Antigen--antibody interactions in the reverse micellar system: quenching of the fluorescence of fluorescein-labeled atrazine by antibodies against atrazine.

This work presents a new method for performing homogeneous fluoroimmunoassay in apolar organic media, quenching fluoroimmunoassay (QFIA). This method is based on utilization of the reverse micellar system of Aerosol OT (AOT) in n-octane as a medium for the analysis of compounds with low water solubility. It is shown using the system for determination of a hydrophobic pesticide atrazine as an example. The conjugate of atrazine with fluorescein (FA) serves as a label for fluorescence detection of antigen-antibody interaction in the reverse micellar system. The fluorescence quantum yield of this compound drastically depends on the micro-environment of the label in the reverse micelle system. Specifically, the binding of this conjugate with the antibodies solubilized in the reverse micelles results in fluorescence quenching. We found that quenching efficiency depends on the properties of the reverse micellar system (surfactant concentration, hydration degree w0, w0 = [water]/[surfactant], etc.). The optimal conditions for quenching of FA fluorescence by antibodies in reverse micelles of AOT in n-octane are low surfactant concentration and hydration degree, allowing one to get large reversed micelles (w0 = 15-20) capable of retaining solubilized antibodies. Addition of free atrazine results in displacement of the conjugate and restoration of its fluorescence. The sensitivity of the analysis to atrazine is only 10 times less than that of the commonly used method of homogeneous immunoassay, polarization fluoroimmunoassay, in aqueous solution using the same antibodies and conjugate. The advantage of QFIA in reverse micelles is that the analyte can be added when dissolved in nonpolar organic solvent.