Determination of binding constants by affinity chromatography

Winzor, Donald J.

doi:10.1016/j.chroma.2003.11.092

Cited by 40 publications

(23 citation statements)

References 49 publications

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“…The dissociation constant of the complex of the screened DP and the SP was determined by frontal affinity chromatography (Shai et al, 1987;Winzor, 2004). The SP immobilized column was equilibrated with 10 mM phosphate buffer plus 150 mM NaCl.…”

Section: Determination Of Affinity Constant Of Immobilized Sp and Scrmentioning

confidence: 99%

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS‐CoV

Zhao

Luo

Xiong

et al. 2008

J of Molecular Recognition

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A combination of high-performance affinity chromatography and antisense peptide based combinatorial peptide libraries was used to screen a potential inhibitor for SARS-CoV. An aromatic-amino acid-rich region within the transmembrane domain at the C terminal of spike (S) protein identified as a membrane-active region was chosen as the target sense peptide (SP) and immobilized as affinity ligand. Four antisense peptides were designed based on the degeneracy of genetic codes. One of them was screened as the lead peptide to construct the extended peptide libraries (EPL). The library screening was carried out at pH 5.5 so as to mimic the low-pH milieu required by virus fusion. After five cycles of screening, a dodecapeptide KKKKYRNIRRPG (DP) was identified to possess the highest binding affinity to the immobilized sense peptide. The dissociation constant of the complex between the DP and the SP was 5.64 x 10(-7) M in a physiological condition. The recognition between the DP and recombinant SARS S protein was demonstrated by ELISA assay to be in a saturable way. The competitive inhibition of the sense peptide in the competitive ELISA reveals the affinity binding between the DP and SARS S protein is specific and directed towards the target SP of the S protein. The results indicate this preferred polypeptide can be used as a lead compound of potent inhibitor of SARS-CoV. The mechanism study suggests the specific recognition between the DP and the target peptide was due to sequence-dependent and multi-modal affinity interaction.

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Section: Determination Of Affinity Constant Of Immobilized Sp and Scrmentioning

confidence: 99%

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS‐CoV

Zhao

Luo

Xiong

et al. 2008

J of Molecular Recognition

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“…Several techniques, such as ultrafiltration [18], affinity chromatography [19,20], surface plasmon resonance [21,22], scintillation proximity assay [23,24], NMR spectroscopy [25], and immunoassay [26], have been used to evaluate the binding properties of ligands for proteins and to develop screening methods with different throughput. Also, affinity CE (ACE) has been successfully applied to study the binding of ligands for receptors and some examples of the application of the technique to the screening of chemical libraries are reported [27][28][29][30].…”

Section: Introductionmentioning

confidence: 99%

Search of ligands for the amyloidogenic protein β2-microglobulin by capillary electrophoresis and other techniques

et al. 2005

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Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.

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“…The change in retention time (Δt) from maximal retention of the reporter molecule reflects the fraction of the sites that is occupied by the competing molecules according to the Langmuir isotherm, assuming a one-to-one interaction (Winzor, 2004):…”

Section: Methodsmentioning

confidence: 99%

Evaluation of a glucose sensing antibody using weak affinity chromatography

Engström¹,

Johansson²,

Koch‐Schmidt³

et al. 2007

Biomedical Chromatography

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Continuous monitoring of drug levels and endogenous molecules in biological fluids is a developing research area with many applications. One example is the need to improve life for millions of diabetes mellitus patients by continuously monitoring the glucose level. In order to have a dynamic response, the recognition molecule in a continuous sensor should preferentially have a fast dissociation rate and a dissociation constant in the millimolar range. We have evaluated the monoclonal antibody (mAb) 3F1E8-A2 for its potential to be used in a future glucose sensor application. The mAb was generated from hybridomas by immunizing mice with 10 kDa dextran (an alpha1,6-glucose polymer) with the aim of obtaining mAbs that can recognize the glucose monomer. The mAb was immobilized to macroporous silica and the interaction with dextran-derived oligosaccharides was evaluated with weak affinity chromatography (WAC). To measure the low affinities between the mAb 3F1E8-A2 and different monosaccharides, a competitive weak affinity chromatography approach was employed. It was found that the mAb had a higher specificity for glucose compared with other monosaccharides and the dissociation constant (K(d)) towards glucose was determined as 18.8 +/- 2.6 mm.

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Determination of binding constants by affinity chromatography

Cited by 40 publications

References 49 publications

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS‐CoV

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS‐CoV

Search of ligands for the amyloidogenic protein β2-microglobulin by capillary electrophoresis and other techniques

Evaluation of a glucose sensing antibody using weak affinity chromatography

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