ISSN 0003-6838, Applied Biochemistry and Microbiology, 2009, Vol. 45, No. 2, pp. 204–209. © Pleiades Publishing, Inc., 2009. Original Russian Text © N.A. Byzova, I.V. Safenkova, S.N. Chirkov, A.V. Zherdev, A.N. Blintsov, B.B. Dzantiev, I.G. Atabekov, 2009, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 2, pp. 225–231.
Development of Immunochromatographic Test Systems for Express Detection of Plant Viruses N. A. Byzovaa, I. V. Safenkovab, S. N. Chirkovb, A. V. Zherdeva, A. N. Blintsovb, B. B. Dzantiev1, and I. G. Atabekovb a
Bakh Institute of Biochemistry, Russian Academy of Science, Moscow, 119071 Russia e-mail
[email protected] b Moscow State University, Biological Faculty, Moscow, 119991 Russia Received January 25, 2008
Abstract—Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rodshaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 µg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions. DOI: 10.1134/S000368380902015X
Viral diseases cause serious crop losses and affect the quality of products, while the use of virus-free seeds and planting stocks results in a substantial increase in agricultural crop production [1, 2]. Due to the lack of effective treatment protocols, the main approach for the production of virus-free seeds and planting stocks is rejection of infected and selection of healthy plants. Thus, the diagnostics of viral infections is a key stage in obtaining virus-free planting material [3]. The effective ELISA- and PCR-based methods have been developed for laboratory detection of most commercially important phytopathogenic viruses [4–7]. In recent years, the DNA microarray diagnostics method has become widely adopted, providing the capability of parallel detection of all pathogens in a single sample of the crop culture tested [8, 9]. All these methods of instrumental analysis possess both high sensitivity and productivity, making them fit for wide use in planting material certification and quarantine control, as well as in monitoring viral infections. However, these methods are complicated and laborious; they require qualified personnel and expensive equipment, limiting their use to well-equipped facilities and laboratories. Thus, the Abbreviations: BS, buffer solution (10 mM K-phosphate buffer, pH 7.4, 0.14 M NaCl and 0,1% Triton X-100); CarMV, carnation mottle virus; BMMV, bean mild mosaic virus; TMV, tobacco mosaic virus; ELISA, Enzyme-Linked ImmunoSorbent Assay; ICA, immunochromatographic assay; CG, colloidal gold; CG-15, preparation of colloidal gold particles of 15 nm nominal diameter; CG-30, preparation of colloidal gold particles of 30 nm nominal diameter; PBS, phosphate buffered saline (50 mM K-phosphate buffer, pH 7,4, 0,14 M NaCl); IgG, immunoglobulin G; IgG-CG, colloidal gold conjugated IgG; SD, standard deviation; PVX, potato virus X; PVY, potato virus Y.
general body of cultivated and imported vegetables, berries, ornamental and horticultural crops and potato is still beyond the scope of phytosanitary and quarantine control. Effective monitoring of viral infections requires rapid and sensitive methods of detection available in both laboratory and field conditions. One of the promising solutions for overcoming this challenge is immunochromatographic assay (ICA), based on the interaction between the target virus and immunoreagents (antibodies and their conjugates with colored colloidal particles) applied on the membrane carriers (teststrips). When the test strip is dipped into the sample being analyzed, the sample liquid flows through membranes and triggers immunochemical interactions resulting in visible coloration in test and reference lines [10, 11]. A number of foreign companies produce test strips for detection of plant viruses: Spot Check LF (Adgen Ltd., UK), Pocket Diagnostic (Forsite Diagnostics Ltd., UK), and Immunostrips (Agdia, United States). Results obtained with these strips indicate that virus infection can be detected within a few minutes. Among the substantial advantages of this approach are its high sensitivity, ease of both sample preparation, and the analysis itself. The aim of this study was the development of immunochromatographic test systems for express detection of plant viruses varying in shape and size of virions: spherical bean mild mosaic virus (BMMV), carnation mottle virus (CarMV), rod-shaped tobacco mosaic virus (TMV), and filamentous potato viruses X and Y (PVX, PVY).
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MATERIALS AND METHODS Reagents and antibodies. Sheep (Imtek, Russia) and goat (Imtek, Russia; Arista Biologicals, United States) antibodies against rabbit IgG, peroxidaselabeled goat antirabbit antibodies immunoglobulins (Medgamal, Russia), Tris, Triton X-100, 3,3',5,5'-tetramethylbenzidine dihydrochloride, sodium azide (Sigma, United States), chloroauric acid (Fluka, Germany), Tween 20, bovine serum albumin (BSA), sodium citrate (MP Biomedicals, UK), glycerol, NaCl, K2CO3 (DiaM, Russia), Na2CO3, NaHCO3, KH2PO4, and KOH (Khimmed, Russia) were used. All the salts used in this study were of analytical or chemical grade. All solutions for the colloidal gold (CG) and its conjugates production were prepared using water and deionized by a Milli-Q system (Millipore, United States). Immunochromatographic test strips were manufactured using the mdi Easypack kit (Advanced Microdevices, India) comprised of the CNPC-SN12 L2-P25 working membrane with a pore size of 15 mkm, PT-R5 conjugate mount, a Type FR1(0.6) sample loading membrane, AP045 absorbent, and an MT-1-based adhesive laminated cover film. Plant Sample Preparation. Leaf extracts were prepared by grinding 200 ± 20 g leaf samples from healthy and virus-infected plants in 2 ml of extraction buffer solution (BS) (10 mM K-phosphate buffer, pH 7.4, 0.14 M NaCl, and 0.1% Triton X-100). Unclear crude leaf extract or leaf extract cleared by centrifugation at 10000 g for 5 min at room temperature was used for virus detection. Viruses and corresponding antibodies. BMMV, TMV, PVX, and PVY were maintained and propagated in a greenhouse in common bean Phaseulus vulgaris L., tobacco Nicotiana tabacum L., thorn apple Datura stramonium L. and tobacco N. tabacum L., respectively. Virus preparations were purified from infected leaves according to [12–15]. Virions of CarMV were purified from infected leaves of carnation Dianthus caryophyllus L. as described earlier [16]. Antisera against the viruses were obtained by immunizing rabbits with purified virus preparations [12–16]. The IgG fractions were obtained by ionexchange chromatography of antisera preparations on DEAE-Sephacel (Pharmacia, United States) in 0.01 M Na-phosphate buffer, pH8.0 [17]. Preparation of colloidal gold particles [18]. To obtain colloidal gold particles of 15 nm nominal diameter (CG-15), a 1% solution of HAuël4 (1.5 ml) was added to deionized water (95.0 ml). The mixture was brought to boil, then a 1% solution of sodium citrate (1.5 ml) was added with constant stirring, and the mixture was boiled for 10 min and then cooled down to room temperature. The protocol for preparation of colloidal gold particles of 30 nm nominal diameter (CG-30) was notable APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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for the use of 97.5 ml of deionized water and 1 ml of 1% solution of HAuël4; the reaction mixture was boiled for 30 min. The obtained CG preparations were stored at 4 to 6°C. Preparation of colloidal gold conjugated IgG [19]. To determine the optimal amount of protein required for conjugation, a CG sol (1 ml) was added to aqueous IgG solution (immunoglobulin concentrations ranging between 5 and 250 µg/ml were used), and the mixture was stirred and incubated for 10 min at room temperature. Then a 10% solution of NaCl (0.1 ml) was added to each sample; the resulting mixtures were stirred, further incubated for 10 min, and then the OD was measured at 580 nm. The IgG concentrations selected based on the data obtained are presented in the next section. To obtain the IgG-CG conjugate, the antibodies were dialyzed against 1000 volumes of 10 mM Na carbonate buffer (pH 9.0) for 2–3 h at 4°ë. The pH of the CG sol was adjusted to 9.0 with 0.2 M solution of ä2ëé3, and then the CG sol was added to the IgG solution at the chosen concentration. The mixture was stirred for 30 min at room temperature, and BSA was added to a final concentration of 0.25%. CG particles with immobilized IgG molecules were separated by centrifugation for 60 min at 20000 g and 8000 g for CG-15 and CG-30, respectively. After aspiration of supernatant, the pellet was resuspended in PBS containing 0.25% BSA. For long term storage of conjugate preparations, a NaN3 solution was added to a final concentration of 0.02%. Electron microscopy. Preparations of CG or its conjugates were applied to 300 mesh copper grids (Pelco International, United States) coated with polyvinyl formal film (formed from a solution of polyvinyl formal in chloroform). Images were acquired at magnifications of 3000000 using a CX-100 electron microscope operating at an accelerating potential of 80 kV. Analysis of digital images was performed using Image Tool software. Preparation of immunochromatographic test strips. Reagents were introduced into the membrane by an IsoFlow dispenser (Imagene Technology, United States). An IgG-CG conjugate (8 µl/cm of strip) was applied at a dilution corresponding to a D520 value of 2.0. For the preparation of the detection and control lines, IgG from antiserum against the corresponding virus and IgG from antiserum against the rabbit IgG were used, respectively. In both cases, 2 µl of 0.75 mg/ml IgG in PBS containing 10% glycerol were introduced per 1 cm of strip. An Index Cutter-I automatic cutter (A-Point Technologies, Inc., United States) was used to cut multimembrane composite strips 3.5 mm in width. Then the strips were sealed in laminated aluminium foil packets containing 0.5 g of silica gel dessicant using an FR-900 continuous band sealer (Wenzhou Dzhingli Packing Machinery Co., Ltd., China). Cutting and packaging
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(‡)
(b)
0.15
Fig. 1. Electron microscopy photographs of CG-15 (a) and CG-30 (b) preparations
operations were done in the specialized chamber at 20– 22°ë at a relative humidity not exceeding 30%. Immunochromatographic assay. was performed at room temperature. The test strip was dipped into a sample analyzed in an upright position for 90 s, then taken out and placed on a horizontal surface. The qualitative result of immunochromatographic assay was estimated by eye after 10 min. Instrumental detection of CG binding was performed using a Reflekom device (Okta-Medika, Russia). Its way of operation is based on videodigital evaluation of the color intensity of lines on a membrane [20]. One standard unit of color intensity was equal to the threshold of reliable visual detection. The degree of potato plant contamination by potato X and Y viruses was quantified by ELISA using Pirotest kits (JSC NVO Immunotekh, Russia) according to the manufacturer’s instructions. RESULTS AND DISCUSSION Parameters of colloidal gold preparations. Colloidal gold preparations, marked as CG-15 and CG-30, respectively (according to the expected diameters of the gold particles, as per [18]) were obtained for use as immunochromatographic markers. Based on electron microscopy data (Fig. 1), the following parameters of colloidal particles preparations were determined: the mean maximum axis, the mean minimal axis, and the Table 1. Average parameters of the colloidal gold preparations (a sample size of 150 particles) Parameter
CG-15
CG-30
Mean maximum axis ± s.d., nm* Mean minimum axis ± s.d., nm* Degree of ellipticity* Mean particle volume, nm3** Mean particle mass, g**
19.0 ± 2.0 16.5 ± 1.5 1.16 ± 0.12 2.34 × 103 4.5 × 10–17
38.7 ± 6.2 30.7 ± 3.7 1.26 ± 0.14 1.75 × 104 3.4 × 10–16
Notes: * According to electron microscopic data. ** Calculated data.
0.10 0.05
0
2
4
6
8
10
12 µg/ml
Fig. 2. Determination of PVX-specific IgG concentration (µg/ml), suitable for the conjugation with CG-30. Zero level of D520 corresponds to IgG-free CG (no 10% NaCl added). The selected IgG concentration (10 µg/ml) is marked with an arrow.
degree of ellipticity (defined as the ratio between maximum and minimum axes). Relying on these parameters, the average mass and volume of particles were calculated. The results obtained (Table 1) demonstrate the high degree of homogeneity of the CG-15 and CG-30 preparations. Preparation of colloidal gold conjugated immunoglobulins. The immunochemical properties of the IgG-CG conjugate depend on the degree of retention of immunoglobulins' reactivity, as well as on their orientation on a CG particle surface. To choose between the CG-15 and CG-30 preparations, we compared the way their anti-TMV IgG conjugates interacted with immobilized ones in the detection and control line of immunoreagents under uniform conditions (CG D520 2.0, 5 µg/ml TMV in PBST, 0.5 mg/ml IgG immobilized in both detection and control lines). The control line color intensity was 5.0 s.u. for CG-15 and 9.4 s.u. for CG-30; for the detection line, 7.3 s.u. and 14.3 s.u., respectively. Based on these data, the CG-30 preparation was used in subsequent experiments due to its ability to provide maximal color intensity in both detection and control lines. The CG-30 particles were conjugated with IgG from the antisera against the five analyzed viruses. To find the IgG concentration optimal for obtaining stable, nonaggregating CG-conjugates, the process was monitored by measuring the optical density at 580 nm in the presence of 10% NaCl [21]. The typical dependence of D580 on IgG concentration for PVX is shown in Fig. 2. Its shape is in good agreement with up-to-date concepts of protein conjugation with colloidal gold [22]. The
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2
1
3
207
(b)
4
5
Ab-CG
Ab
Ab2
(d) (c) 6
(e) 7
8
Fig. 3. The principle of immunochromatographic assay. a—test-system composition: 1—plastic mount, 2—absorption membrane; 3—conjugate mount; 4—working membrane; 5—absorption pad. b—reagents introduced: Ab—virus-specific antibodies; Ab2— antibodies against rabbit IgG; Ab-CG - colloidal gold conjugated to virusspecific antibodies. c—A schematic representation of analysis: 6—spreading of liquid front; 7—test line; 8—control line. d—Test result in the absence of the virus: one colored band in the control line area. e—Test result in the presence of the virus: two colored bands in both test and control line areas.
increase in the number of IgG molecules leads to CG particle stabilization, preventing their aggregation in solutions of high ionic strength; as well, D580 rises up to a maximum value and then descends to a plateau. Based on the correlations observed, IgG concentrations exceeding by 10–15% that of the exit point of the plateau for D580 were selected (as recommended by [22] to ensure the maximum stability of the conjugates). The following concentrations were found to be optimal: 10, 10, 14, 20, and 16 µg/ml of IgG from antisera against TMV, PVX, PVY, CarMV and BMMV, respectively. Development of plant virus detection immunochromatographic test system. Since viruses are polyvalent antigens, the sandwich-type immunochromatographic assay was used; the scheme of the assay is shown in Fig. 3. Test-systems were optimized by choosing the proper concentrations of reagents (IgG, IgG-CG conjugates), providing maximal color intensity in both detection and control lines and the lowest detection limit with no background staining of the membrane. Analysis of IgG-CG accumulation in the detection and control lines revealed that among IgG-CG preparations with the optical density at 520 nm between 0.25 and 3.0 the variant with D520 = 2.0 was the optimal one with the intensities of both lines reaching the plateau level. The criterion for the choice of an IgG concentration was the color intensities in the detection and control lines. The maximal color intensity of the detection line was achieved when a 0.75 mg/ml solution of IgG was APPLIED BIOCHEMISTRY AND MICROBIOLOGY
loaded on the membrane; higher IgG concentrations did not produce any further increase of the detection line intensity. Therefore, an IgG concentration of 0.75 mg/ml was used in subsequent experiments. The choice of antispecies antibodies for the immobilization at the test line was made based on a comparison between goat and sheep antibodies against rabbit IgG (Imtek, Russia) and goat antibodies against rabbit IgG (Arista Biologicals, United States). The color intensities of the detection line under uniform conditions (D520 = 2.0, antispecies IgG concentration 0.5 mg/ml) for these preparations were 8.0, 3.9 and 13.9 s.u., respectively. Therefore, antispecies antibodies from “Arista Biologicals” were chosen for the teststrips preparation. Antispecies antibodies were introduced to the membrane as 0.75 mg/ml solution, providing, like the virus-specific IgG, the maximal line intensity. The results of videodigital evaluation demonstrate that color intensity in the detection and control lines reached its peak value in 10 min after the sample application to the test strip (Fig. 4). Therefore, 10 min analysis time was chosen as the optimal one. Based on the results of experiments with all five test-systems, the optimal conditions for immunoreagent obtaining, test-strips preparation and the analysis execution were established. Determination of detection limits of the test systems. The results of immunochromatographic detection of PVY standard preparations (dilutions in BS) are shown in Fig. 5. As evident from these data, PVY can
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s.u. 12 10 8 6 4 2 0
2
4
6
8
10 min
Fig. 4. The dependence of color intensity in the test line (s.u.) on the time of analysis of PVY (1.5 µg/ml) in BS.
be reliably analyzed by ICA method at concentrations starting at 0.08 µg/ml. To evaluate the applicability of ICA test for the biological samples analysis, the purified PVY preparation was diluted with either BS, crude or cleared extracts from healthy potato leaves. The results of the analysis are shown in Table 2. It is noteworthy that the color intensity of the detection line during the analysis of virus in leaf extract was lower than that of the virus detection in buffer solution. At the same time, there was no noticeable difference in results of the analysis between crude and cleared extracts. This last point should be emphasized because the lysate clearing is not always feasible for out-of-laboratory test-strip analyses.
Similar results were obtained for the other four viruses. Virus concentrations, corresponding to the limit of reliable detection of test line coloration, were (in µg/ml): 0.08 for PVX, 0.2 for CarMv and TMV, 0.5 for BMMV. Based on the reagents prepared, the immunochromatographic test-system, capable for parallel detection of three potato-infecting viruses: PVX, PVY and TMV should be developed. This system should carry three detection lines loaded with antibodies against PVX, PVY and TMV, and the control line containing antispecies antibodies. As compared with the test systems for the individual detection of the viruses, the duration of multianalysis does not increase, remaining within 10 min, thus providing the substantial increase in test productivity. Test-strips examination using the plant material samples. The sensitivity of PVX and PVY detection by immunochromatographic method complies with the average level of accumulation of these viruses in infected potato leaves [4]. Taking into account these features of test-systems, their examination has been performed on the plant material samples. Twenty PVYinfected potato plants of Lugovskii cultivar and five PVX-infected potato plants of Nevskii cultivar grown from tubers in a greenhouse and tested for the presence of the corresponding viruses by ELISA using “Pirotest” kits were analyzed by immunochromatography using a “Reflekom” device. Twenty PVX-negative potato plants of Lugovskii cultivar and five PVY-negative potato plants of Nevskii cultivar were used as a negative control. The absence of infection was also confirmed by ELISA assay. The results of the ELISA assay for the viral infections are presented in Table 3. The incidence of PVY false positive results and false negative results was 5% and 10%, respectively,
(‡)
(b) s.u. 10 2 1
8 6 4 2 0
0.08
0.15
0.30
0.60
1.25
2.50 µg/ml
10–1
1
µg/ml
Fig .5. Immunochromatographic detection of PVY in BS: the overview of test-strips after the analysis (a; 1—test line, 2—control line) and dependence of color intensity (s.u.) in the test line on the PVY concentration (µg/ml, b) APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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DEVELOPMENT OF IMMUNOCHROMATOGRAPHIC TEST SYSTEMS Table 2. Dynamics of PVY binding in the immunochromatographic system PVY concentration, mkg/ml 0.2 0.2 0.5 0.5 0.5 1.5 1.5 1.5 5.0 5.0
PVY preparation diluent
Mean intensity of the test line, s.u. ± s.d., (n = 10) 6.4 ± 0.8 2.1 ± 0.6 7.0 ± 1.3 4.3 ± 0.6 4.5 ± 0.7 9.6 ± 1.4 6.0 ± 1.7 5.2 ± 1.6 10.9 ± 1.4 7.8 ± 1.9
BS Cleared lysate BS Cleared lysate Crude extract BS Cleared lysate Crude extract BS Cleared lysate
Table 3. Analysis of PVX and PVY in potato leaf extracts
Plants
Total
According to ICA assay data: infected
noninfected
PVY infected
20
18
2
PVY noninfected
20
1
19
PVX infected
5
5
0
PVX noninfected
5
0
5
while in the case of PVX the ELISA and ICA results agreed completely. The data obtained indicate that immunochromatographic test-systems developed may by used for express-diagnostics of plant virus diseases, in particular in field and out-of-laboratory conditions. ACKNOWLEDGMENTS The authors are grateful to Dr. V. A. Shtein-Margolina (A.N. Bakh Institute of Biochemistry, RAS) for the help in electron microscopy and Dr Yu. A. Varitsev (Lorkh All-Russian Research Institute of Potato Growing, RAAS) for kindly providing purified PVY preparation. This work was supported by the Russian Foundation for Basic Research (project no. 06-04-08290-ofi-a).
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