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 IC(50) 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.