel-filtration experiments indicated the formation of a 1:1 ovine or human leptin–chLBD complex with a molecular mass of approx. 41 kDa. Gel-filtration experiments yielded 1:1 complexes with those mutants in which affinity had decreased, but not with the six mutants, which had totally lost their binding capacity. Modelling the leptin–chLBD complex indicated that the binding domain of the latter is located mainly in the L3 loop, which contributes nine amino acid residues interacting with leptin. Contact-surface analysis identified the residues having the highest contribution to the recognition site to be Phe73, Phe77 and Leu79.

scholarly journals Association of Model Neurotransmitters with Lipid Bilayer Membranes

2019 ◽  
Author(s):  
B. Josey ◽  
F. Heinrich ◽  
V. Silin ◽  
M. Lösche
Keyword(s):  
Surface Plasmon Resonance ◽  
Ion Channels ◽  
Lipid Bilayer ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Dissociation Constants ◽  
Measurement Techniques ◽  
Neutron Reflectometry ◽  
Membrane Association ◽  
Lipid Bilayer Membranes

AbstractAimed to reproduce the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neuro-transmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neuro-transmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflectometry on artificial membranes reveals the structural aspects of neurotransmitters association with zwitterionic and anionic bilayers. We establish that serotonin interacts non-specifically with the membrane at physiologically relevant concentrations whilst GABA (γ-aminobutyric acid) does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity and neutron reflectometry shows that it penetrates the membrane deeply whereas GABA is excluded from the bilayer.SignificanceReceptor ion channels in the postsynaptic membrane and their neurotransmitter agonists enable fast communication between neuronal cells. Electrophysiology studies reveal surprisingly complex kinetics that apparently require a variety of protein conformational states for their quantitative interpretation, but an alternate hypothesis invoking neurotransmitter membrane association reduces the complexity of the underlying reaction schemes significantly. While their affinity may be low, and is hard to quantify experimentally, neurotransmitter membrane association can be relevant because of their large temporary concentration in the synaptic cleft. With thermodynamic and structural measurements we quantify membrane-bound states of serotonin, establishing this neurotransmitter as membrane-affine, whereas the affinity of the more hydrophilic GABA is too low to register in our sensitivity-optimized measurement techniques.

Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor

1997 ◽  
Vol 12 (8) ◽  
pp. 765-778 ◽  
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Stefania Moscato ◽  
Alessandra Guidi ◽  
Stefania Viganò ◽  
Paolo Rovero ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Epitope Mapping ◽  
Endothelin 1
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