Ligand Choice Strategy for the Purification of Polyclonal Antibodies Used for Improved Immunochemical-Based Analytical Methods for the Herbicide Isoproturon

Abstract

A common problem in developing immunochemical-based analytical methods is that the desired antibodies represent only a small portion of the heterogeneous antibody population. Affinity purification of polyclonal serum requires the correct choice of ligands used to extract antibodies featuring similar characteristics in their recognition properties (similar paratop group and dissociation constants). This paper describes several strategies of ligand design and synthesis used to purify polyclonal anti-serum generated against the herbicide isoproturon. A synthetic ligand mimicking the analyte isoproturon produced a 2.6 fold increase in isoproturon antibody concentration compared to an extraction using an IgG specific commercial gel. Another strategy involving the use of herbicide-protein conjugates resulted in affinity gels efficient in extracting 6 times more specific antibodies than the IgG commercial gel. The performances of immunochemical-based analytical methods using these refined antibodies were significantly improved. An indirect enzyme immunoassay gave an IC₉₀ of 0.01 ng/mL, when assayed with river water samples and a better correlation with HPLC analyses was also reached (r = 0.998) for those samples. Immobilization of such antibodies on activated silica afforded a high capacity immunoaffinity column, which retained an average of 8.2 μg of isoproturon for 20 mg of immobilized antibodies per gram of silica.

Key Words:

• Affinity chromatography
• antibody
• purification
• isoproturon
• enzyme immunoassay
• immunoaffinity chromatography