Effect of organic solvents on immunoassays of environmental pollutants studied using a piezoelectric biosensor Original Research Article

The affinity interactions of haptens with corresponding antibodies were studied in the presence of organic solvents. As the model haptens, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), and 4,4′-dichlorobiphenyl (DCB, belonging to the group of polychlorinated biphenyls) were used. The piezoelectric quartz crystal was used for real-time monitoring of immunoaffinity interactions without any additional labels. Affinity binding reactions were followed in the presence of water/methanol mixtures. Kinetic studies indicated the decrease of the kinetic association equilibrium constants KA and of the measured resonant frequency changes Δf with increasing methanol contents. To evaluate this influence on the determination of haptens, the competitive immunoassays of 2,4-D and DCB were carried out in the presence of 5, 10 and 25% methanol. The influence of solubility of organic solvent in water on the immunochemical reaction between 2,4-D and monoclonal anti-2,4-D antibody was further characterized in the presence of different organic solvents (10% contents in the assay mixture). The parameter log P was used as a measure of hydrophobicity. Higher values of log P resulted in increased affinity reflected by higher values of KA. Finally, the existence of affinity interaction in the pure organic solvent (toluene) was proved. For this purpose, 2,4-D and DCB were modified with polystyrene moiety functioning as a mass label; thus obtained conjugates were allowed to interact with the corresponding immunoglobulins immobilized at the sensing surfaces. The competition of the DCB-polystyrene conjugate and free DCB for the immobilized IgG was observed in toluene.