Introduction:
Immunochemical assays are widely used to detect various compounds in complex media. Their conduction under homogeneous conditions provide simple, rapid and accurate testing. However, realization of immunoassays in a solution without separation of reactants needs a parameter changing after immune complexes formation. It is known that binding of antibodies to a fluorescently labeled hapten often leads to modulation (quenching or enhancement). of registered fluorescence. By this way, direct measurement of fluorescence can provide information on the formation of immune complexes in solution. In the presented work this approach was used to detect aflatoxin B1 (AFB1), an important toxic contaminant of food stuffs, in grain samples.

Methods:
AFB1 was conjugated with a fluorescein derivative (FAM5) using 3-(O-carboxymethyl)oxime as a bridge molecule. The conjugate was purified by thin-layer chromatography. Its ability to interact with antibodies was confirmed by fluorescence polarization immunoassay (FPIA). For immunoassay with modulated fluorescence of the label, competition between the conjugate and AFB1 in the sample for binding to antibodies was used. AFB1 was extracted from ground wheat grain with acetonitrile, filtered, and the filtrate was evaporated at room temperature by a factor of 6. Then, the filtrate was diluted to the original volume with phosphate-buffered saline, pH 7.4, containing monoclonal antibodies to AFB1 and AFB1-FAM5 conjugate, and fluorescence of FAM5 was recorded (ex - 495 nm, em - 520 nm).

Results:
Mixing the AFB1-FAM5 conjugate (5 nM) with antibodies to AFB1 (3 nM) resulted in an approximately 2.3-fold increase in fluorescence. Thus, the bound form of AFB1-FAM5 fluoresces significantly more strongly than the free form. Addition of free AFB1 to the mixture resulted in restoration of the fluorescence level up to the initial one. This effect was used to determine the AFB1 content in grain extracts. For contaminated grain samples, the possibility of determining AFB1 at concentrations up to 0.25 ng/mL was demonstrated, which is significantly lower than the established MRL value of 2 ppb. The degree of AFB1 revealing varies from 70 to 97%.

Conclusions:
The proposed method is characterized by methodical simplicity and high sensitivity. Conducting a homogeneous analysis allows reducing the time of results obtaining and excluding the stages of separation of bound and free reagents, typical for heterogeneous immunoassays, such as ELISA. Measuring fluorescence requires simpler and cheaper equipment than fluorescence polarization in the case of FPIA.