Development of Kinetic Ligand-binding Assays Using a Fiber Optic Sensor

Smith, Richard H.; Lemon, William J.; Erb, Judith L.; Erb-Downward, John R.; Downward, James G.; Ulrich, Otho E.; Wittliff, James L.

doi:10.1093/clinchem/45.9.1683

Cited by 10 publications

(4 citation statements)

References 9 publications

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“…The immobilized PSA in these assays was exploited to separate and quantify the fraction of soluble antibody binding sites that remained unoccupied in the equilibrium reaction mixtures of soluble antibody, antigen, and antibody–antigen complexes. Unlike surface plasmon resonance and other automated instrumentation devoted to the study of protein binding interactions in which the interaction to be quantified is that between a soluble and an immobilized binding partner, − the equilibrium binding data determined by kinetic exclusion assays are those obtained for the binding reaction in homogeneous solution. The equilibrium binding of total PSA to a limiting concentration of antibody M612165 is shown in Figure A.…”

Section: Resultsmentioning

confidence: 99%

Electrospray Ionization–Ion Mobility Spectrometry Identified Monoclonal Antibodies that Bind Exclusively to Either the Monomeric or a Dimeric Form of Prostate Specific Antigen

Blake

2012

Anal. Chem.

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Macroion mobility spectrometry was used to distinguish between a monoclonal antibody (clone M612165) that bound exclusively to monomeric prostate specific antigen and a different monoclonal antibody (clone M612166) that bound exclusively to a dimeric form of the antigen that only comprised 6.8% of the total protein. In the presence of excess antigen, the mobility spectrum of M612165 was replaced by a composite spectrum that represented a mixture of antibodies that included either one or two equivalents of the protein antigen. In similar circumstances, the mobility spectrum of M612166 was replaced by a composite spectrum that represented a mixture of antibodies that included either two or four equivalents of the protein antigen. When exposed to either of the two antibodies, the mobility spectrum of the prostate specific antigen showed a concomitant decrease in the monomeric antigen in one case and in the dimeric antigen in the other case. While sensitive kinetic exclusion assays demonstrated large differences in the antigen binding behavior of the two antibodies, these functional studies alone were insufficient to reveal the likely structural origins of the observed differences. Macroion mobility measurements were shown to be a useful and informative complement to functional studies in understanding complex macromolecular interactions.

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Section: Resultsmentioning

confidence: 99%

Electrospray Ionization–Ion Mobility Spectrometry Identified Monoclonal Antibodies that Bind Exclusively to Either the Monomeric or a Dimeric Form of Prostate Specific Antigen

Blake

2012

Anal. Chem.

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“…This allows us to measure the binding kinetics of interaction between the target antigen and the detection antibody, using an LSPCF-FOB by recording the fluorescent signal as a function of time. In addition, to calculate the kinetic association and dissociation rate constants, the following conditions are assumed to be satisfied experimentally: (1) the interaction of antigen/detection antibody has first-order kinetics; (2) there are a limited number of antigen binding sites, and there is no interaction between the binding sites; (3) the dissociation between the antigen and immobilized capture antibody is ignored under conditions where the surface density of the antigen is lower than that of the capture antibody; (4) the rebinding of antigen and detection antibody can be disregarded because the surface density of the antigen is lower than that of the detection antibody. , …”

Section: Methodsmentioning

confidence: 99%

Binding Kinetics of Biomolecule Interaction at Ultralow Concentrations Based on Gold Nanoparticle Enhancement

Chang²,

Chou

et al. 2011

Anal. Chem.

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Measuring the kinetic constants of protein-protein interactions at ultralow concentrations becomes critical in characterizing biospecific affinity, and exploring the feasibility of clinical diagnosis with respect to detection sensitivity, efficiency and accuracy. In this study, we propose a method that can calculate the binding constants of protein-protein interactions in sandwich assays at ultralow concentrations at the pg/mL level, using a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB). We discuss a two-compartment model to achieve reaction-limited kinetics under the stagnant conditions of the reaction chamber. The association rate constant, dissociation rate constant, and the equilibrium dissociation constant, that is, k(a), k(d), K(D), respectively, of the kinetics of binding between total prostate-specific antigen (t-PSA) and anti-t-PSA at concentrations from 0.1 pg/mL to 1 ng/mL, were measured either in PBS or in human serum. This is the first time that k(a), k(d), and K(D) have been measured at such a low concentration range in a complex sample such as human serum.

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“…The increased sensitivity of this flow fluorimeter compared with those of other instrumental methods for studying protein-ligand binding interactions, such as isothermal titration calorimetry, permits one to easily study antibody-antigen interactions with equilibrium dissociation constants in the low nanomolar range. Further, the KinExA assays yield data for binding interactions between soluble partners in a homogeneous solution, unlike other heterogeneous methods that quantify binding reactions with an immobilized reaction partner (17)(18)(19). The general KinExA assay procedures have been described in detail elsewhere (5,20,21).…”

Section: Methodsmentioning

confidence: 99%

Covalent and Noncovalent Modifications Induce Allosteric Binding Behavior in a Monoclonal Antibody

Blake

et al. 2007

Biochemistry

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Detailed equilibrium binding studies were conducted on a monoclonal antibody (8A11) directed against UO22+ complexed with 2,9-dicarboxy-1,10-phenanthroline (DCP-UO22+). Covalent modification of 8A11 with amine-reactive derivatives of Cy5 or Alexa 488 altered the binding curves obtained with DCP-UO22+ from hyperbolic to sigmoidal, the latter characterized by Hill coefficients of 1.5-1.6. Binding curves obtained with DCP-UO22+ and the bivalent (Fab)2 or the monovalent Fab fragments derived from limited proteolysis of the covalently modified 8A11 were characterized by Hill coefficients of 1.2 and 1.0, respectively. Incubation of 8A11 with saturating concentrations of the Fab fragments of goat antibodies directed against the Fc portion of mouse IgG increased the affinity of the native 8A11 for DCP-UO22+ by 3-fold. Conversely, incubation of the 8A11-Cy5 covalent conjugate with saturating concentrations of protein G (which likewise binds to the constant regions of mouse IgG) decreased the affinity of the primary antibody for DCP-UO22+ by 4-fold. In addition, the binding curves obtained with 8A11-Cy5 and DCP-UO22+ species changed from sigmoidal to hyperbolic at high concentrations of protein G. The presence of the antigen had a reciprocal effect on the binding of protein G to the 8A11-Cy5 conjugate; incubation of the 8A11-Cy5 conjugate with saturating concentrations of DCP-UO22+ decreased the affinity of the conjugate for protein G by 20-fold. These complex binding data were interpreted in terms of a free energy binding model in which (i) 2 mol of DCP-UO22+ and 1 mol of protein G bind to each mole of the 8A11-Cy5 conjugate, (ii) binding of the first equivalent of DCP-UO22+ to the antibody promotes the binding of the second equivalent of antigen in the absence of protein G, and (iii) DCP-UO22+ and protein G oppose each other's binding to the antibody. This is the first detailed description of the energetic balance of reciprocal binding events among the antigen binding sites and distant points on the constant portion of an immunoglobulin.

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Development of Kinetic Ligand-binding Assays Using a Fiber Optic Sensor

Cited by 10 publications

References 9 publications

Electrospray Ionization–Ion Mobility Spectrometry Identified Monoclonal Antibodies that Bind Exclusively to Either the Monomeric or a Dimeric Form of Prostate Specific Antigen

Electrospray Ionization–Ion Mobility Spectrometry Identified Monoclonal Antibodies that Bind Exclusively to Either the Monomeric or a Dimeric Form of Prostate Specific Antigen

Binding Kinetics of Biomolecule Interaction at Ultralow Concentrations Based on Gold Nanoparticle Enhancement

Covalent and Noncovalent Modifications Induce Allosteric Binding Behavior in a Monoclonal Antibody

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