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

Abstract

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 K A and of the measured resonant frequency changes Δ f 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 K A. 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.