PURIFIED POLYCLONAL ANTI-PHENYLUREA ANTIBODIES FOR AN IMPROVED IMMUNOAFFINITY CHROMATOGRAPHY

A one-step technique for the purification of isoproturon antibody has been developed to overcome the common elution problems encountered whe n high-affinity polyclonal antibodies are used in immunoaffinity colum ns. Because of the non-uniform affinity, desorption of the analyte can be problematic. Two herbicide derivatives were coupled to Sepharose C L solid support using a flexible linker, beta-lactoblobulin. Using a s tructurally-related chlortoluron ligand showed a lower affinity to the target antibody rather than the isoproturon derivative ligand and res ulted in an improved capacity of the affinity chromatography gel. The recovery of the specifically adsorbed antibodies was achieved using a carboxylic derivative of the analyte isoproturon in a water-ethylene g lycol buffer (70/30). The purification efficiences of this procedure w ere characterized by dot blot, fast protein liquid chromatography and ELISA. The chlortoluron-beta-lactoglobulin purified IgG showed a six t imes higher titer than the pure isoproturon-specific Ige fraction obta ined using a commercial IgG purification gel. This one-step purificati on procedure was reproducible in isolating the pesticide-specific anti bodies. Immobilization of such antibodies on activated silica afforded a high capacity immunosorbent which retained an average of 8.2 mu g o f isoproturon for 20 mg of immobilized antibodies per gram of silica.