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Use of an immunoaffinity column for tetrachlorodibenzo-p-dioxin serum sample cleanup.

Posted by on in 1995-1999
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 Shelver WL, Larsen GL, Huwe JK
J Chromatogr B Biomed Sci Appl 1998 Feb 705:261-8


Covalently linking 1-amino-3,7,8-trichlorodibenzo-p-dioxin with either keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) provided antigens that generated antibodies in chickens. Competitive ELISA analysis demonstrated that the antibodies isolated from egg yolk (IgY) bound with 1,3,7,8-tetrachlorodibenzo-p-dioxin (1,3,7,8-TCDD). The antibodies were linked to CNBr-Sepharose to generate an immunoaffinity column. Radiolabeled 1,3,7,8-TCDD in a 0.05% Tween 20 solution was retained by the column and could be eluted by increasing the Tween 20 concentration. The binding efficiency for 10.7 ng per ml gel matrix ranged from 85 to 97%. Immunoaffinity columns generated by this method did not effectively bind 14C-1,3,7,8-TCDD from serum samples. Diluting the serum 1:20 with 0.05% Tween 20 increased the binding efficiency. Alternately, ethanol-hexane extraction followed by solid phase extraction on a carbon column using a fat removal protocol also provided an appropriate preaffinity column cleanup for serum samples. After this preaffinity column cleanup, spiked serum samples applied to the immunoaffinity column showed binding efficiencies of over 90%.

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