Investigation of binding site density: Effects on the interaction between cibacron blue–dextran conjugates and lysozyme

Mayes, Andrew G.; Moore, John; Eisenthal, Robert; Hubble, John

doi:10.1002/bit.260361103

Cited by 13 publications

(9 citation statements)

References 23 publications

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“…Recently, a series of hydrogels was fabricated from CM-dextran in our laboratories using carbodiimide chemistry (Zhang, 2005;Zhang et al, 2005). This approach is quicker and easier than the triazine method that we previously employed (Mayes et al, 1990;Tang et al, 2003Tang et al, , 2004, and allows greater control over charge density and the degree of cross-linking in the gel. Thus the balance between degradation rates and mechanical properties is very easily controlled.…”

Section: Resultsmentioning

confidence: 98%

“…The principle of using competitors of internal affinity cross-links as a permeability switch is a general one. For example, based on earlier work (Mayes et al, 1990), Tang et al (2004) have fabricated dextran hydrogel membranes with additional internal affinity interactions based on Cibacron blue/lysozyme. The permeability of these membranes to solute diffusion can be switched from a closed to an open state in the presence of free NAD, a competitive ligand of the internal affinity interactions.…”

Section: Introductionmentioning

confidence: 99%

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A smart membrane based on an antigen‐responsive hydrogel

Zhang

Bowyer

Eisenthal

et al. 2007

Biotech & Bioengineering

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Hydrogel membranes have been fabricated that incorporate antibody/antigen moieties. The permeability of large solutes through these membranes is dependent on the presence of soluble antigen that can compete with the internal interactions between antibody and antigen leading to an increase in gel mesh size. Specifically, the membrane's structure is based on a dextran backbone grafted with a fluorescein isothiocyanate (FITC) antigen and a sheep anti-FITC IgG antibody. The backbone is covalently cross-linked by conjugated divinyl sulfone (DVS) groups. The gel structure is additionally stabilized by affinity crosslinks formed by biospecific interactions between the bound IgG and FITC. FTIR spectra of the gel are consistent with formation of covalent bonds between cysteine groups in the IgG and DVS groups in the dextran. Results obtained using isothermal titration calorimetry (ITC) confirmed the competitive interaction binding between IgG-FITC-dextran and free sodium fluorescein at pH 5.0. Scanning electron microscopy (SEM) of samples prepared using cryofixation and cryofracturing techniques showed that observed changes in permeability correlate with free fluorescein-dependent structural changes in the gel. Three-dimensional images obtained from confocal laser scanning microscopy show that these changes occur throughout the gel and indicate that SEM results are not artifacts of sample preparation. The permeability of these gels, as shown by blue-dextran (12 kDa) diffusion, increases in response to the presence of free fluorescein of the external medium, which causes competitive displacement of the affinity cross-links. Sequential addition and removal of sodium fluorescein showed that these permeability changes are reversible.

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Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

A smart membrane based on an antigen‐responsive hydrogel

Zhang

Bowyer

Eisenthal

et al. 2007

Biotech & Bioengineering

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“…This suggests that, as capacity increases, average binding strength decreases, indicating the possibility of an increasing contribution of weaker, ion‐exchange based binding at higher dye loadings. This effect was also observed by Mayes et al [10] in a study on the effects of ligand density on the affinity interaction between soluble CB–dextran conjugates and lysozyme that showed both high‐ and low‐affinity contributions to binding. In the study described here, estimated K d values are in the one‐tenth‐millimolar range, consistent with an affinity interaction.…”

Section: Resultsmentioning

confidence: 99%

“…It has been shown that, with highly substituted CB-based adsorbents, less than 20 % of the ligand groups may be available for affinity interactions. [10]. However, even with an ideal ligand density, it is still possible for weak specific interactions to occur between CB and contaminant proteins, which will potentially decrease the purity of the final product.…”

Section: Introductionmentioning

confidence: 99%

An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme

Wang

Zhang

Eisenthal

et al. 2006

Biotech and App Biochem

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The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle.

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“…Subsequently, the degree of cross‐linking decreases and drugs can be released. Examples in literature are glucose‐responsive dextrane hydrogels with concanavalin A‐glucose affinity cross‐links and nicotinamide adenine dinucleotide‐responsive dextrane hydrogels with cibacron blue/lysozyme affinity cross‐links . Likewise interesting are antigen‐responsive hydrogels, where antibody–antigen pairs, chemically grafted to the hydrogel network, serve as reversible cross‐linkers.…”

Section: Introductionmentioning

confidence: 99%

In vitro characterization of polyacrylamide hydrogels for application as implant coating for stimulus‐responsive local drug delivery

Minrath

Arbeiter

Schmitz

et al. 2014

Polymers for Advanced Techs

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The improvement of well‐established local drug delivery (LDD) systems, often providing drug release independently of their need, is a challenge of current implant technology. Systems that sense signals caused by a certain disease, judge the magnitude of the signal and act to release the right amount of drug in response are hence searched for. Specific responsive hydrogels with antibody–antigen cross‐links, whose non‐covalent interaction is broken in the presence of the corresponding antigen and reclosed in its absence, are a promising candidate. However, the challenging implementation of such antigen‐responsive hydrogels as implant coating and their applicability for implant‐associated LDD systems have not been explored so far. In this context and in order to define adequate gel properties for the envisaged application, we prepared a matrix at examples of polyacrylamide hydrogels of different gel architectures, gel contents and cross‐linking degrees and investigated their impact on the permeation of the differently sized model proteins bovine serum albumin (BSA), protein A and myoglobin in a specifically designed chamber simulating an implant‐associated LDD system and the mechanical stability of the polymeric network. Water uptake, as estimated by Karl Fischer titration, which was mainly influenced by gel content, seems to primarily govern drug permeability and mechanical stability. Within the investigated matrix, a medium gel content of 15.0 w% acrylamide and N,N′‐methylenebisacrylamide evidenced low drug permeability, enough tensile strength and flexibility for integration of antibody–antigen cross‐links, which we could successfully evidence via fluorescence microscopy. A stimulus‐triggered release of BSA through such a hydrogel could be moreover demonstrated. Copyright © 2014 John Wiley & Sons, Ltd.

show abstract

Investigation of binding site density: Effects on the interaction between cibacron blue–dextran conjugates and lysozyme

Cited by 13 publications

References 23 publications

A smart membrane based on an antigen‐responsive hydrogel

A smart membrane based on an antigen‐responsive hydrogel

An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme

In vitro characterization of polyacrylamide hydrogels for application as implant coating for stimulus‐responsive local drug delivery

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