Alina V. Petrakova

Rapid Immunoenzyme Assay of Aflatoxin B1 Using Magnetic Nanoparticles

The main limitations of microplate-based enzyme immunoassays are the prolonged incubations necessary to facilitate heterogeneous interactions, the complex matrix and poorly soluble antigens, and the significant sample dilutions often required because of the presence of organic extractants. This study presents the use of antibody immobilization on the surface of magnetic particles to overcome these limitations in the detection of the mycotoxin, aflatoxin B1. Features of the proposed system are a high degree of nanoparticle dispersion and methodologically simple immobilization of the antibodies by adsorption. Reactions between the immobilized antibodies with native and labeled antigens are conducted in solution, thereby reducing the interaction period to 5 min without impairing the analytical outcome. Adsorption of immunoglobulins on the surface of magnetic nanoparticles increases their stability in aqueous-organic media, thus minimizing the degree of sample dilution required. Testing barley and maize extracts demonstrated a limit of aflatoxin B1 detection equal to 20 pg/mL and total assay duration of 20 min. Using this method, only the 3-fold dilution of the initial methanol/water (60/40) extraction mixture in the microplate wells is necessary. The proposed pseudo-homogeneous approach could be applied toward immunodetection of a wide range of compounds.

Rapid Multiple Immunoenzyme Assay of Mycotoxins

Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

Application of magnetite nanoparticles for the development of highly sensitive immunochromatographic test systems for mycotoxin detection

Highly sensitive immunochromatographic test systems were developed for the detection of zearalenone (ZEA) and T-2 toxin (T2T) using magnetite nanoparticles (MNPs) for the labeling. In order to detect an analyte with high sensitivity, the competitive reaction was performed with free specific antibodies, while immune complexes were detected by the reaction with label-conjugated anti-species antibodies. The conditions for the synthesis of magnetite nanoparticles conjugated to anti-species antibodies were optimized. The concentrations of specific reagents that provided the highly sensitive detection of T-2 toxin and zearalenone were found. The instrumental detection limit for the determination of T-2 toxin and zearalenone in baby food samples (oat flakes) was 0.1 and 0.05 ng/mL (2.0 and 1.0 ng/g), respectively. The assay time was 15 min. The results of the present study confirm the possibility of the practical use of magnetite nanoparticles for immunochromatographic assay labeling.

Magnetic ELISA of aflatoxin B1 – pre-concentration without elution

While immunoenzyme assay (ELISA) is widely used for the detection of various compounds, its use is significantly limited by the considerable duration (determined by the heterogeneous reaction to form detectable immune complexes) and the restricted detection limit. This study proposes an ELISA variant based on the application of highly dispersed (average diameter – 10 nm) magnetic iron oxide nanoparticles as carriers for the adsorbable antibodies. In conducting the proposed ELISA, the antibodies react with the detectable compound within the sample volume; the formed complexes are preconcentrated by precipitation in a magnetic field and are used for immunoenzyme detection in the wells of a microplate. This approach has been implemented for the detection of aflatoxin B1, a low molecular weight compound that needs to be controlled at extremely low concentrations due to its high toxicity. Using magnetic nanoparticles provided a 10-fold lowering of the detection limit and cut the test duration by half, compared to conventional ELISA. Immobilized antibodies exhibited high resistance to methanol when testing aqueous/methanol extracts of contaminated vegetable feed stocks (corn kernels), making it possible to identify aflatoxin B1 at concentrations as low as 2 pg ml−1 (40 pg g−1). The proposed approach is universal and can be used for the immunodetection of various compounds.

Application of Magnetic Nanoparticles in Immunoassay

Complexes of magnetic nanoparticles with antibodies are used for the selective and highly sensitive detection in medical diagnostics, environmental monitoring, and the product quality and safety control. Magnetic nanoparticles are applied as controllable carriers to concentrate samples, as a solid phase in assays, or as labels detected by virtue of their magnetic, optical, or other properties. This review covers the results of recent studies, including methods for the preparation of analytical reagents, new assays, and techniques for the detection of magnetic nanoparticles.

Magnetic Nanopartices as Carriers for Immunoassays

Magnetic nanoparticles (MNP) are efficient molecular carriers for affine molecules. MNP complexes with antibodies can be used for the selective concentration and highly sensitive detection of various compounds. In this paper, development steps of the enzyme immunoassay using MNP are considered in details. Simple method of MNP synthesis and production of conjugates of antibodies with the aggregated MNP using physical sorption is presented. Simple, yet effective, formats of enzyme immunoassay are given. Using aflatoxin B1 detection as an example, possibility of decreasing detection limit up to 2 pg/mL, with a considerable decrease in the assay time, and performing immune interaction in the media with high organic content is shown.

Immunochromatographic assay of T-2 toxin using labeled anti-species antibodies

A new scheme of immunochromatographic assay was developed for the highly sensitive detection of low-molecular-weight analytes. This scheme includes the following two steps: the formation of complexes of free specific antibodies with an antigen and their detection by anti-species antibodies conjugated to gold nanoparticles as the label. This scheme was tested with mycotoxin T-2 toxin in maize extracts. The use of specific antibodies and a label as two individual components made it possible to independently vary their concentrations with a simultaneous decrease in the detection limit and an increase in the color intensity. The assay did not require additional reagents and manipulations. The instrumental and visual detection limits of the designed test system were 0.1 and 5.0 ng/mL, respectively (2 and 90 ng per gram of analytes), which are two orders of magnitude lower compared to conventional immunochromatography using the same reagents.

Comparative study of strategies for antibody immobilization onto the surface of magnetic particles in pseudo-homogeneous enzyme immunoassay of aflatoxin B1

Pseudo-homogeneous enzyme immunoassay (EIA) of aflatoxin B1 (AFB1) was accomplished using anti-AFM1 monoclonal antibodies conjugated with magnetic particles (MPs). The assay includes the concentration of AFB1 from the test sample on the surface of the MP-antibody conjugate, the binding of the AFB1-peroxidase conjugate to free sites of the antibodies, the separation of the complexes that formed from unreacted components by means of magnetic field, and the evaluation of the enzymatic activity of MP-bound peroxidase. A comparative study of antibody conjugates, which were prepared by three different methods, namely, by physical adsorption on native MP and covalent binding to oleic acidor polystyrene-coated MPs, was performed. For these conjugates, the detection limits of EIA for AFB1 are 2.6, 0.4, and 0.6 ng/mL, respectively. The advantages of the pseudo-homogeneous EIA format as a tool for the highly sensitive control of toxic contaminants in food are the shorter time of incubation of immunoassay reagents (5 min; the total assay time is 20 min) and the possibility of concentrating the analyte from the test samples.

Addendum: Urusov, A.E.; Zherdev, A.V.; Petrakova, A.V.; Sadykhov, E.G.; Koroleva, O.V.; Dzantiev, B.B. Rapid Multiple Immunoenzyme Assay of Mycotoxins. Toxins, 2015, 7, 238-254.

This work was supported by the Russian State Targeted Program «R esearch and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014–2020» (contract 14.607.21.0015 from 5 June 2014; unique identifier of applied research: RFMEFI60714X0015), Russian Foundation for Basic Research (grant 14-03-00753).”© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).