Diphenylurea hapten sensing with a monoclonal antibody and its Fab fragment: Kinetic and thermodynamic investigations

Monoclonal antibodies against a diphenylurea compound (a potential pesticide) were characterized with biospecific interaction analysis (BIA). Kinetic binding rates and affinities were obtained for the IgG and its Fab fragment. Hapten was immobilized onto the sensor chip and antibody binding was detected. Affinity in solution was determined by preincubating antibody with hapten and the amount of `freeʹ antibodies was measured by binding to an extensively haptenated flow cell surface. The spacer used for immunogen synthesis and immobilization of the hapten was shown to improve the KD from 9 to 0.23 nM for the Fab–hapten binding in solution at 25°C. Protonation of the hapten aromatic amino group at pH below 4.5 prevented binding, whereas the hapten derivative carrying the spacer group was nearly insensitive to pH variations between 4 and 10. The affinity increased with decreasing temperature, and the binding was enthalpy driven below 37°C. Addition of 10 to 20% ethanol to the medium weakened the binding of the hapten to the Fab fragment more than the binding of the hapten derivative. The BIACORE® 2000 instrument allowed the detection of less than 0.1 μg l−1 of the hapten derivative within 15 min. Memory effects were observed at high hapten concentrations. Therefore, extended regeneration of the sensor chip was necessary.