Enhanced Emission of Fluorescein Label in Immune Complexes Provides for Rapid Homogeneous Assay of Aflatoxin B1

Homogeneous fluorescence immunoassays are in high demand due to their simplicity, rapidity, sensitivity, and specificity. These methods typically utilize immune-induced changes in the rotational mobility of the fluorophore with depolarization of plane-polarized excitation light (FPIA, etc.) or fluorescence quenching based on intramolecular energy transfer (FRET, etc.). This article presents an immunoassay based on enhanced emission of the fluorescein label in immune complexes. Over the entire history of fluorescence immunoassay research, this effect has been described in a few papers, while it allows overcoming the limitations of prevailing approaches. We discovered the assay for detecting aflatoxin B1 (AFB1), a widespread toxic contaminant of agricultural products. The one-step assay procedure consists of mixing the sample with antibodies and fluorescently labeled AFB1, accompanied by fluorescence measurement. This method enables the detection of AFB1 at concentrations up to 200 pg/mL in 10 min, including measurements in complex samples (corn extracts). Minimal manipulations in the course of the testing also provide high accuracy. The AFB1 revealed in contaminated corn samples was in the range of 76-136%. The influence of immune complex formation on the fluorescent label's emission can be easily tested and serve as a basis for applying this principle to other diverse analytes and various kinds of samples.