Cyanogen bromide activated supports

Immunoaffinity chromatography cleanup has also been applied as an ideal and reliable strategy for residue analysis. Immunoaffinity columns prepared by coupling the antibodies to a cyanogen bromide-activated support were used to analyze avermectin BI residues in cattle tissues (359) and ivermectin in sheep serum (376). An immunoaffinity column prepared by an alternative activation/ coupling procedure with carbonyl diimidazole was also employed to analyze ivermectin residues in swine liver (361) since the earlier-reported methods did not work well in the analysis of this matrix. This recent work demonstrated the high specificity of tire antibody-mediated cleanup, but also showed that the immunoaffinity procedures could not always or completely eliminate matrix interference of samples. Therefore, application of additional cleanup steps before or after these procedures is often inevitable. 

Coupling of affinants on cyanogen bromide-activated support... 

Antibodies are affinity purified by binding them to immobilized antigen. The most common solid support for immobilizing antigen is cyanogen bromide-activated Sepharose. Almost all antigens containing amino... 

Macromolecules bearing reactive groups in the repeat units along their chains are capable of multiple interaction with the matrix. As early as 1973, Wilchek prepared Sepharose-based supports chemically modified by chemisorbed polylysine and polyvinylamine [41]. The leakage of dyes covalently bonded to these supports was reduced remarkably as compared to non-modified Sepharose activated by cyanogen bromide. Thus, stable and high capacity affinity adsorbents could be prepared by the introduction of macromolecular spacers between a matrix and a biospecific ligand. 

Cyanogen bromide can be used to activate hydroxyl groups on particles to create reactive cyanate esters, which then can be coupled to amine-containing ligands to form an isourea bond (Figure 14.17). CNBr activation also can produce cyclic imidocarbonate groups, which are less reactive than the cyanate ester, but can form imidocarbonate bonds. The exact reactive species formed by the reaction is dependent on the structure of the hydroxylic support being activated (Kohn and Wilchek, 1982). 

The most commonly used biopolymers, such as agarose, contain alcoholic hydroxyl groups which can be activated with cyanogen bromide however, better methods have recently been developed including activation with sulfonyl chlorides (17), 2-fluoro-l-methylpyridinium toluene sulfonate (FMP) (10), and chlorocarbonates (18). The first two are commercially available as activated supports tresyl-activated Sepharose (Pharmacia) and FMP-Trisacryl (BioProbe International). The newer methods yield more stable bonds, which preclude leaching of the enzyme from the matrix. Most of these activated supports are too expensive for commercial use in a large process bioreactor however, they may be extremely useful for preparing analytical bioreactors. 

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