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Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses
Author’s Accepted Manuscript Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses Irina V. Safenkova, Vasily G. Panferov, Natalia A. Panferova, Yuri A. Varitsev, Anatoly V. Zherdev, Boris B. Dzantiev www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)31271-2 https://doi.org/10.1016/j.talanta.2018.12.004 TAL19352
To appear in: Talanta Received date: 29 September 2018 Revised date: 2 December 2018 Accepted date: 3 December 2018 Cite this article as: Irina V. Safenkova, Vasily G. Panferov, Natalia A. Panferova, Yuri A. Varitsev, Anatoly V. Zherdev and Boris B. Dzantiev, Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses, Talanta, https://doi.org/10.1016/j.talanta.2018.12.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses
Irina V. Safenkovaa, Vasily G. Panferova, Natalia A. Panferovaa, Yuri A. Varitsevb, Anatoly V. Zherdeva, Boris B. Dzantieva*
a
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy
of Sciences, 119071 Moscow, Russia. b
A.G. Lorch All-Russian Potato Research Institute, 140051 Kraskovo, Moscow region, Russia.
*
Corresponding author at: A.N. Bach Institute of Biochemistry, Research Centre of
Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, 119071 Moscow, Russia. Tel.: +7-495-954-3142. [email protected]
Abstract This study presents new type of the lateral flow immunoassay (LFIA) for multi-target analysis. A test, named alarm-LFIA, has an essentially new function that consists in notice (signaling the danger) about the presence at least one target from the controlled list without identification. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies, and multi-specific conjugate that binds the several targets. The alarm test is based on the novel conjugate with broaden specificity due to the immobilization of a mix of antibodies, specific to several structurally different targets, on the surface of gold nanoparticles. For proof of concept, multi-specific conjugate to five important potato viruses (potato virus X, -M, -S, -Y and potato leaf roll virus) was fabricated using five antibodies with different specificity. The alarm-LFIA was developed for rapid detection of the total infection caused by up to five viruses. Detection limits of the viruses in potato leaf extracts are from 10 to 30 ng/mL. The alarm-LFIA was successfully used for viruses’ detection in potato leaves; results were confirmed by enzymelinked immunosorbent assay. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples.
Keywords: Lateral flow immunoassay; Gold nanoparticles; Multi-specific conjugate; Multitarget detection; Screening testing
1. Introduction Multi-target analysis is one of the main trends of immunoassay evolution. To date, simultaneous detection of multiple analytes in a single test is an emerging approach for different practical areas such as medicine, veterinary, food quality and safety and environmental analysis [1-3]. Multi-target analysis comes in a variety of immunomethods and analytical approaches [3]. The most attractive rapid, simple and cost-effective method is lateral flow immunoassay (LFIA) [4-7]. One of the main advantages associated with LFIA is its ease of use since all reagents are applied to the test strip by the manufacturer. Contact of reagents with a tested sample initiates specific interactions, which are assessed by a visible result (i.e. the presence or absence of colour in defined areas of the strip). In general, a large number of developments for multi-analytic applications are known for LFIA [8-14]. In a recent review, Li and Macdonald [15] highlighted biosensors with multiple lines (or dots) and multi-coloured labels in the lateral flow biosensors for multiple analytes detection on a single device. The general approach in all cases is the separated information about each target due to different signals or spatial separation. By increasing the number of test zones or labels, more targets will be differentiated and identified. The most commonly used approach for multiplex LFIA is based on the application of multiple test zones with immunoreagents with different specificity [10, 15]. However, often the detection in the sample of any target from the controlled list leads to the same further actions, for example the sample rejection. In immunoassay, the test showing one signal for many targets usually involves antibodies with broad specificity [3]. However, such tests can be realised only for the analyses of closely related analytes. There are many analytical tasks associated with the presence of various structurally different compounds need to control. Finding the solution for non-closely related analytes without using the antibody with broaden-specificity is an urgent task of multi-analysis. The easy and rapid LFIA is an appropriate method to get answers about disease if at least one pathogen presents in the sample. The LFIA with one binding (test) zone for detecting a set of non-related pathogens without identification will have two coloured lines at the presence of even one pathogen in the sample. This approach is simpler than the spatial separation of the detection
within one test. The approach is similar to determining the temperature of a sick person in that it shows the disease but does not identify its reasons. Several papers proposed the use of one test zone formed by a mixture of immunoreagents to identify several analytes [16-19]. However, the aims of these studies were also in the recognition and differentiation of individual analytes due to the use of a mixture of conjugates with different labels specific to different target analytes. Recognition of individual analytes occurs by processing with special equipment or colour data analysis. Thus Wang et al. proposed for the simultaneous detection of two tumor markers (alpha fetoprotein and carcinoembryonic antigen) a single test zone comprising a mixture of antibodies and a mixture of the antibody’s conjugates with quantum dots [16]. The similar paper by Wang et al. described LFIA with one test zone and two antibody conjugates with different enzymes (horseradish peroxidase and alkaline phosphatase) providing time-resolved chemiluminescence detection of two β-agonists in the test zone [18]. Detection of three viruses (dengue, yellow fever, and Ebola viruses) in single test zone using multicolour silver nanoparticles with different sizes was described by Yen et al. [17]. For antibody – gold nanoparticle conjugates, a similar LFIA was developed by Di Nardo et al. with the single test zone and multicolour GNPs for detection of aflatoxin B1 and type-B fumonisins [19]. The idea proposed in this paper is different both in purpose and in implementation. The approach is similar to determining the temperature of a sick person in that it shows the disease but does not identify its reasons. We refused the use of mixture of several mono-specific conjugates, which has already been reported and chose to utilize one multi-specific conjugate to solve the specific tasks. In our knowledge, a multi-target LFIA with one test zone design and multi-specific gold nanoparticles (GNP) – antibody conjugate, was not earlier described. Thus, in this study we have realised alarm-LFIA. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies. As a proof of concept, the idea was realised with five antibodies. Therefore, we propose the alarm-LFIA for five targets. Commonly, the detecting reagent for multi-analyte LFIA is a mixture of individual GNP – antibody conjugates of different specificity [20-22]. However, for our purposes the mixture is not optimal because only a part of the conjugated GNPs in the mixture can bind a specific target and provide coloration. Therefore, we have done it another way through the conjugation of GNPs with a mixture of antibodies of different specificity. The concentration of the immune complexes in the test zone will be about five times higher (in the case of five target pathogens for one multispecific conjugate). Therefore, the multispecific GNP antibody conjugate was used in the alarm-LFIA. In the control zone of the test strips, protein A was immobilised because it is a universal reagent binding antibody.
Complexes of protein A GNP conjugate are formed in the control zone, regardless of the presence of any antigen in the sample. The schemes of the alarm test strip and a common LFIA monotest strip are shown on Fig. 1. For proof of concept, the set of five important potato viruses, potato virus X (PVX), potato virus M (PVM), potato virus S (PVS), potato virus Y (PVY) and potato leaf roll virus (PLRV) were chosen as targets to aim the alarm test. These viruses cause large yield losses and related economic losses [23, 24]. It is crucial to have efficient methods of disease detection so that the damage caused by pathogens can be reduced. A one alarm test is more useful than five monotests as it solves the main task of monitoring (the divide of healthy and infected seeds), reducing the cost and labour intensity. Herein, we propose an alarm-LFIA for rapid nonlaboratory detection of total viral infection caused the five major potato viruses. 2. Materials and methods 2.1. Materials Potato viruses (PVX, PVM, PVS, PVY, PLRV) and their specific polyclonal antibodies as partly described in previous works [20, 25] were provided by Y. A. Varitsev. Tris(hydroxymethyl)aminomethane (Tris), Tween 20, Triton X-100 (Sigma-Aldrich, USA); chloroauric acid (Fluka, Germany); bovine serum albumin (BSA), sodium citrate, and dimethyl sulfoxide (MP Biomedicals, UK); glycerol, sodium chloride and potassium carbonate (DiaM, Russia); protein A from Staphylococcus aureus (Imtek, Russia); and sodium carbonate, sodium bicarbonate, potassium dihydrogen phosphate and potassium hydroxide (Khimmed, Russia) were used for this study. All the chemicals were of analytical reagent or chemical reagent grade. The nitrocellulose membranes (CNPH-N-200) adhering to the surface of the laminated card, conjugate release matrix (PT-R5), sample pads (GFB-R4, 0.35) and absorbent pads (AP045) were obtained from Advanced Microdevices (India). 2.2. Leaf extract preparation Infected (14 samples) and healthy (3 samples) potato leaves were used to prepare the extracts. The potato leaves were homogenised in 50 mM Tris-HCl containing 0.05% Triton X100 and 100 mM NaCl (1:10 w/v) for a duration of 5 min in a porcelain mortar and stored at 4 °C before use. 2.3. Gold nanoparticle synthesis A Frens method [26] was used for GNP synthesis. First, 1 mL of 1% HAuCl4 was added to 95 mL of deionised water and heated to the boiling point, and then 4 mL of 1% sodium citrate was added while the mixture was stirred. The mixture was boiled for 25 min, cooled and stored at 4 °C.
2.4. Determining the antibody concentration for the conjugation (flocculation method) To determine the optimal amount of the antibodies for the conjugation, according to Hermanson [27], we added a GNPs (1.0 mL portions at pH 9.0; optical density at 520 nm (OD520) = 1.0) to antibody (0.1 mL at pH 9.0; concentrations from 1 to 250 mg/mL), incubating the mixture at room temperature for 10 min. 10% NaCl (0.1 mL) was added to each sample, stirred for 10 min, and measured OD580. Dependence of OD580 in the presence of 10% NaCl against the antibody concentration (flocculation curves) was plotted. The concentration corresponding to the beginning of the plateau in the flocculation curve was determined as a minimum ratio to prevent the aggregation of GNPs. 2.5. Synthesis of GNP-antibody conjugates Antibody at a chosen concentration by flocculation curve was added to the GNP solution and mixed for an hour. Free GNP surface was blocked with BSA (final concentration of 0.25%). The conjugates were separated through centrifugation at 20,000 g for 30 min at 4 °C. The synthesised conjugates were suspended in 10 mM Tris buffer, pH 7.4, containing 0.25% BSA, 0.25% Tween 20 detergent and 1% sucrose. The conjugates were stored at 4 °C. 2.6. Transmission electron microscopy (TEM) Preparations of the GNPs and their conjugates were adsorbed on cooper grid coated with a poly(vinyl formal) for 10 min. Images were obtained using a JEM CX-100 electron microscope (JEOL, Japan) operating at 80 kV. The digital images were analysed with Image- Tool (UTHSCSA, USA). 2.7. Preparation of mono LFIA and alarm-LFIA test strips Test and control zones were formed on the nitrocellulose membrane (CNPH-N-200) using specific antibodies and protein A, respectively. Antibodies specific to viruses were dispensed in the test zone at 0.5 mg/mL for monotest strips and 2.5 mg/mL (5 types of specific antibodies, per 0.5 mg/mL of each antibody) for alarm test strips, whereas protein A (0.5mg/mL) was dispensed in the control zone. An IsoFlow dispenser (Hanover, NH, USA) dispensed all reagents at 0.15 mL per mm membrane width in PBS containing 5% glycerol and 0.03% NaN 3. The GNP-antibody conjugates (mono- or multi-specific) were deposited onto glass-fibre membranes (conjugate release fiberglass membrane PT-R5) from a solution with OD520 = 4; the conjugate load was 1.6 µL per mm of strip width. All membranes were dried at 37 °C for eight hours. Following this, we attached glass-fibre membranes, sample pads (GFB-R4, 0.35), and adsorbed pads (AP045) to plastic supports with the nitrocellulose membrane (CNPH-N-200). The obtained multimembrane composites were cut into strips 3.5 mm in width by an Index Cutter-1 (A-Point Technologies, USA). Test strips were hermetically packed into laminated aluminum foil bags containing silica gel at 20-22 °C and a relative humidity of less than 30%.
2.8. Lateral flow immunoassay The assay was performed at room temperature. The test strip was vertically submerged in the test sample for 1.5 min before it was removed and placed on a horizontal surface. The formations of coloured test and control zones were visually detected in 10 min. The limits of the visual detection of the LFIA tests were determined as virus concentrations (ng/mL) when the test line appeared. For the quantitative analysis, the test strips were scanned using a Canon 9000F Mark II scanner (Canon, Tokyo, Japan), and digital images were analysed with a TotalLab TL120 (Nonlinear Dynamics, UK). Linear approximations for the initial sections of the dependence of the colour intensity of the LFIA test line on the viruses concentration were then constructed by Origin Pro 9.0 (OriginLab, USA). 2.9. Enzyme-linked immunosorbent assay (ELISA) of the viruses in potato leaf extracts The potato leaf extracts were tested using ELISA kits for PVX, PVM, PVS, PVY, PLRV (Test Potato, Ltd, Russia) according to the manufacturer's protocols. The statistical processing and linear regression analysis were performed by Origin Pro 9.0 (Origin Lab, USA). To calculate the content of the viruses, linear ranges of the calibration curves were used. 3. Results and discussion 3.1. Synthesis of GNPs and multi‐specific GNP – antibody conjugate At the first stage, we synthesised and characterised GNPs by TEM (Data are provided in Supplementary Information, Fig. S1). The GNPs had a narrow size distribution with a mean diameter of 18.6 ±3.8 nm. Our previous experiences in the synthesis of GNP conjugates with the same antibodies specific to these viruses confirm that spherical GNPs with the same diameters provide high stability of the conjugate and high sensitivity of the LFIAs [20, 28]. The maximum number of IgG that can be immobilised on the surface of one nanoparticle is ~60 molecules (100% coverage area of GNP) taking to account the 5–7 nm of hydrodynamic diameter of IgG (data from the Protein Data Bank). This is a sufficient number to simultaneously immobilise five antibodies with different specificity. We used dynamic light scattering to verify homogeneity and stability of GNP preparation (Data are provided in Supplementary Information, Fig. S2). No aggregates were observed, and dispersion of GNPs was confirmed. Electrophoretic light scattering showed the GNP zeta potential of −31.6 mV that confirmed stability of the GNPs [29]. The synthesised GNPs were used for conjugation with antibodies specific to PVX, PVM, PVS, PVY and PLRV. To synthesise the different monospecific conjugates and multi-specific conjugates with broaden-specificity to the viruses, physical adsorption was used as the most
common technique for synthesis of the GNP conjugates. We used the flocculation method to estimate the adsorption ability of five antibodies to the GNP surface. The minimum ratio of each antibody preventing the aggregation of GNPs was about 30–35 antibodies per one nanoparticle, and the maximum coverage of the GNP surface accorded to the range 55–80 antibodies per GNP (Fig. 2). This coincidence of flocculation curves means that the immobilisation of a mixture of antibodies with a predetermined equal ratio, the content of the antibodies of different specificity on the GNP surface is close to each other. We used a larger concentration of antibodies (57 antibodies per GNP) corresponding to the maximum coverage of the nanoparticle surface. To evaluate the effect of increase of the total antibodies’ concentration, we obtained multi-5-specific conjugate (50 μg/mL total concentration, with 10 μg/mL (57 antibodies per GNP) of each antibody) and five mono-specific conjugates (10 μg/mL total concentration of one antibody). For all synthesised conjugates, the absorption peaks have the same narrow width and a slight shift of the maxima (not more than 5 nm) as compared with the native GNPs, which indicates the adsorption of antibodies on the GNP surface [30]. Certainly, the surface coverage of specific antibodies isn’t the same for multi- and monospecific conjugates. However, the functional properties of individual virus recognition are not worsened. Based on previous works [31-33], we could assume that 20% coverage of IgG molecules of each antibody type on the GNP surface is already sufficient for good binding to the target. 3.2. Optimization and characterization of the alarm test We have optimised concentration of all reagents for the alarm test strip. We used a nitrocellulose membrane with the highest protein binding that guaranteed the sorption capacity in the required range. The concentration of antibodies immobilised on nitrocellulose membrane in the test zone was 2.5 μg/μL (five types of specific antibodies, per 0.5 μg/μL of each antibody). It should be noted that the concentrations are slightly lower (~2–3 times) than those commonly used in LFIA [25], but we used them to reduce nonspecific reactions. The optimal amount of the multi-5-specific conjugate in the LFIA test system corresponded to OD520 = 4 (16 µL per centimetre of the pad with) (Fig. 3) to provide the highest signal to noise ratio and absence of non-specific reactions. Probably, non-specific reactions with a total OD520 of the conjugate more than 4 are largely related to the mixture composition of the antibodies in the test zone. Similar nonspecific effects were also noted by Shu et al. [34]. Therefore, we adhered to the conditions that the total OD520 of the conjugate(s) in the lateral flow systems, especially in the system with a mixture of antibodies in the test zone, should not exceed 4. Under these conditions, the replacement of the multi-5-specific conjugate by five mono-specific conjugates will cause additional negative factors. This means that OD520 of each conjugate would be equal to 0.8,
which doesn’t provide high sensitivity [25]. Moreover, the colorimetric signal in test zone for one virus will be formed with the participation of all GNPs for multi-5-specific conjugate and with participation of one-fifth of the GNPs for mixture of five mono-specific conjugates. This is an additional reason for the use of the multi-5-specific conjugate. To determine the influence of the simultaneous immobilisation of several antibodies on the limit of detection (LOD), the test strips with multi-5-specific conjugates and test strips with monospecific conjugates were compared. At first, the testing was realised in a model buffer solution (0.05 M potassium phosphate, pH 7.4, 0.1 M NaCl, 0.05% Tween-20; PBST) containing different concentrations of viruses. The multi-test demonstrated LODs in the range 10–30 ng/mL depending on the virus. Data are provided in Supplementary Information, Fig. S3. Three monospecific LFIA test systems for detection of PVX, PVM and PVY were fabricated according to the scheme on Fig. 1A and under the optimised conditions described previously [25]. Concentrations of the mono-specific GNP – antibody conjugates accorded concentration of the multi-5-specific conjugate (16 µL per cm, OD520 = 4). The LOD of mono-LFIA for each virus was 8 ng/mL. The analysis time for multi- and mono-LFIA was the same, 10 min. The results show that LODs of PVX, PVM and PVY test strips with multi-5-specific conjugate to be slightly higher than the LODs of monotest strips. The obtained small differences of LODs are no more than four times and are not critical for diagnosis. The alarm-LFIA with multi-5-specific conjugate was used to analyse potato leaf extracts spiked with different concentrations of PVX, PLRV, PVY, PVS and PVM. Fig. 4 shows the typical test strips after the different viruses determination and the plotted dependences of colour intensities of the test zone on virus concentration. The LOD for the five viruses in extracts coincided to the LOD in the buffer (10 ng/mL of PVX, 20 ng/mL of PVY and PVM 30 ng/mL of PLRV and PVS). We would like to note that there are no exact threshold values in diagnostics of potato viruses. The data about the accumulation of viruses [23, 24] and the detection limits achieved by ELISA and LFIA [20, 35, 36] allow to conclude that the reached LODs are relevant to assess the total level of virus infection. At the same time, the multi-conjugate system has great potential for further developments due to the ability to control and modulate the sensitivity of the analysis. This ability is mostly associated with the change in ratio of specific antibodies with different affinities in the multi-conjugate. As shown in [31, 32], the dependence of LOD on the number of antibodies immobilized on the GNP surface is not linear, but the LOD can be adjusted if necessary to satisfy practical demands. 3.3. Approbation with potato leaf samples The final task, approbation with real samples, was conducted for the diagnostics of the potato plants (17 leaf extracts, each in duplicate). The developed alarm-LFIA was compared with
ELISA. To calculate the viruses content by ELISA, linear ranges of the calibration curves were used. Table 1 provides the test results. Twelve leaf samples showed the same results of the infection by alarm-LFIA and ELISA. Three leaf samples showed the same results of health. These results confirm that the alarm-LFIA could be used as simple, rapid (10 min) and effective method for potato virus detection without differentiation. Certainly, the developed alarm test assumes only a qualitative analysis. The colour intensity in the test zone can’t be recalculated into concentration; different viruses cause a different signal. For clarity, Table 2 shows the test zones after the analysis of samples (1 and 4) with the same total viral infection with different colour intensities. At the same time, a sample 15 with a lower total infection has a larger signal than sample 4. This is explained by the different content of individual viruses in the sample (see Table 1).
Conclusions The obtained results show that one alarm-LFIA for rapid non-laboratory detection replaces five tests and can be used to determine total viral infection caused the five major potato viruses. The result is largely due to the synthesised multi-specific GNP conjugate with broadenspecificity to five major potato viruses. The developed alarm-LFIA can be easily used for multiple-pathogen analysis and total virus infection determination, which greatly improves the agrotechnical control. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples. These cases include pathogens dangerous to humans, animals or plants, or pesticides, antibiotics and mycotoxins, whose level should not exceed a present threshold. If at least one from the controlled list is detected, the sample will be singled out for special processing. A special attention deserves the concept of a multi-specific reagent promising for the simultaneous analysis of several structurally different targets.
Acknowledgements This study was financially supported by the Russian Science Foundation (grant 16-1604108).
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Fig. 1. Schemes of test strips for LFIAs: (A) a common monotest strip with a monospecific GNP conjugate and one type of specific antibody in the test zone; (B) the alarm test strip with a multispecific GNP conjugate and several types of antibodies in the test zone. 1 indicates the membrane with the conjugate applied, 2 indicates the test zone and 3 indicates the control zone. Fig. 2. Flocculation curves of antibodies’ immobilisation on the GNP’s surface. Concentration dependencies of the absorbance of GNPs at 580 nm in the presence of excess salt (10% NaCl) after the addition of different concentrations of antibodies (from 5 to 90 antibodies per one GNP). Arrows indicate the number of antibodies for the stabilisation of the GNP’s surface (stop flocculation) and the maximum number of antibodies according the calculations. Grey areas schematically illustrate surface coverage at different ratios of antibodies to GNP. Fig. 3. Colour intensity of the test zone of LFIA test strips with multi-5-specific conjugate for PVX, PLRV, PVY, PVS or PVM detection (concentration of each virus is 50 ng/mL). Fig. 4. Alarm-LFIA for virus detection in leaf extracts: Test zones of the strips after detection of 500, 250 and 60 ng/mL of each virus and dependences of colour intensities of the test zone on virus concentration. 1–5 accord to PVX, PLRV, PVY, PVS and PVM, accordingly. The dashline indicates the average colour intensity of the negative control.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Graphical Abstract
Table 1 Results of alarm-LFIA and ELISA with potato leaf extracts. Sample №
Alarm-LFIA
ELISA results (ng/mL) PVX
PLRV PVY PVS
PVM
1
infected
≥1000 –
–
–
–
2
infected
≥1000 –
150
15
–
3
infected
160
–
125
20
–
4
infected
–
10
770
110
140
5
infected
115
850
60
–
–
6
infected
100
–
–
22
–
7
non- infected
–
–
–
–
–
8
non- infected
–
-–
–
–
–
9
infected
115
380
125
35
–
10
infected
20
–
–
–
130
11
infected
≥1000 –
–
–
–
12
non- infected
–
–
–
–
13
infected
≥1000 20
–
160
–
14
infected
390
70
–
–
–
15
infected
200
–
–
–
85
16
infected
≥1000 125
–
230
–
17
infected
–
–
–
80
–
–
Table 2 Results of alarm-LFIA for samples with the same and different total viral infections. Sample number
1
4
15
Total viral infection, ng/mL
1000
1030
285
18.5
5.8
10.5
Test strips after analysis
Colour intensity, arb. units
Highlights
The alarm lateral flow immunoassay (LFIA) is proposed for multi-target analysis The concept of the test is based on the antibody’s conjugate with broaden-specificity Gold nanoparticle conjugate specific to 5 structurally different targets is obtained One test line of the alarm LFIA indicates total infection caused from 1 to 5 viruses The alarm LFIA is easy tool for a multi-target analysis by one test strip
PII: DOI: Reference:
S0039-9140(18)31271-2 https://doi.org/10.1016/j.talanta.2018.12.004 TAL19352
To appear in: Talanta Received date: 29 September 2018 Revised date: 2 December 2018 Accepted date: 3 December 2018 Cite this article as: Irina V. Safenkova, Vasily G. Panferov, Natalia A. Panferova, Yuri A. Varitsev, Anatoly V. Zherdev and Boris B. Dzantiev, Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses, Talanta, https://doi.org/10.1016/j.talanta.2018.12.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses
Irina V. Safenkovaa, Vasily G. Panferova, Natalia A. Panferovaa, Yuri A. Varitsevb, Anatoly V. Zherdeva, Boris B. Dzantieva*
a
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy
of Sciences, 119071 Moscow, Russia. b
A.G. Lorch All-Russian Potato Research Institute, 140051 Kraskovo, Moscow region, Russia.
*
Corresponding author at: A.N. Bach Institute of Biochemistry, Research Centre of
Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, 119071 Moscow, Russia. Tel.: +7-495-954-3142. [email protected]
Abstract This study presents new type of the lateral flow immunoassay (LFIA) for multi-target analysis. A test, named alarm-LFIA, has an essentially new function that consists in notice (signaling the danger) about the presence at least one target from the controlled list without identification. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies, and multi-specific conjugate that binds the several targets. The alarm test is based on the novel conjugate with broaden specificity due to the immobilization of a mix of antibodies, specific to several structurally different targets, on the surface of gold nanoparticles. For proof of concept, multi-specific conjugate to five important potato viruses (potato virus X, -M, -S, -Y and potato leaf roll virus) was fabricated using five antibodies with different specificity. The alarm-LFIA was developed for rapid detection of the total infection caused by up to five viruses. Detection limits of the viruses in potato leaf extracts are from 10 to 30 ng/mL. The alarm-LFIA was successfully used for viruses’ detection in potato leaves; results were confirmed by enzymelinked immunosorbent assay. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples.
Keywords: Lateral flow immunoassay; Gold nanoparticles; Multi-specific conjugate; Multitarget detection; Screening testing
1. Introduction Multi-target analysis is one of the main trends of immunoassay evolution. To date, simultaneous detection of multiple analytes in a single test is an emerging approach for different practical areas such as medicine, veterinary, food quality and safety and environmental analysis [1-3]. Multi-target analysis comes in a variety of immunomethods and analytical approaches [3]. The most attractive rapid, simple and cost-effective method is lateral flow immunoassay (LFIA) [4-7]. One of the main advantages associated with LFIA is its ease of use since all reagents are applied to the test strip by the manufacturer. Contact of reagents with a tested sample initiates specific interactions, which are assessed by a visible result (i.e. the presence or absence of colour in defined areas of the strip). In general, a large number of developments for multi-analytic applications are known for LFIA [8-14]. In a recent review, Li and Macdonald [15] highlighted biosensors with multiple lines (or dots) and multi-coloured labels in the lateral flow biosensors for multiple analytes detection on a single device. The general approach in all cases is the separated information about each target due to different signals or spatial separation. By increasing the number of test zones or labels, more targets will be differentiated and identified. The most commonly used approach for multiplex LFIA is based on the application of multiple test zones with immunoreagents with different specificity [10, 15]. However, often the detection in the sample of any target from the controlled list leads to the same further actions, for example the sample rejection. In immunoassay, the test showing one signal for many targets usually involves antibodies with broad specificity [3]. However, such tests can be realised only for the analyses of closely related analytes. There are many analytical tasks associated with the presence of various structurally different compounds need to control. Finding the solution for non-closely related analytes without using the antibody with broaden-specificity is an urgent task of multi-analysis. The easy and rapid LFIA is an appropriate method to get answers about disease if at least one pathogen presents in the sample. The LFIA with one binding (test) zone for detecting a set of non-related pathogens without identification will have two coloured lines at the presence of even one pathogen in the sample. This approach is simpler than the spatial separation of the detection
within one test. The approach is similar to determining the temperature of a sick person in that it shows the disease but does not identify its reasons. Several papers proposed the use of one test zone formed by a mixture of immunoreagents to identify several analytes [16-19]. However, the aims of these studies were also in the recognition and differentiation of individual analytes due to the use of a mixture of conjugates with different labels specific to different target analytes. Recognition of individual analytes occurs by processing with special equipment or colour data analysis. Thus Wang et al. proposed for the simultaneous detection of two tumor markers (alpha fetoprotein and carcinoembryonic antigen) a single test zone comprising a mixture of antibodies and a mixture of the antibody’s conjugates with quantum dots [16]. The similar paper by Wang et al. described LFIA with one test zone and two antibody conjugates with different enzymes (horseradish peroxidase and alkaline phosphatase) providing time-resolved chemiluminescence detection of two β-agonists in the test zone [18]. Detection of three viruses (dengue, yellow fever, and Ebola viruses) in single test zone using multicolour silver nanoparticles with different sizes was described by Yen et al. [17]. For antibody – gold nanoparticle conjugates, a similar LFIA was developed by Di Nardo et al. with the single test zone and multicolour GNPs for detection of aflatoxin B1 and type-B fumonisins [19]. The idea proposed in this paper is different both in purpose and in implementation. The approach is similar to determining the temperature of a sick person in that it shows the disease but does not identify its reasons. We refused the use of mixture of several mono-specific conjugates, which has already been reported and chose to utilize one multi-specific conjugate to solve the specific tasks. In our knowledge, a multi-target LFIA with one test zone design and multi-specific gold nanoparticles (GNP) – antibody conjugate, was not earlier described. Thus, in this study we have realised alarm-LFIA. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies. As a proof of concept, the idea was realised with five antibodies. Therefore, we propose the alarm-LFIA for five targets. Commonly, the detecting reagent for multi-analyte LFIA is a mixture of individual GNP – antibody conjugates of different specificity [20-22]. However, for our purposes the mixture is not optimal because only a part of the conjugated GNPs in the mixture can bind a specific target and provide coloration. Therefore, we have done it another way through the conjugation of GNPs with a mixture of antibodies of different specificity. The concentration of the immune complexes in the test zone will be about five times higher (in the case of five target pathogens for one multispecific conjugate). Therefore, the multispecific GNP antibody conjugate was used in the alarm-LFIA. In the control zone of the test strips, protein A was immobilised because it is a universal reagent binding antibody.
Complexes of protein A GNP conjugate are formed in the control zone, regardless of the presence of any antigen in the sample. The schemes of the alarm test strip and a common LFIA monotest strip are shown on Fig. 1. For proof of concept, the set of five important potato viruses, potato virus X (PVX), potato virus M (PVM), potato virus S (PVS), potato virus Y (PVY) and potato leaf roll virus (PLRV) were chosen as targets to aim the alarm test. These viruses cause large yield losses and related economic losses [23, 24]. It is crucial to have efficient methods of disease detection so that the damage caused by pathogens can be reduced. A one alarm test is more useful than five monotests as it solves the main task of monitoring (the divide of healthy and infected seeds), reducing the cost and labour intensity. Herein, we propose an alarm-LFIA for rapid nonlaboratory detection of total viral infection caused the five major potato viruses. 2. Materials and methods 2.1. Materials Potato viruses (PVX, PVM, PVS, PVY, PLRV) and their specific polyclonal antibodies as partly described in previous works [20, 25] were provided by Y. A. Varitsev. Tris(hydroxymethyl)aminomethane (Tris), Tween 20, Triton X-100 (Sigma-Aldrich, USA); chloroauric acid (Fluka, Germany); bovine serum albumin (BSA), sodium citrate, and dimethyl sulfoxide (MP Biomedicals, UK); glycerol, sodium chloride and potassium carbonate (DiaM, Russia); protein A from Staphylococcus aureus (Imtek, Russia); and sodium carbonate, sodium bicarbonate, potassium dihydrogen phosphate and potassium hydroxide (Khimmed, Russia) were used for this study. All the chemicals were of analytical reagent or chemical reagent grade. The nitrocellulose membranes (CNPH-N-200) adhering to the surface of the laminated card, conjugate release matrix (PT-R5), sample pads (GFB-R4, 0.35) and absorbent pads (AP045) were obtained from Advanced Microdevices (India). 2.2. Leaf extract preparation Infected (14 samples) and healthy (3 samples) potato leaves were used to prepare the extracts. The potato leaves were homogenised in 50 mM Tris-HCl containing 0.05% Triton X100 and 100 mM NaCl (1:10 w/v) for a duration of 5 min in a porcelain mortar and stored at 4 °C before use. 2.3. Gold nanoparticle synthesis A Frens method [26] was used for GNP synthesis. First, 1 mL of 1% HAuCl4 was added to 95 mL of deionised water and heated to the boiling point, and then 4 mL of 1% sodium citrate was added while the mixture was stirred. The mixture was boiled for 25 min, cooled and stored at 4 °C.
2.4. Determining the antibody concentration for the conjugation (flocculation method) To determine the optimal amount of the antibodies for the conjugation, according to Hermanson [27], we added a GNPs (1.0 mL portions at pH 9.0; optical density at 520 nm (OD520) = 1.0) to antibody (0.1 mL at pH 9.0; concentrations from 1 to 250 mg/mL), incubating the mixture at room temperature for 10 min. 10% NaCl (0.1 mL) was added to each sample, stirred for 10 min, and measured OD580. Dependence of OD580 in the presence of 10% NaCl against the antibody concentration (flocculation curves) was plotted. The concentration corresponding to the beginning of the plateau in the flocculation curve was determined as a minimum ratio to prevent the aggregation of GNPs. 2.5. Synthesis of GNP-antibody conjugates Antibody at a chosen concentration by flocculation curve was added to the GNP solution and mixed for an hour. Free GNP surface was blocked with BSA (final concentration of 0.25%). The conjugates were separated through centrifugation at 20,000 g for 30 min at 4 °C. The synthesised conjugates were suspended in 10 mM Tris buffer, pH 7.4, containing 0.25% BSA, 0.25% Tween 20 detergent and 1% sucrose. The conjugates were stored at 4 °C. 2.6. Transmission electron microscopy (TEM) Preparations of the GNPs and their conjugates were adsorbed on cooper grid coated with a poly(vinyl formal) for 10 min. Images were obtained using a JEM CX-100 electron microscope (JEOL, Japan) operating at 80 kV. The digital images were analysed with Image- Tool (UTHSCSA, USA). 2.7. Preparation of mono LFIA and alarm-LFIA test strips Test and control zones were formed on the nitrocellulose membrane (CNPH-N-200) using specific antibodies and protein A, respectively. Antibodies specific to viruses were dispensed in the test zone at 0.5 mg/mL for monotest strips and 2.5 mg/mL (5 types of specific antibodies, per 0.5 mg/mL of each antibody) for alarm test strips, whereas protein A (0.5mg/mL) was dispensed in the control zone. An IsoFlow dispenser (Hanover, NH, USA) dispensed all reagents at 0.15 mL per mm membrane width in PBS containing 5% glycerol and 0.03% NaN 3. The GNP-antibody conjugates (mono- or multi-specific) were deposited onto glass-fibre membranes (conjugate release fiberglass membrane PT-R5) from a solution with OD520 = 4; the conjugate load was 1.6 µL per mm of strip width. All membranes were dried at 37 °C for eight hours. Following this, we attached glass-fibre membranes, sample pads (GFB-R4, 0.35), and adsorbed pads (AP045) to plastic supports with the nitrocellulose membrane (CNPH-N-200). The obtained multimembrane composites were cut into strips 3.5 mm in width by an Index Cutter-1 (A-Point Technologies, USA). Test strips were hermetically packed into laminated aluminum foil bags containing silica gel at 20-22 °C and a relative humidity of less than 30%.
2.8. Lateral flow immunoassay The assay was performed at room temperature. The test strip was vertically submerged in the test sample for 1.5 min before it was removed and placed on a horizontal surface. The formations of coloured test and control zones were visually detected in 10 min. The limits of the visual detection of the LFIA tests were determined as virus concentrations (ng/mL) when the test line appeared. For the quantitative analysis, the test strips were scanned using a Canon 9000F Mark II scanner (Canon, Tokyo, Japan), and digital images were analysed with a TotalLab TL120 (Nonlinear Dynamics, UK). Linear approximations for the initial sections of the dependence of the colour intensity of the LFIA test line on the viruses concentration were then constructed by Origin Pro 9.0 (OriginLab, USA). 2.9. Enzyme-linked immunosorbent assay (ELISA) of the viruses in potato leaf extracts The potato leaf extracts were tested using ELISA kits for PVX, PVM, PVS, PVY, PLRV (Test Potato, Ltd, Russia) according to the manufacturer's protocols. The statistical processing and linear regression analysis were performed by Origin Pro 9.0 (Origin Lab, USA). To calculate the content of the viruses, linear ranges of the calibration curves were used. 3. Results and discussion 3.1. Synthesis of GNPs and multi‐specific GNP – antibody conjugate At the first stage, we synthesised and characterised GNPs by TEM (Data are provided in Supplementary Information, Fig. S1). The GNPs had a narrow size distribution with a mean diameter of 18.6 ±3.8 nm. Our previous experiences in the synthesis of GNP conjugates with the same antibodies specific to these viruses confirm that spherical GNPs with the same diameters provide high stability of the conjugate and high sensitivity of the LFIAs [20, 28]. The maximum number of IgG that can be immobilised on the surface of one nanoparticle is ~60 molecules (100% coverage area of GNP) taking to account the 5–7 nm of hydrodynamic diameter of IgG (data from the Protein Data Bank). This is a sufficient number to simultaneously immobilise five antibodies with different specificity. We used dynamic light scattering to verify homogeneity and stability of GNP preparation (Data are provided in Supplementary Information, Fig. S2). No aggregates were observed, and dispersion of GNPs was confirmed. Electrophoretic light scattering showed the GNP zeta potential of −31.6 mV that confirmed stability of the GNPs [29]. The synthesised GNPs were used for conjugation with antibodies specific to PVX, PVM, PVS, PVY and PLRV. To synthesise the different monospecific conjugates and multi-specific conjugates with broaden-specificity to the viruses, physical adsorption was used as the most
common technique for synthesis of the GNP conjugates. We used the flocculation method to estimate the adsorption ability of five antibodies to the GNP surface. The minimum ratio of each antibody preventing the aggregation of GNPs was about 30–35 antibodies per one nanoparticle, and the maximum coverage of the GNP surface accorded to the range 55–80 antibodies per GNP (Fig. 2). This coincidence of flocculation curves means that the immobilisation of a mixture of antibodies with a predetermined equal ratio, the content of the antibodies of different specificity on the GNP surface is close to each other. We used a larger concentration of antibodies (57 antibodies per GNP) corresponding to the maximum coverage of the nanoparticle surface. To evaluate the effect of increase of the total antibodies’ concentration, we obtained multi-5-specific conjugate (50 μg/mL total concentration, with 10 μg/mL (57 antibodies per GNP) of each antibody) and five mono-specific conjugates (10 μg/mL total concentration of one antibody). For all synthesised conjugates, the absorption peaks have the same narrow width and a slight shift of the maxima (not more than 5 nm) as compared with the native GNPs, which indicates the adsorption of antibodies on the GNP surface [30]. Certainly, the surface coverage of specific antibodies isn’t the same for multi- and monospecific conjugates. However, the functional properties of individual virus recognition are not worsened. Based on previous works [31-33], we could assume that 20% coverage of IgG molecules of each antibody type on the GNP surface is already sufficient for good binding to the target. 3.2. Optimization and characterization of the alarm test We have optimised concentration of all reagents for the alarm test strip. We used a nitrocellulose membrane with the highest protein binding that guaranteed the sorption capacity in the required range. The concentration of antibodies immobilised on nitrocellulose membrane in the test zone was 2.5 μg/μL (five types of specific antibodies, per 0.5 μg/μL of each antibody). It should be noted that the concentrations are slightly lower (~2–3 times) than those commonly used in LFIA [25], but we used them to reduce nonspecific reactions. The optimal amount of the multi-5-specific conjugate in the LFIA test system corresponded to OD520 = 4 (16 µL per centimetre of the pad with) (Fig. 3) to provide the highest signal to noise ratio and absence of non-specific reactions. Probably, non-specific reactions with a total OD520 of the conjugate more than 4 are largely related to the mixture composition of the antibodies in the test zone. Similar nonspecific effects were also noted by Shu et al. [34]. Therefore, we adhered to the conditions that the total OD520 of the conjugate(s) in the lateral flow systems, especially in the system with a mixture of antibodies in the test zone, should not exceed 4. Under these conditions, the replacement of the multi-5-specific conjugate by five mono-specific conjugates will cause additional negative factors. This means that OD520 of each conjugate would be equal to 0.8,
which doesn’t provide high sensitivity [25]. Moreover, the colorimetric signal in test zone for one virus will be formed with the participation of all GNPs for multi-5-specific conjugate and with participation of one-fifth of the GNPs for mixture of five mono-specific conjugates. This is an additional reason for the use of the multi-5-specific conjugate. To determine the influence of the simultaneous immobilisation of several antibodies on the limit of detection (LOD), the test strips with multi-5-specific conjugates and test strips with monospecific conjugates were compared. At first, the testing was realised in a model buffer solution (0.05 M potassium phosphate, pH 7.4, 0.1 M NaCl, 0.05% Tween-20; PBST) containing different concentrations of viruses. The multi-test demonstrated LODs in the range 10–30 ng/mL depending on the virus. Data are provided in Supplementary Information, Fig. S3. Three monospecific LFIA test systems for detection of PVX, PVM and PVY were fabricated according to the scheme on Fig. 1A and under the optimised conditions described previously [25]. Concentrations of the mono-specific GNP – antibody conjugates accorded concentration of the multi-5-specific conjugate (16 µL per cm, OD520 = 4). The LOD of mono-LFIA for each virus was 8 ng/mL. The analysis time for multi- and mono-LFIA was the same, 10 min. The results show that LODs of PVX, PVM and PVY test strips with multi-5-specific conjugate to be slightly higher than the LODs of monotest strips. The obtained small differences of LODs are no more than four times and are not critical for diagnosis. The alarm-LFIA with multi-5-specific conjugate was used to analyse potato leaf extracts spiked with different concentrations of PVX, PLRV, PVY, PVS and PVM. Fig. 4 shows the typical test strips after the different viruses determination and the plotted dependences of colour intensities of the test zone on virus concentration. The LOD for the five viruses in extracts coincided to the LOD in the buffer (10 ng/mL of PVX, 20 ng/mL of PVY and PVM 30 ng/mL of PLRV and PVS). We would like to note that there are no exact threshold values in diagnostics of potato viruses. The data about the accumulation of viruses [23, 24] and the detection limits achieved by ELISA and LFIA [20, 35, 36] allow to conclude that the reached LODs are relevant to assess the total level of virus infection. At the same time, the multi-conjugate system has great potential for further developments due to the ability to control and modulate the sensitivity of the analysis. This ability is mostly associated with the change in ratio of specific antibodies with different affinities in the multi-conjugate. As shown in [31, 32], the dependence of LOD on the number of antibodies immobilized on the GNP surface is not linear, but the LOD can be adjusted if necessary to satisfy practical demands. 3.3. Approbation with potato leaf samples The final task, approbation with real samples, was conducted for the diagnostics of the potato plants (17 leaf extracts, each in duplicate). The developed alarm-LFIA was compared with
ELISA. To calculate the viruses content by ELISA, linear ranges of the calibration curves were used. Table 1 provides the test results. Twelve leaf samples showed the same results of the infection by alarm-LFIA and ELISA. Three leaf samples showed the same results of health. These results confirm that the alarm-LFIA could be used as simple, rapid (10 min) and effective method for potato virus detection without differentiation. Certainly, the developed alarm test assumes only a qualitative analysis. The colour intensity in the test zone can’t be recalculated into concentration; different viruses cause a different signal. For clarity, Table 2 shows the test zones after the analysis of samples (1 and 4) with the same total viral infection with different colour intensities. At the same time, a sample 15 with a lower total infection has a larger signal than sample 4. This is explained by the different content of individual viruses in the sample (see Table 1).
Conclusions The obtained results show that one alarm-LFIA for rapid non-laboratory detection replaces five tests and can be used to determine total viral infection caused the five major potato viruses. The result is largely due to the synthesised multi-specific GNP conjugate with broadenspecificity to five major potato viruses. The developed alarm-LFIA can be easily used for multiple-pathogen analysis and total virus infection determination, which greatly improves the agrotechnical control. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples. These cases include pathogens dangerous to humans, animals or plants, or pesticides, antibiotics and mycotoxins, whose level should not exceed a present threshold. If at least one from the controlled list is detected, the sample will be singled out for special processing. A special attention deserves the concept of a multi-specific reagent promising for the simultaneous analysis of several structurally different targets.
Acknowledgements This study was financially supported by the Russian Science Foundation (grant 16-1604108).
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Fig. 1. Schemes of test strips for LFIAs: (A) a common monotest strip with a monospecific GNP conjugate and one type of specific antibody in the test zone; (B) the alarm test strip with a multispecific GNP conjugate and several types of antibodies in the test zone. 1 indicates the membrane with the conjugate applied, 2 indicates the test zone and 3 indicates the control zone. Fig. 2. Flocculation curves of antibodies’ immobilisation on the GNP’s surface. Concentration dependencies of the absorbance of GNPs at 580 nm in the presence of excess salt (10% NaCl) after the addition of different concentrations of antibodies (from 5 to 90 antibodies per one GNP). Arrows indicate the number of antibodies for the stabilisation of the GNP’s surface (stop flocculation) and the maximum number of antibodies according the calculations. Grey areas schematically illustrate surface coverage at different ratios of antibodies to GNP. Fig. 3. Colour intensity of the test zone of LFIA test strips with multi-5-specific conjugate for PVX, PLRV, PVY, PVS or PVM detection (concentration of each virus is 50 ng/mL). Fig. 4. Alarm-LFIA for virus detection in leaf extracts: Test zones of the strips after detection of 500, 250 and 60 ng/mL of each virus and dependences of colour intensities of the test zone on virus concentration. 1–5 accord to PVX, PLRV, PVY, PVS and PVM, accordingly. The dashline indicates the average colour intensity of the negative control.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Graphical Abstract
Table 1 Results of alarm-LFIA and ELISA with potato leaf extracts. Sample №
Alarm-LFIA
ELISA results (ng/mL) PVX
PLRV PVY PVS
PVM
1
infected
≥1000 –
–
–
–
2
infected
≥1000 –
150
15
–
3
infected
160
–
125
20
–
4
infected
–
10
770
110
140
5
infected
115
850
60
–
–
6
infected
100
–
–
22
–
7
non- infected
–
–
–
–
–
8
non- infected
–
-–
–
–
–
9
infected
115
380
125
35
–
10
infected
20
–
–
–
130
11
infected
≥1000 –
–
–
–
12
non- infected
–
–
–
–
13
infected
≥1000 20
–
160
–
14
infected
390
70
–
–
–
15
infected
200
–
–
–
85
16
infected
≥1000 125
–
230
–
17
infected
–
–
–
80
–
–
Table 2 Results of alarm-LFIA for samples with the same and different total viral infections. Sample number
1
4
15
Total viral infection, ng/mL
1000
1030
285
18.5
5.8
10.5
Test strips after analysis
Colour intensity, arb. units
Highlights
The alarm lateral flow immunoassay (LFIA) is proposed for multi-target analysis The concept of the test is based on the antibody’s conjugate with broaden-specificity Gold nanoparticle conjugate specific to 5 structurally different targets is obtained One test line of the alarm LFIA indicates total infection caused from 1 to 5 viruses The alarm LFIA is easy tool for a multi-target analysis by one test strip