Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring.

The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In the present study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density of two different class of pesticides (atrazine and 2,4-dichlorophenoxyacetic acid in this study) coupled to carrier protein. The study proved useful for monitoring the course of hapten-protein conjugation for the production of specific antibodies against small molecules. Well-characterized hapten-protein conjugates enabled obtaining highly sensitive anti-atrazine and anti-2,4-D antibodies with IC50 values equal to 12 and 70 ng mL-1 for atrazine and 2,4-D respectively. These antibodies were used for developing a fluorescence-based immunoassays format demonstrating a detection limit of atrazine and 2,4-D in standard water samples 2 and 7 ng mL-1, respectively. The developed immunoassay format could be used as convenient quantitative tools for sensitive and specific screening of pesticides in samples.