Support Materials for Affinity Chromatography

Gustavsson, Per-Erik; Larsson, Per‐Olof

doi:10.1201/9780824751982.ch2

Cited by 15 publications

(47 citation statements)

References 1 publication

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“…For instance, covalent immobilization can produce improper orientation, steric hindrance or multi-site attachment for an immobilized binding agent if proper coupling conditions are not selected. These effects, in turn, can lead to a change in actual or apparent activity for an immobilized protein [14,16,19,25,29,30]. …”

Section: Introductionmentioning

confidence: 99%

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

Jackson

Vargas-Badilla

et al. 2016

Journal of Chromatography B

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A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (± 0.5) × 105 M−1, which agreed with a previously reported value of 1.0 (± 0.1) × 105 M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

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Section: Introductionmentioning

confidence: 99%

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

Jackson

Vargas-Badilla

et al. 2016

Journal of Chromatography B

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“…This support was selected because of its good mechanical stability, fast mass transfer properties, ease of chemical modification for ligand attachment, and low non-specific binding for antibodies and many biological substances that are found in samples such as serum or plasma [15-17,29,36,41]. Silica with a nominal pore size of 300 Å was chosen to help maximize the amount of HSA (i.e., the immobilized protein analog) that could be immobilized to the support while also allowing good accessibility of the labeled antibodies to this immobilized analog [29,41,42]; for higher mass proteins, a larger pore size could also have been employed [41,43]. Silica with a particle diameter of 7 μm was used to provide good mass transfer rates for the binding of the labeled antibodies to the immobilized HSA [33,34].…”

Section: Resultsmentioning

confidence: 99%

“…For instance, the binding of one HSA with an antibody may have blocked the remaining site on the antibody and prevented, or at least slowed down, the interaction of this second region with immobilized HSA in the microcolumn. It is also possible that the size of the HSA-antibody complex (~140-150 Å) versus the pore size of the support (300 Å, prior to protein immobilization) may have created restricted diffusion or prevented this complex from reaching most of the immobilized HSA [41]. Such effects could have resulted in pseudo-monovalent behavior and a response that was still consistent with that predicted by Eqn.…”

Section: Resultsmentioning

confidence: 99%

Development of microcolumn-based one-site immunometric assays for protein biomarkers

Pfaunmiller

Anguizola

Milanuk

et al. 2014

Journal of Chromatography A

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One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1 mm i.d. × 5 mm and contained 8-9 nmol of immobilized HSA. These microcolumns were used from 0.10-1.0 mL/min and gave results within 35 s to 2.8 min of sample injection. Limits of detection down to 0.10-0.28 ng/mL (1.5-4.2 pM) or 25-30 pg/mL (0.38-0.45 pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ± 0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.

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“…The IAC matrix should be easily modified for antibody attachment, and should be macroporous with uniform particle and pore size and good flow properties (Urh et al, 2009). A compromise should be achieved between pore size and surface area, as supports with small pore size have a large surface area, much of which may not be available for immobilization of antibody (Gustavsson and Larsson, 2006). In contrast, large pore support systems do not have accessibility problems, but may result in a low level of antibody attachment due to the small surface area.…”

Section: Solid Matrixmentioning

confidence: 99%