Assays affinity column chromatography

One of the original clones secreted anti-forskolin antibody which was purified by protein A affinity column chromatography and its purity was confirmed by MALDl mass spectrometry. The MAh was bound to polystyrene microtitration plates precoated with a forskolin-human serum albumin (HAS). The full measuring range of the assay extended from 6 ng to 200 ng mP of forskolin [68, 76]. The ELISA system established in this study was more sensitive compared to TEC [47], GLC [74] or HPLC [47, 77]. 

HPLC affinity columns (such as ProAnaMabs from Hyclone) offer a rapid assay for measuring antibodies in serum-free culture medium and they could be used instead of ELISA. Affinity chromatography can also be used for large-scale antibody extraction, although the preparative Protein A or G columns are expensive. 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

For mycotoxin analyses radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs) and affinity chromatography are the principal immunochemical methods in commercial application. Immunoaffinity columns or cartridges for specific mycotoxins are now being increasingly used in preliminary clean-up of extracts prior to final analysis by HPLC or GLC methods. 

Several milliliters of affinity medium used repeatedly can be sufficient for the development and preliminary testing of an assay. Large-scale trials or extensive optimizations may require significant amounts of purified antibody. Affinity purification on that scale may benefit from standard chromatography equipment (columns, pumps, a UV/pH monitor and a fraction collector). 

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