Affinity solid phase

Godfrey, M. A., Kwasowski, P., Clift, R., and Marks, V. (1993). Assessment of the suitability of commercially available SpA affinity solid phases for the purification of murine monoclonal antibodies at process scale. J. Immunol. Methods 160, 97-105. 

Besides choosing the appropriate Ab, the assay method can be designed or manipulated to improve assay specificity using (a) protein precipitation, (b) liquid/liquid or solid-phase extraction, (c) HPLC separation of the analyte from the interfering compounds, (d) sample dilution with buffer or control matrix, or (e) an affinity solid phase (e.g., antibody-coated microtiter plate or polystyrene beads) to capture the analyte followed by wash steps. Affinity-purified antibodies and protein blockers are used in EIA to decrease nonspecific binding in plate assays. Increasing incubation time to reach equilibrium also improves binding specificity. 

Stevenson D (2000) Immuno-affinity solid-phase extraction. Journal of Chromatography B Biomedical Sciences and Applications 745 39-48. 

Kozutsumi, D. Kawashima, A. Adachi, M. Takami, M. Takemoto, N. Yonekubo, A. Determination of docosahex-aenoic acid in milk using affinity solid phase purification with argentous ions and modified thin layer chromatography. Inti. Dairy J. 2003,13 (12), 937-943. 

The affinity of A and B for the solid phase are different, B is more retained than A. Thus, it is possible to choose flowrates in order to make A move upward and B to move downward, leading to a spatial separation. This system requires inlet lines for the feed and eluent and outlet lines for the raffinate A and extract B. 

Streptavidin-single-stranded DNA covalent conjugates were described as the building blocks for assembling nanostructured scaffolds [31], The amount and type of biotinylated ligands were used to modulate the affinity of duplex formation between solid-phase-bound nucleic acid templates and DNA-streptavidin conjugates. This system has been proposed for the design of fine-tuned sequence detection systems. 

This method was used, for example, for the solid-phase immunoassay of thyroxine (affinity chromatography). Various activation methods (CDI, periodate, and cyanogen bromide procedures) were compared with each other for coupling antibodies to magnetizable cellulose/iron oxide solid-phase particles. 211]... 

Several methods have been used to separate the lanthanides chemically solvent extraction, ion exchange chromatography, HPLC using Q-hydroxyisobutyric acid and, in limited cases, selective reduction of a particular metal cation.40-43 The use of di(2-ethylhexyl)orthophosphoric acid (HDEHP) for the separation of various rare-earth elements via solvent extraction has also been reported.44 16 This separation method is based on the strong tendency of Ln3+ ions to form complexes with various anions (i.e., Cl- or N03 ) and their wide range of affinities for com-plexation to dialkyl orthophosphoric acid. When the HDEHP is attached to a solid phase resin, the lanthanides can be selected with various concentrations of acid in order of size, with the smallest ion being the most highly retained. 

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

Many of the methods developed to study protein interactions use the bait/prey model to detect interacting partners (Phizicky and Fields, 1995 Archakov et al., 2003 Piehler, 2005). The bait protein is a purified protein (often recombinant) that is used to lure and capture a putative interacting protein or biomolecule. The bait protein may be immobilized to a solid phase for affinity separations or be used in solution. It also may be fusion tagged (i.e., GST or 6X His) or labeled with a detectable molecule, such as a fluorescent probe. It often is the case... 

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