Currently, enzyme-linked immunosorbent assay (ELISA) is widely used for the detection of target analytes in medical diagnostics [1], food industry [2], veterinary medicine [3], and environmental monitoring [4, 5]. ELISA is the most widely used in a heterogeneous microplate format. The use of solid support allows to carry out the separation of free and bound immunoreagents by washing its wells. Complexes of antigens and specific antibodies formed during ELISA are identified using immunoreagent conjugates with enzymes that are detected by their catalytic activity. The most widely used label in ELISA is horseradish peroxidase (HRP) [6].

However, despite the high catalytic activity of HRP, in some cases the limit of detection of ELISA, which for polyvalent antigens (proteins, viruses and cells) is limited by the sensitivity of label detection, is insufficient. For highly sensitive ELISA, an approach based on the detection of the optical properties of nanoparticles (the wavelength of the absorption maximum and the value of the maximum absorption) that change during their interactions and modifications can be used [7]. Another approach to lower the detection limit of ELISA is based on the amplification of the detectable molecules by the method of polymerase chain reaction (immuno-PCR) [8].

This paper presents an ELISA scheme based on multiple covalent introduction of tyramine derivatives (biotin-tyramine). As shown in [9], HRP is capable of activating tyramine to form a highly active free radical that binds to tyrosine residues of proteins, leading to covalent binding of tyramine to protein molecules. The structural formula of tyramine is shown in Fig. 1. The formation of active radicals is implemented by a free hydroxyl group in its molecule, whereas the synthesis of conjugates with biotin (or other labels)—by the amino group [10].

Fig. 1.
figure 1

Structural formula of tyramine molecule.

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Amplification is based on the introduction of a large number of tyramine molecules into protein molecules, which ensures the inclusion of a much larger amount of a label into the immune complexes—fluorophore [11], enzyme [12], etc. Tyramine amplification was used to lower the detection limits of various immunoassay formats [13–17].

The aim of the work is to develop an ELISA with tyramine amplification for highly sensitive detection of potato virus X (PVX), one of its main pathogens [18], and optimization of conditions of its carrying out for plant samples with a high level of endogenous peroxidases that provide minimal non-specific signal.

MATERIALS AND METHODS

PVX was isolated from infected potato plants by the method of extraction/precipitation followed by gradient centrifugation, as described in [19]. The preparation and characterization of monoclonal antibodies of clone 3G4 specific for PVX are provided in [20], and polyclonal antibodies to PVX in [18]. In this work, dimethylformamide (DMF), 3,3',5,5'-tetramethylbenzidine (TMB), conjugate of streptavidin-polyperoxidase, sodium periodate, triethylamine, tyramine hydrochloride, sodium cyanoborohydride and horseradish peroxidase (Sigma, USA), dimethylsulfoxide (DMSO), bovine serum albumin (BSA) and biotin-N-hydroxysuccinimide (MP Biomedicals, UK), egg albumin (EA) (Bioreba, Switzerland), ethanolamine (ICN Pharmaceuticals, USA) and hydrogen peroxide (Himmed, Russia) were used. All salts, acids and solvents were of chemical purity.

For carrying out the ELISA, 96-well transparent polystyrene microplates Costar 9018 (Corning Costar, USA) were used. Microplate wells were washed from unrelated reagents using an automatic Biochrom Anthos Fluido 2 washing device (Biochrom, UK), and the color intensity in the wells at the end of the ELISA was measured using a PerkinElm microplate spectrophotometer (PerkinElmer, USA). To obtain the absorption spectra, a Biochrom Libra S80 spectrophotometer (VBW, UK) was used.

Synthesis of conjugates of polyclonal antibodies with horseradish peroxidase. HRP was conjugated to antibodies using the periodate oxidation method [21]. Sodium periodate was added to a solution of HRP (10 mg/mL in 50 mM potassium phosphate buffer, pH 7.4, containing 0.1 M NaCl, PBS) to a final concentration of 8 mM and incubated for 20 min. The oxidized HRP was separated using Centricons (Merck Millipore, USA) with a cut-off threshold of 15 kDa, and polyclonal antibodies against PVX (in 200 mM sodium carbonate buffer, pH 9.5) were added to it. The molar ratio of HRP:antibodies was 5 : 1. The resulting mixture was incubated for 3 h, then sodium cyanoborohydride was added to a final concentration of 50 μM and the mixture was incubated for 1 h. To block unreacted oxidized HRP groups, ethanolamine was added to a final concentration of 50 mM and the mixture was incubated for another 1 h. The resulting conjugate was dialyzed three times against PBS, glycerol was added to a final concentration of 50% and the mixture was stored at –24 °C.

Synthesis of tyramine-biotin conjugate. For conjugation, tyramine (55 mM) and biotin-N-hydroxysuccinimide (66 mM) were dissolved in anhydrous DMSO and their equal volumes were mixed. 30 mM triethylamine was added to the reaction mixture that was kept with constant stirring at room temperature for 3 h. To block the excess of activated groups, ethanolamine was added to a final concentration of 20 mM and the mixture was incubated for 1 h. The resulting conjugate solution was mixed with an equal volume of ethanol and stored at 4°C [22].

Preparation of extracts of potato leaves. Freshly cut leaves of healthy and PVX-infected potato plants were placed in PBS with 0.05% Triton X-100 (PBS-T). The mass ratio of leaves : buffer was 1 : 15. The leaves were thoroughly crushed in a mortar for 30 s and used for assay without further sample preparation. For the preparation of artificially contaminated extracts, the extracts of leaves of healthy plants were mixed with PVX at known concentrations.

Conducting ELISA. Monoclonal antibodies of clone 3G4 (2 µg/mL in PBS) were sorbed onto the surface of the microplate wells for 2 h at 37°C. The microplate was washed five times with PBS-T. To block the surface, a solution of BSA or EA (3 mg/mL) was added to the microplate wells and kept for 1 h at 37 °C. The microplate was washed five times with PBS-T. Then, samples containing PVX at concentrations from 500 to 0.008 ng/mL (or an extract with an unknown content of PVX) were added to the microplate wells (100 µL per well), incubated for 1 h at 37°C and then the wells were washed with PBS-T five times. Next, 100 μL of HRP conjugate with antibodies against PVX in PBS-T were added to the microplate wells at various dilutions (1 : 7000, 1 : 5000, 1 : 2000 and 1 : 1000), incubated for 1 h at 37°C and the wells were washed with PBS-T. A substrate solution containing TMB (0.4 mM) and H2O2 (3 mM) in 40 mM Na-citrate buffer, pH 4.0, was added to the microplate wells. The mixture was incubated for 10 min at room temperature. The enzymatic reaction was stopped by the addition of 50 μL of 1 M sulfuric acid, and the optical density was measured at 450 nm (A450). The dependence of optical density on the concentration of PVX was constructed using the program OriginPro 9.0 (Origin Lab, USA). The limit of detection (LOD) of ELISA was defined as the amount of PVX for which the A450 value exceeded the sum of optical density for negative control (Blank450) and three standard deviations (SD): LOD ELISA = Blank450 + 3SD.

Conducting ELISA with tyramine amplification. Antibody sorption, blocking of the surface, and addition of the sample with PVX and antibody—HRP conjugate were performed as described above. After washing five times with PBS-T, a solution containing the tyramine—biotin conjugate at a concentration of 20 μM (at the optimization stage, the concentration varied from 100 to 0.1 μM) and hydrogen peroxide (0.08%) was added and the wells were incubated for 10 min at room temperature. The microplate was washed five times with PBS-T, streptavidin–polyperoxidase conjugate (1 : 7000 in PBS-T) was added and incubated for 40 min at 37°C. After washing the microplate five times with PBS-T, the substrate solution was added and the results of the assay were recorded as described in the previous section.

RESULTS AND DISCUSSION

Optimization of ELISA conditions. The system with the direct introduction of the peroxidase label had fewer stages, which made it possible to reduce the non-specific background. The assay is based on the formation of a sandwich complex containing adsorbed antibodies–PVX–conjugate of HRP–polyclonal antibodies against PVX. For the system without tyramine amplification, TMB was added at stage 3 (Fig. 2) and A450 was recorded.

Fig. 2.
figure 2

ELISA schemes used in the work: without tyramine amplification (upper row; 1–3—successive stages of assay) and with tyramine amplification (middle row; 4, 5—successive stages of assay). The bottom row shows the graphic symbols of the components of the immune complexes formed during the ELISA. Letter symbols are given in the text of the article.

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To lower LOD and reduce non-specific binding, optimization of the ELISA conditions without/with tyramine amplification was carried out. At each stage of ELISA, the concentrations of the reagents, the incubation time, and the washing conditions were successively varied. Parameters that provided the lowest LOD of assay and the lowest non-specific background were considered optimal. The results are summarized in Table 1.

Table 1. Results of optimization of ELISA without/with tyramine amplification
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The experiments showed that in order to achieve the maximum specific and minimum non-specific signals, it is necessary to block the microplate surface with EA (3 mg/mL). At selected conditions, non-specific binding of immunoreagents was not observed at subsequent stages of the assay. The blocking made it possible to increase A450 due to the greater binding of the tyramine label activated by HRP with the tyrosine residues of the blocking EA [23]. All subsequent experiments were performed under optimized conditions, including five-time washing the microplate with PBS-T, 15 μM concentration of the tyramine – biotin conjugate, 10 min incubation time with the tyramine – biotin conjugate, and 1 : 7 × 103 dilution of the streptavidin – polyperoxidase conjugate (Table 1).

ELISA for PVX in the buffer with tyramine amplification. To determine the analytical characteristics of ELISA, PVX was assayed in a buffer at different dilutions of the conjugate of HRP-antibodies – from 1 : 104 to 1 : 105. The dependence of A450 on the concentration of PVX for ELISA without and with tyramine amplification is shown in Fig. 3.

Fig. 3.
figure 3

Dependence of A450 on PVX concentration when conducting ELISA in a buffer without tyramine amplification, conjugate of HRP-antibodies diluted 1 : 104 (1), and with tyramine amplification, conjugate of HRP-antibodies diluted 1 : 104, 1 : 5 × 104, 1 : 105 (2–4). a—Calibration curves; b—sections of the curves at low concentrations of PVX. Levels of A450 corresponding to the detection limits of the ELISA for calibration curves 2–4 are shown by dashed lines.

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As can be seen from the obtained results, the detectable signal for ELISA without tyramine amplification was observed only at a 1 : 104 dilution of the conjugate of HRP–antibody (Fig. 3a, curve 1). The PVX LOD in ELISA without tyramine amplification was 100 ng/mL. When using high dilutions of the conjugate (1 : 5 × 104 and 1 : 105), no staining of the substrate solution was observed even at high concentrations of the virus (more than 200 ng/mL).

For ELISA with tyramine amplification, the greater the amount of HRP was administered at the stage of formation of the sandwich complex (Fig. 2, stage 3), the more activated the tyramine label was and the more streptavidin-polyperoxidase conjugate was introduced into the immune complex (Fig. 2, stages 4 and 5). Because of this, the LOD of ELISA with tyramine amplification depended on the dilution of the HRP conjugate. For instance, when the HRP conjugate was diluted 1 : 104, 1 : 5 × 104 and 1 : 105, the PVX LOD in the buffer was equal to 3, 10 and 20 ng/mL, respectively (Fig. 3b). The use of lower dilutions of HRP conjugate was limited to non-specific staining even in the scheme without tyramine amplification.

Detection of PVX in extracts of potato leaves. Potato leaves are characterized by high endogenous peroxidase activity, which in the used ELISA can lead to a high background signal due to non-specific introduction of the tyramine label. The selected ELISA conditions (surface blocking and optimized concentrations of reagents, see Table 1) mitigated endogenous peroxidase activity, which made it possible to carry out ELISA in plant extracts. The dependencies of A450 on the virus content in the plant extract are shown in Fig. 4.

Fig. 4.
figure 4

Dependence of A450 on PVX concentration when conducting ELISA in extract of potato leaves without tyramine amplification, conjugate of HRP-antibodies diluted 1 : 104 (1), and tyramine amplified, conjugate of HRP-antibodies diluted 1 : 104 (2). a—Calibration curves; b—sections of the curves at low concentrations of PVX. Levels of A450 corresponding to the detection limits of the ELISA for calibration curves 1 and 2 are shown by dashed lines.

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As seen from the obtained results (Fig. 4b), tyramine amplification lowered the PVX LOD in potato leaf extracts by more than 30 times—from 100 to 3 ng/mL.

ELISA with and without tyramine amplification were used for the detection of PVX in the extract of potato leaves. 9 extracts before and after dilution (10, 50, 100, 200 times) with a healthy extract were tested. The results are presented in Table 2.

Table 2.   Results of ELISA on extracts of potato leaves without/with tyramine amplification
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The use of tyramine amplification in ELISA made it possible to detect PVX in highly diluted extracts as compared to the ELISA format without amplification. Extract of leaves of a healthy plant (no. 9) showed a negative result in ELISA both with and without tyramine amplification.

Comparison of detection limits of the assay without/with tyramine amplification. Tyramine amplification was shown to have varying efficacy (Table 3). The ELISA with tyramine amplification developed in this study made it possible to lower the LOD by 30 times, which is comparable with the previously described implementations of this approach. It should be noted that until now tyramine amplification has not been used in the assaying of samples with high endogenous peroxidase activity.

Table 3.   Efficiency of tyramine amplification for different immunoassay systems
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The proposed ELISA format was used to detect PVX in extracts of potato leaves. The high sensitivity of phytopathogen detection made it possible to discover infection at the initial stages and to diagnose latent infections that are accompanied by the absence of external symptoms and the accumulation of small amounts of the pathogen.

Thus, we developed a method to lower the PVX LOD by ELISA based on the multiple administration of tyramine label. When analyzing the extract of potato leaves, tyramine amplification using HRP made it possible to lower the PVX LOD from 100 to 3 ng/mL. Tyramine amplification which is a universal method of lowering the LOD of immunoassay can be used for highly sensitive detection of other antigens.