ISSN 00036838, Applied Biochemistry and Microbiology, 2015, Vol. 51, No. 6, pp. 688–694. © Pleiades Publishing, Inc., 2015. Original Russian Text © A.V. Petrakova, A.E. Urusov, M.V. Voznyak, A.V. Zherdev, B.B. Dzantiev, 2015, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2015, Vol. 51, No. 6, pp. 616–623.

Immunochromatographic Test System for the Detection of T2 Toxin1 A. V. Petrakovaa, A. E. Urusova, M. V. Voznyakb, A. V. Zherdeva, and B. B. Dzantieva a

A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia b IL TestPuschino Ltd., Puschino, Moscow region, 142290 Russia email: [email protected] Received June 8, 2015

Abstract—An immunochromatographic test system was developed for the detection of T2 toxin (T2T), which is one of priority contaminants of cereals. The detection is based on the competition between T2T in the sample and the T2Tprotein conjugate immobilized on the test strip for the binding to the complexes of antiT2T antibodies with gold nanoparticles serving as the marker. The results of the competition are recorded as the coloration in the test zone of the test strip produced by the marker. The optimum dilution of the sample for the reliable highsensitivity analysis corresponds to the final methanol concentration equal to 20%. The deceleration of the movement of reactants along the test strip due to the use of additional mem branes impregnated with 10% BSA resulted in the decrease in the detection limit of T2T. The test system was examined for the detection of T2T in watermethanol extracts of maize grains. The disappearance of the color in the test zone, which attests to the presence of mycotoxin, was observed for grain samples containing T2T at a concentration of 53 µg/kg or more (the final T2T concentration in the immunochromatorgaphic assay is 3 ng/mL). The videodigital detection limit of T2T is 16 µg/kg (0.9 ng/mL). The duration of the assay is 15 min. The results of the present study suggest that the developed test system is suitable for the control of the maximum allowable T2T content. Keywords: immunochromatography, T2 toxin, plant extracts, waterorganic mixtures, food safety DOI: 10.1134/S0003683815060113 1

Immunochromatographic assay (ICA) is a method based on the use of labeled immunoreactants and a test strip (multimembrane composite), in certain regions of which antibodies and/or antigens are immobilized before the assay. The contact of a test strip with an ana lyte provides the flow of the absorbed fluid and washedout immunoreactants along the test strip accompanied by the formation of immune complexes, which can be monitored based on the label binding. Immunochromatography has principal advantages, such as the rapid (within 15 min) and simple detection technique, which does not require additional reactants and equipment [1, 2]. However, immunochromatography is generally less sensitive than alternative immunoanalytical techniques, such as enzyme immunoassays (EIA), etc. This is asso ciated with the facts that the reactions are carried out under nonequilibrium conditions and their duration is limited by the movement of reactants along the test strip, as well as with the necessity of using rather high concentrations of a marker in order to observe its bind ing with a naked eye [2, 3]. In recent years, extensive research has been carried out in order to overcome these shortcomings of ICA. These approaches include the development of various ways of enhancing the detected 1 The article was translated by the authors.

signal, the application of alternative markers detected at low concentrations, the optimization of the composi tion of immunoreactants, and so on [4–7]. Meanwhile, along with these general approaches, studies aimed at developing techniques allowing for the improvement of ICA characteristics for particular classes of compounds are of considerable interests. Thus, in order to detect compounds poorly soluble in watersalt media, the ini tial samples are prepared as extracts in organic solvents. These samples cannot be directly used for ICA due to the denaturing effect of the organic solvents on proteins (including antibodies), whereas high dilutions of the samples lead to a proportional decrease in the sensitiv ity. Therefore, methodological solutions are required for waterinsoluble antigens to minimize the adverse effect of the extractant and to increase the time of the interaction of immunoreactants during their movement along the test strip without reducing the rapidity of the assay (not longer than 15 min). We chose T2 toxin (4,15diacetoxy8(3methyl butyryloxy)12,13epoxytrichothec9en3ol, T2T) as the model compound. This toxin belongs to myc otoxins, which are metabolites of mold fungi parasit izing plants, including many agricultural crops. Main T2 producers are fungi from the genus Fusarium (F. sporotrichioides, F. acuminatum, F. culmorum, F. equiseti, F. graminearum, F. moniliforme, F. myroth

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ecium, F. poae, etc.), although other fungi are also known to be able to synthesize T2T. Toxin T2T and other structurally similar trichothecene mycotoxins belong, along with zearalenone, to mycotoxins found in highlatitude regions. Agricultural production con taminated with T2T is most frequently found in Rus sia, Japan, Argentina, and Brazil. The spectrum of damaged agricultural crops is rather broad. The main agricultural crops to be monitored are oats, wheat, barley, rye, rice, and sorghum. The toxin T2T occurs in particularly high abundance in crops used for farm animal feed [8–12]. The adverse biological effect of T2T is character ized by the damage of the hematopoietic system and immunocompetent organs, as well as of the gas trointestinal tract, and also be leukopenia, anemia, and the development of the hemorrhagic syndrome. This toxin has high acute toxicity (LD50 assessed for different kinds of mammals varies from 3 to 10 mg/kg of body weight) [13, 14]. Like many other mycotox ins, T2T is characterized by high stability both in bio logical media and in the manufacture of food prod ucts [15]. When getting into the agricultural food chain, T2T travels along this chains, thus creating serious risks for human health [16, 17]. The toxicity and abundance of T2T generate a need for the development of efficient highthroughput methods for the detection of this compound [2, 18]. In the step of the sample preparation of solid foodstuff and feed products, the latter should be milled and the mycotoxin should be extracted. Since the solubility of T2T, like that of most other mycotoxins, in water is very low, waterorganic mixtures are used as extract ants; a 70 : 30 (vol/vol) methanol–water mixture is most commonly used for this purpose [19, 20]. When testing samples containing a large percentage of organic solvents, immunoreactants comprising the test system undergo denaturing inactivation [21]. No general solution of this problem has been proposed so far. There is no agreement on the optimum concentra tion of methanol in the sample, which should be used for ICA. In available publications concerning ICA of T2T [22–24] and commercial test systems (manufac tured by Rbiopharm AG, Neogen Corporation, Charm Sciences, AC Diagnostics, and Quicking Bio tech Co), the level of an organic solvent varies from 4 to 14%, but the protocols used are not substantiated. The aim of the present study is to develop an immunochromatographic test system for the detection of T2 toxin, including the selection of the optimum concentration of the organic extractant in the sample and the assessment of the possibilities of varying the duration of immunochemical interactions in the course of the assay. METHODS Chemicals. Chloroauric acid, sodium citrate, sodium azide, 3,3',5,5'tetramethylbenzidine dihydro chloride (TMB), and the surfactants Triton X100 and APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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Tween20 were from SigmaAldrich (USA); T2 toxin and bovine serum albumin (BSA), from MP Biomedi cals (USA); Tris and sucrose, from Reakhim (Russia). The T2TBSA conjugate and mouse monoclonal anti bodies against T2T were provided by the IL Test Puschino Ltd. (Russia). The standard T2T sample (state standard sample, 100 μg/mL) was manufactured by Khromresurs (Russia). Goat antimouse immuno globulin (IgG) antibodies were purchased from Arista Biologicals (USA). Peroxidaselabeled antimouse immunoglobulin (IgG) antibodies were produced in the N.F. Gamaleya Scientific Research Institute of Epide miology and Microbiology (Russia). All auxiliary chemicals (salts, acids, alkalis, organic solvents) were of analytical or reagent grade. Solutions for the production of gold nanoparticles (GNPs) and antibodyGNP conjugates were pre pared using deionized water (Simplicity System, Millipore, USA; the resistivity at 25°C was not lower than 18.2 MΩ cm). The extracts were obtained using methanol from Fluka (USA) and then filtered through paper filters (grade 589/2, 125 mm diameter; Schleicher and Schuell, Germany). Immunochromatographic test strips were fabri cated using the following components: 1—nitrocellulose working membranes based on CNPC10 and CNPC15 (Advanced Microdevices, India), HF240 and HF75 (Millipore, USA), FF80HP, FF170HP, Immunopore SP, and Immunopore RP (General Electric, USA) solid supports; 2—a glass fiber membrane PTR7 (Advanced Microdevices); 3—a sample pad GFBR7L (0.6) (Advanced Microdevices); 4—a final adsorption pad AP045 (Advanced Microdevices). Enzyme immunoassays were performed in Costar 9018 96well clear polystyrene microplates (Corning Costar, USA). Detection of T2T by competitive EIA. The EIA tech nique was proposed based on the results of our previous studies [25, 26]. The T2TBSA conjugate at a concen tration of 1.0 μg/mL was adsorbed in microplate wells from 100 μL of a 50 mM phosphate buffer (PB), pH 7.4, at +4°C overnight. After the fourfold washing of the microplate wells with PB containing 0.05% Triton X100 (PBT), 50μL portions of a T2T solution diluted to 100–0.005 ng/mL in PBT were pipetted into the wells, and then 50μL portions of a solution of antiT2T antibodies at a concentration of 100 ng/mL were added. The incubation was performed at 37°C for 1 h. After the fourfold washing with PBT, 100μL portions of a solu tion of peroxidaselabeled antimouse immunoglobulin antibodies (commercial antibodies diluted by 1 : 3000 in PBT) was injected into the wells, and the incubation was performed at 37°C for 1 h. Then the microplate was washed four times with PBT.

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To determine the peroxidase activity of the result ing immune complexes, 100μL portions of a substrate solution containing 0.42 mM TMB and 1.8 mM H2O2 in a 0.1 M sodium citrate buffer, pH 4.0, were injected into the microplate wells. After the incubation at room temperature for 15 min, the reaction was terminated by adding 1 M H2SO4 (50 μL). The absorbance of the reaction product was read at 450 nm using a Zenyth 3100 microplate reader (Anthos Labtec Instruments, Austria). The plot of the absorbance (y) versus the antigen concentration in the sample (x) was approximated with the Origin 7.5 software (Origin Lab, USA) by the fourparameter sigmoidal function y = (A – D)/(1 + (x/c)B) + D. The value of the parameter c corresponds to the concentration of the free antigen (that is present in the sample) causing 50% inhibition of the antibody bind ing to the immobilized conjugated antigen (IC50). The antigen concentrations providing 10, 20, and 80% inhibition of the antibody binding (IC10, IC20, and IC80, respectively) were calculated using the resulting function. In accordance with the practice of the inter pretation of EIA results [27], these values were consid ered as the detection limit and the lower and upper limits of the working range for the quantitative detec tion, respectively. Preparation of gold nanoparticles. Nanoparticles were prepared as described in [28]. A 1.0% aqueous HAuCl4 solution (1.0 mL) was added to water (100 mL), the mixture was heated to boiling, and then a 1.0% aqueous sodium citrate solution (1.5 mL) was added with vigorous stirring. The solution was refluxed for 30 min, cooled, and stored at +4°C. The GNP preparation can be used for the immobilization of antibodies for at least one year. Immobilization of antibodies on the surface of gold nanoparticles. The immobilization of antibodies was performed as described in [29, 30]. Monoclonal anti bodies against T2T were transferred into a 10 mM Tris buffer solution, pH 9.0. A GNP solution was brought to pH 8.9 by the addition of potassium carbonate. Then solutions of antibodies at concentrations from 5 to 30 μg/mL were added with vigorous stirring to a GNP solution (D520 = 1.0). The solution was incu bated for 30 min, a 10% BSA solution was added to the conjugate (vconjugate : vBSA = 40 : 1), and the incubation was carried out with vigorous stirring for 15 min. Gold nanoparticles with immobilized antibodies were pre cipitated by centrifugation at 13000 g and 4°C for 15 min. The supernatant was removed, the precipitate was transferred into a 50 mM Tris buffer solution, pH 7.5, containing 1.0% BSA and 1.0% sucrose, sodium azide was added to the final concentration of 0.05%, and the preparation was stored at 4°С. The preparation can be used for fabricating test strips for at least one year. Application of reagents onto membranes and the assembly of test strips. A solution of the T2TBSA con

jugate at a concentration of 0.5 mg/mL was blotted onto a nitrocellulose working membrane (0.1 μL per mm of the membrane width) using an IsoFlow dispenser (Ima gene Technology, USA). The antibodyGNP conjugate characterized by D520 = 3.0 (or a 10% BSA solution) was applied on a glass fiber membrane (3.2 μL per mm of the membrane). Then the membranes were dried at room temperature overnight. A multimembrane com posite was assembled from a working membrane, glass fiber membranes (from one to three), a sample pad, and a final adsorption pad. Then 3.5mmwide test strips were fabricated using an Index Cutter1 automatic guil lotine cutter (APoint Technologies, USA). The manu factured test strips were hermetically packed in bags made of an aluminum foilbased combined material, which contained 0.6 g of silica gel as the desiccant, using a packing machine with a FR900 miniconveyor (Wenzhou dingli packing machinery, China). The cut ting and packing were carried out at 20–22°C in a spe cial room with a relative humidity under 30%. The expi ration period of the test strips was at least one year. Preparation of plant extracts. Extracts were prepared according to the Russian State Standard method GOST 2800188 with some modifications. A sample of maize grains was milled using an electric miller and then mixed with an extracting solution (vmethanol : vwater= 70 : 30) in a ratio of 1 : 5. The incubation was performed with stirring at room temperature overnight. The extract was filtered through a filter paper and then separated from solid components by centrifugation at 15000 g and 4°C for 15 min. The supernatant was collected and stored at 4°C for no more than two weeks. Immunochromatographic assay for the detection of T2 toxin. A standard solution of T2T was added to a plant extract to the final mycotoxin concentrations in the range of 100–0.02 ng/mL. Then the extract was mixed with PB containing 0.1% Triton X100 (vextract : vPB = 1 : 2.5). Immunochromatography was per formed at room temperature. The test strip taken from the bag was dipped strictly vertically to the depth of 5– 7 mm into the solution of the sample (100 μL) for 10 min. Then the test strip was withdrawn and placed on a horizontal surface. After 5 min, the test strip was scanned on a Lide 90 scanner (Canon, Japan) at 600 dpi without automatic contrast enhancement and color balance. The color intensity in the test zone of the test strip was calculated with the TotalLab v2.01 software (Nonlinear Dynamics, UK) as described in [29], and its dependence on the T2T concentration in the sample was determined using the Origin 7.5 soft ware. As in the case of EIA, the working range for the quantitative detection of T2T (IC20 and IC80) was cal culated using the fourparameter sigmoidal function. The visual detection limit of the GNP binding in the test zone corresponded to the recorded integral color intensity (I) equal to 5 arb. units, and the instrumental detection limit corresponded to the value of IC10. The relation between the concentration of T2T in the sample (С, ng/mL) and its content is the contam

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inated grain (С0, μg/kg or ng/g) was determined from the equation: С = С0a1/a2, where a1 = 0.2 g/mL is the coefficient that accounts for the transformation of the solid sample into the liq uid one upon extraction, and a2 = 3.5 characterizes the dilution of the extract before the immunochro matographic assay. RESULTS AND DISCUSSION Optimization of the components of the test system. Monoclonal antibodies against T2T were characterized by EIA. The detection limit of T2T was 0.13 ng/mL; IC50 = 0.55 ng/mL. In order to obtain antibody conjugates, 30 μg of antibodies were added to 1.0 mL of GNPs (D520 = 1.0). According to [30], this provides saturation of adsorp tion sites on the particle surface. Based on our previous results [31], the test zone was formed by immobilizing the conjugate from a solution with a concentration 0.5 mg/mL, which was applied in an amount of 0.1 μL per mm of the working membrane width. The antibodyGNP conjugate was applied on a glass fiber membrane from a solution with D520 = 3.0 in an amount of 3.2 μL per mm. Eight nitrocellulose working membranes from three leading manufacturers—Millipore, General Electric, and Advanced Microdevices—were com pared in the ability to bind the marker and in the detection limit of T2T achieved in the assay of maize extracts 3.5fold diluted in PBT. The Immunopore RP and FF170HP membranes appeared to be unsuitable for immunochromatography under the conditions used, because the fluid front did not reach the test zone. In assays using the Immunopore SP membrane, only very high concentrations of T2T were detected, i.e., the test system proved to be insufficiently sensi tive. At the selected concentrations of the reactants, Millipore 240, FF80SP, CNPC10, and CNPC15 membranes did not provide the color intensity in the test zone of the test strip sufficient for the unambigu ous qualitative interpretation of the results. Therefore, subsequent experiments were performed with the Mil lipore 75 working membrane, because it provided the highest color intensity in the test zone and the lowest detection limit of T2T. Variation of the duration of the immunochemical reaction in ICA. In immunochromatographic assays, reactions between immunoreactants occur through the kinetic mechanism [1]. In this case, a considerable portion of molecules of the toxin to be detected have no time to bind to the antibodyGNP conjugate, which interferes with the detection of this analyte at low concentrations. To overcome these shortcomings, in some works samples were incubated with the antibodyGNP con jugate before chromatographic processes on the test strip [32, 33]. However, the use of additional steps (the APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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I, arb. units 120 100 80

1 3

60

2

40 20 0 0.1

1

10

100

T2T, ng/mL Fig. 1. Concentration dependences of the color intensity in the test zone of the test strip (I, arb. units) for ICA of T2T measured for different numbers of membranes impregnated with BSA (from 0 to 2 in curves 1–3, respec tively). The detection limits of T2T are indicated by arrows.

cancellation of the principle “all reagents are applied on the test strip in advance”) is an evident drawback of this technique. An alternative approach is to increase the time of soaking of the sample solution into the membrane and to decelerate the fluid movement along the test strip. Thus, Lutz and coworkers [34] fabricated a test strip from several working membranes which met in the test zone. These membranes were immersed in blocking solutions of sucrose at different concentrations, which retard the fluid movement. Depending on the sucrose concentration, the reactants that move along different membranes reach the test zone within 2–27 min. As a result, the degree of binding of the marker in the test zone was 2.6 times higher for the 30min duration of the assay. A simpler composition of the test strip described in our previous study [35], implied the use of an addi tional glass fiber membrane impregnated with a block ing solution (10% BSA). The time required for the fluid front to reach the test zone increased from 10 s to one min, which was accompanied by a sixfold increase in the degree of binding of the marker. In the present work, we studied the scope of this approach in more detail. For this purpose we com pared test strips fabricated without an additional membrane, as well as with one or two sequentially arranged glass fiber membranes impregnated with 10% BSA. For these three test systems, the time dur ing which the maximum color intensity of the test zone was achieved, was 7, 10, and 15 min, and the detection limit of T2T was 3.2, 1.8, and 1.0 ng/mL, respectively (Fig. 1). Therefore, the inclusion of additional glass fiber membranes impregnated with BSA in the test strip

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I, arb. units 120

1 5

100

6 7

2

80 60 40 20 0

3

70

35

4 3

20 10 CH3OH, % (b)

5

0

made it possible to increase the duration of the inter action of the analyte in the sample with the antibody GNP conjugate, which move toward the test zone. The optimum configuration with the use of two addi tional membranes (Fig. 2) resulted in a threefold decrease in the detection limit of T2T. A further increase in the number of membranes has no sense, because in this case ICA does not meet the require ments of rapidity, i.e., the time of the assay becomes longer than 15 min. Variation of the methanol concentration in the tested sample. A 70 : 30 (vol/vol) methanolwater mix ture is usually used for the extraction of T2T [36]. A high methanol concentration has an adverse effect on the stability of antibodies and their ability to interact with the antigen, thus decreasing the color intensity in the test zone, whereas high dilutions of the initial extract interfere with the detection of low concentra tions of T2T [37]. Therefore, we performed the quantitative compar ison of the results of ICA of T2T obtained for different dilutions of the extract. The highest color intensity in the test zone was achieved at a methanol concentra tion of 5%, whereas a sharp decrease in the color intensity was observed at concentrations higher than 20% (Fig. 3a). The detection limit of T2T in the sam ple (the dilute extract) remained unchanged up to 20% (Fig. 3b). These results show that it is expedient to test samples containing 20% of methanol (3.5fold diluted extracts) providing the lowest detection limit of T2T per 1 gram of maize. For more dilute samples, the

Detection limit, ng/mL

Fig. 2. Composition of the immunochromatographic test strip for the detection of T2 toxin: 1, control zone; 2, test zone; 3, glass fiber membrane impregnated with 10% of BSA; 4, glass fiber membrane containing an antiT2T antibodyGNP conjugate; 5, nitrocellulose working mem brane; 6, adsorption pad; 7, sample pad.

Detection limit, ng/mL

120 100 2 80 60 1

40 20

0

0 35

20

10 CH3OH, %

5

0

Fig. 3. Plot of the methanol concentration in the sample versus the ICA characteristics of T2T: a, color intensity in the test zone in the absence of T2T in the sample; b, detec tion limits of T2T in the reaction mixture (empty bars) and calculated per gram of maize (striped bars).

detection limit of T2T in the grain is higher than 100 ng/g. It should be noted that the recommended dilutions of extracts are smaller than the values proposed in other publications dealing with ICA of T2T [22–24]. For instance, Molinelli and coworkers suggested to dilute plant extracts by a factor of 5 [22]; Lattanzio and coworkers, by a factor of 10–20 [23]. Examination of the immunochromatographic test system. The immunochromatographic assay of T2T was examined for 3.5folddiluted maize extracts, i.e., for extracts diluted to the methanol concentration of 20%. The lower concentration limit of T2T visually detected based on the disappearance of the color in the test zone is 3 ng/mL. In the case of the videodigital detection, down to 0.9 ng/mL of T2T can be detected (Fig. 4). The working range of the quantitative detec tion of T2T is 1–5 ng/mL (18–88 ng/g).

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As can be seen in the table, the detection limit pro vided by the developed test system is lower compared to most of similar systems, and the new test system is comparable with other systems in the duration of the assay. The record detection limit was achieved by Zhang et al. [24] due to the use of polystyrene micro spheres containing europiurm Eu(III) as the marker, as well as owing to the preliminary 12min incubation of the Eu(III)antibody conjugate with the sample. The immunochromatographic assay based on fluores cent markers, although being more sensitive compared to the conventional technique based on GNPs [38], can be performed only by employing an additional source of excitation light, which limits the use of such test systems under field conditions.

I, arb. units 120 100 80 60 40 20 0

It should also be noted that the test system devel oped for the detection of T2T is characterized by the lowest dilution of the starting extracts. The maximum allowable T2T concentrations in foodstuff and infant food specified in the Sanitary and Epidemiological Regulations of the Russian Federa tion (SanPiN 2.3.2.10781) and in accordance with the laws of the Customs Union (Technical Regulation on Food Safety TS 021/2011) are 100 and 50 ng/g, respectively. In the case of the visual detection, the proposed immunochromatographic test system is suit able for the first requirement control; in the case of the instrumental detection, for both requirements. Due to the methodological simplicity of the use and high per formance, this system can be efficiently applied for the preliminary screening of a large number of samples in the framework of the food safety program.

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1 T2T, ng/mL

10

Fig. 4. Calibration curve for the quantitative immunochro matographic detection of T2T in contaminated maize extracts.

An immunochromatographic test system was developed for the detection of T2 toxin in plant extracts with a visual detection limit of 3 ng/mL (53 μg/kg of grain) and an instrumental detection limit of 0.9 ng/mL (16 μg/kg). The time of detection is 15 min. The inclusion of additional membranes impregnated with BSA in the test system leads to an increase in the duration of the incubation of the sam ple and the antibodygold nanoparticle conjugate, resulting in a threefold decrease in the detection limit.

Comparison of immunochromatographic test systems for the detection of T2T Source

Detection limit Dilution Time of assay, Mode of T2T, ng/g of extract, times min of detection

This study

60

3.5

15

Visual

This study

20

3.5

15

Instrumental

5

10

Visual

30

Instrumental

7

15

Instrumental

Molinelli et al. [22]

100

Lattanzio et al. [23] (determination of the total content of T2 and HT2 toxins)

400

Zhang et al. [24]

10–20

0.09

“Rbiopharm AG”, RIDA“QUICK T2/HT2 RQS (Germany)

50

10

5

Instrumental

“Neogen Corporation”, Reveal“ Q+ for T2/HT2 (USA)

50

5

6

Instrumental

“Charm Sciences”, ROSA T2/HT2 Quantitative Test Kit (USA)

10

10

10

Instrumental

“AC Diagnostics”, T2 Toxin Lateral Flow Device Test Kit (USA)

100

–*

5–10

Visual

“Quicking Biotech Co.”, T2 Toxin Rapid Test (China)

100

–*

5–10

Visual

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APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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2015