Post-column affinity detection

Immunoaffinitv assay The Irnmunoaffinity assay which Involves the use of an antibody column that traps the mycotoxins has been used for AFBl, AFMl and OTA (8, 9, 13, 36, 83,84). The toxin can be then eluted from the column for subsequent analysis or adsorbed In a solid-phase to which the fluorescence Is then read directly. Thus, the affinity column serves as a specific cleanup and concentration tool for the analysis. Recent advances In Improvement of Instrumentation of fluorescence detection and post-column derlvatlzatlon have led to a wider application of this method for AF detection. An AOAC collaborative study showing good result has been completed (85). In such an assay, AF extracted from the sample Is first diluted with buffer at pH 7.0 and then subjected to a disposable affinity column containing antl-AF antibody Sepharose gel. After washing, AF Is removed from the column with methanol, subjected to treatment with Iodine solution, and the fluorescence determined. Nevertheless, this method cannot be used for mycotoxins, such as TCTCs, which do not have high fluorescence or a chromophore. 

Characterization of Displaced Protein. With labelled antithrombin III, chromatography of the displaced radioactivity on heparin-Sepharose revealed that the bulk of the displaced radioactive material did not bind to heparin-Sepharose (Table II). With arvinized plasma as the displacing eluent, 65% of the antithrombin III eluted in the void volume, compared with 49% of the control I-antithrombin III (diluted in citrated plasma) that had not previously been used to inactivate thrombin the latter unbound fraction was likely labelled impurities or inhibitor modified by radiolabelling to lose its heparin affinity. With 5% (w/v) albumin used as a displacing eluent, 78% of the I-antithrombin III came out in the void volume. This increase in material that did not bind to heparin after displacement from heparin-PVA was attributed to post-complex antithrombin III, a modification of the original inhibitor resulting from the inactivation of thrombin. Neither thrombin-antithrombin III complex nor free antithrombin III were detected in the 5% (w/v) albumin displaced fractions while there was a barely detectable amount of complex (6%) and free antithrombin III (4%) in the material displaced by arvinized plasma. With the control I-antithrombin III, 25% of the radioactivity was determined to be free antithrombin III and 2% as complex. The remainder (22-27%) was not recovered from the column. 

---