Steric hindrance effects on surface reactions: applications to BIAcore

Edwards, David A.

doi:10.1007/s00285-007-0093-7

Cited by 15 publications

(29 citation statements)

References 31 publications

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“…We treat the reacting zone in the BIAcore as a thin dextran gel layer occupying the region −H g ỹ 0. This is in contrast to the work in Edwards (2007), where the reacting zone is taken to be a surface. In the gel, the bound state is created when an available reacting site reacts with a ligand molecule (concentrationC g ).…”

Section: General Modellingmentioning

confidence: 93%

“…A naive model which does not include the effect will underestimate the rate constantk a (where the subscript 'a' refers to 'association') because receptors which are merely occluded will be counted as receptors which are available, but do not bind because of the kinetics. Edwards (2007) proposed a model where the reacting zone is treated as a surface (the limit H g → 0 in our terminology). In this work, we extend this model to include the case of a reacting zone of finite width.…”

Section: Introductionmentioning

confidence: 99%

“…When Da = O(1), only short-time solutions can be obtained. Again, since an external supply of ligand is not fully developed, both association and dissociation kinetics become straightforward extensions of the work in Edwards (2001Edwards ( , 2007. The changes occur only in certain dimensionless parameters in the problem.…”

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confidence: 99%

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Steric hindrance effects in thin reaction zones: applications to BIAcore

Edwards¹

2007

IMA Journal of Applied Mathematics

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Many biological and industrial processes have reactions which occur in thin zones of densely packed receptors. Understanding the rate of such reactions is important, and the BIAcore surface plasmon resonance biosensor for measuring rate constants has such a geometry. However, interpreting biosensor data correctly is difficult since large ligand molecules can block multiple receptor sites, thus skewing the kinetics. General mathematical principles are presented for handling this phenomenon, and a receptor layer model is presented explicitly. An integro-partial differential equation results. Using perturbation techniques, the problem can be simplified somewhat. In the limit of small Damköhler number, the non-local nature of the system becomes evident in the association problem, while other experiments can be modelled using local techniques. Explicit and asymptotic solutions are constructed for large-molecule cases motivated by experimental design. The analysis provides insight into surface-volume reactions occurring in various contexts. In particular, this steric hindrance effect can often be quantified with a single dimensionless parameter.

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Section: General Modellingmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

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Steric hindrance effects in thin reaction zones: applications to BIAcore

Edwards¹

2007

IMA Journal of Applied Mathematics

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“…where A is antigen, B is the bound antibody, C is the (bound) product, while k on and k off represent the association and dissociation rate constants, respectively. This can be cast as the following differential equation (see, for example, [1], [3], [8])…”

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confidence: 99%

Modelling random antibody adsorption and immunoassay activity

Mackey¹,

Kelly²,

Nooney³

2016

MBE

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One of the primary considerations in immunoassay design is optimizing the concentration of capture antibody in order to achieve maximal antigen binding and, subsequently, improved sensitivity and limit of detection. Many immunoassay technologies involve immobilization of the antibody to solid surfaces. Antibodies are large molecules in which the position and accessibility of the antigen-binding site depend on their orientation and packing density. In this paper we propose a simple mathematical model, based on the theory known as random sequential adsorption (RSA), in order to calculate how the concentration of correctly oriented antibodies (active site exposed for subsequent reactions) evolves during the deposition process. It has been suggested by experimental studies that high concentrations will decrease assay performance, due to molecule denaturation and obstruction of active binding sites. However, crowding of antibodies can also have the opposite effect by favouring upright orientations. A specific aim of our model is to predict which of these competing effects prevails under different experimental conditions and study the existence of an optimal coverage, which yields the maximum expected concentration of active particles (and hence the highest signal).

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“…Direct immobilization of α-bgtx to the CM-5 chip surface eliminates potential binding artifacts and elevated backgrounds due to streptavidin. Although binding of AChBP to α-bgtx did not appear to be substantially hindered by the direct chemical immobilization of α-bgtx to the chip, caution should be taken as other protein-protein interactions may encounter steric effects adversely affecting the binding of the analyte to the ligand when using BIAcore technology [15]. …”

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confidence: 99%

A radioisotope label-free α-bungarotoxin-binding assay using BIAcore sensor chip technology for real-time analysis

Paulo

Hawrot

2009

Analytical Biochemistry

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Abstractα-Bungarotoxin (α-bgtx) binding proteins, including certain nicotinic acetylcholine receptors and acetylcholine-binding proteins (AChBPs), are frequently characterized with radioisotope-labeled α-bgtx-binding assays. Such assays, however, preclude investigations of binding interactions in realtime and are hampered by the inconveniences associated with radioisotope-labeled reagents. We have used surface plasmon resonance-based technology (BIAcore) to investigate the binding of recombinant AChBP to CM-5 sensor chip surfaces with directly immobilized α-bgtx. We have validated our BIAcore results by comparing the same biological samples using the traditional [ 125 I]-α-bgtx-binding assay. An α-bgtx sensor chip, as described here, enables detailed, real-time, radioisotope-free interaction studies that can greatly facilitate the characterization of novel α-bgtxbinding proteins and complexes. Keywordsα-bungarotoxin; BIAcore; surface plasmon resonance; AChBP; acetylcholine-binding proteinThe use of surface plasmon resonance (SPR)-based instrumentation for investigating proteinprotein interactions has grown substantially over the past decade [1]. Instruments, such as the BIAcore T100 (GE Healthcare, Piscataway, NJ), can be used to determine binding parameters by measurement of the changes in the refractive index at the surface of a sensor chip to which a protein or peptide (ligand) is covalently bound and over which a potential binding partner (analyte) is perfused [2]. Interactions between the analyte and the ligand are detected through the increase in mass on the surface of the sensor chip [3].The most commonly used strategies for detecting soluble α-bungarotoxin (α-bgtx)-binding proteins require radioisotope-labeled α-bgtx and are not appropriate for real-time binding analysis [4]. We have developed a radioisotope-free method for measuring the binding activity of recombinant, acetylcholine-binding protein (AChBP) that is secreted by Pichia pastoris into yeast culture medium. We utilized SPR-based technology which incorporates sensor chips directly immobilized with α-bgtx as an alternative to the traditional [ 125 I]-α-bgtx-binding assay for AChBP detection. This radioisotope-free methodology introduces a convenient and innovative tool for the investigation of α-bgtx-binding proteins in real-time.

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Steric hindrance effects on surface reactions: applications to BIAcore

Cited by 15 publications

References 31 publications

Steric hindrance effects in thin reaction zones: applications to BIAcore

Steric hindrance effects in thin reaction zones: applications to BIAcore

Modelling random antibody adsorption and immunoassay activity

A radioisotope label-free α-bungarotoxin-binding assay using BIAcore sensor chip technology for real-time analysis

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