DIPHENYLUREA HAPTEN SENSING WITH A MONOCLONAL-ANTIBODY AND ITS FAB FRAGMENT - KINETIC AND THERMODYNAMIC INVESTIGATIONS

Monoclonal antibodies against a diphenylurea compound (a potential pes ticide) were characterized with biospecific interaction analysis (BIA) . Kinetic binding rates and affinities were obtained for the IgG and i ts Fab fragment. Hapten was immobilized onto the sensor chip and antib ody binding was detected. Affinity in solution was determined by prein cubating antibody with hapten and the amount of 'free' antibodies was measured by binding to an extensively haptenated flow cell surface. Th e spacer used for immunogen synthesis and immobilization of the hapten was shown to improve the K-D from 9 to 0.23 nM for the Fab-hapten bin ding in solution at 25 degrees C. Protonation of the hapten aromatic a mino group at pH below 4.5 prevented binding, whereas the hapten deriv ative carrying the spacer group was nearly insensitive to pH variation s between 4 and 10. The affinity increased with decreasing temperature , and the binding was enthalpy driven below 37 degrees 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 B IACORE(R) 2000 instrument allowed the detection of less than 0.1 mu g l(-1) of the hapten derivative within 15 min. Memory effects were obse rved at high hapten concentrations. Therefore. extended regeneration o f the sensor chip was necessary. (C) 1998 Elsevier Science B.V.