USE OF AN IMMUNOAFFINITY COLUMN FOR TETRACHLORODIBENZO-P-DIOXIN SERUMMAPLE CLEANUP

Covalently linking 1-amino-3,7,8-trichlorodibenzo-p-dioxin with either keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) provide d antigens that generated antibodies in chickens. Competitive ELISA an alysis demonstrated that the antibodies isolated from egg yolk (IgY) b ound with 1,3,7,8-tetrachlorodibenzo-p-dioxin (1,3,7,8-TCDD). The anti bodies were linked to CNBr-Sepharose to generate an immunoaffinity col umn. Radiolabeled 1,3,7,8-TCDD in a 0.05% Tween 20 solution was retain ed by the column and could be eluted by increasing the Tween 20 concen tration. The binding efficiency for 10.7 ng per mi gel matrix ranged f rom 85 to 97%. Immunoaffinity columns generated by this method did not effectively bind C-14-1,3,7,8-TCDD from serum samples. Diluting the s erum 1:20 with 0.05% Tween 20 increased the binding efficiency. Altern ately, ethanol-hexane extraction followed by solid phase extraction on a carbon column using a fat removal protocol also provided an appropr iate preaffinity column cleanup for serum samples. After this preaffin ity column cleanup, spiked serum samples applied to the immunoaffinity column showed binding efficiencies of over 90%. 1998 Published by Els evier Science B.V.