Purified polyclonal anti-phenylurea antibodies for an improved immunoaffinity chromatography

• Published in: Food and agricultural immunology Vol. 10; no. 3; pp. 203 - 213
• Main Authors: Rejeb, Samy Ben, Fischer-Durand, Nathalie, Martel, Annie, Daniel, Régis, Jolivalt, Claude, Le Goffic, François, Lawrence, James F., Yeung, Jupiter M.
• Format: Journal Article
• Language: English
• Published: London Taylor & Francis Group 01.09.1998; Taylor and Francis; Taylor & Francis Ltd; Taylor & Francis
• Subjects: affinity chromatography; Biochemistry; Biochemistry, Molecular Biology; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; immunosorbent; Life Sciences; Methods and techniques. Experimental designs. Plant protection equipments; phenylurea; Phytopathology. Animal pests. Plant and forest protection; Polyclonal antibodies; purification; Immunoaffinity chromatography; Isoproturon; Molecular model; Ureas; FPLC chromatography; ELISA assay; Herbicide; Quantitative analysis; Immunological method
• ISSN: 0954-0105; 1465-3443
• DOI: 10.1080/09540109809354984

A one-step technique for the purification of isoproturon antibody has been developed to overcome the common elution problems encountered when high-affinity polyclonal antibodies are used in immunoaffinity columns. Because of the non-uniform affinity, desorption of the analyte can be problematic. Two herbicide derivatives were coupled to Sepharose CL solid support using a flexible linker, β-lactoblobulin. Using a structurally-related chlortoluron ligand showed a lower affinity to the target antibody rather than the isoproturon derivative ligand and resulted 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 glycol buffer (70/30). The purification efficiences of this procedure were characterized by dot blot, fast protein liquid chromatography and ELISA. The chlortoluron-β-lactoglobulin purified IgG showed a six times higher titer than the pure isoproturon-specific IgG fraction obtained using a commercial IgG purification gel. This one-step purification procedure was reproducible in isolating the pesticide-specific antibodies. Immobilization of such antibodies on activated silica afforded a high capacity immunosorbent which retained an average of 8.2 µg of isoproturon for 20 mg of immobilized antibodies per gram of silica.