RU2744705C1 - Method of indirect competitive immune-enzyme analysis for determining the content of tetrodotoxin and its analogues in extracts of marine organisms - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, namely, to a method for performing indirect competitive immune-enzyme analysis to determine the content of tetrodotoxin (TTX), 4-epiTTX, 4.9-anhydroTTX, 5-deoxiTTX, 11-deoxiTTX and 5,6,11-trideoxiTTX in extracts of marine organisms. According to this method, a tablet is sensitized by incubation with a solution of bovine serum albumin conjugate with TTX in a carbonate buffer, followed by washing and incubation in a blocking buffer; extracts of marine organisms and a solution of polyclonal antibodies, got by immunization of mice with phytohemagglutinin conjugate with TTX, are added to the wells, followed by incubation and washing of the tablet; a conjugate of secondary antibodies with horseradish peroxidase is added, followed by incubation and washing, orthophenylenediamine is added as a substrate; the solution is kept, and then the reaction is stopped and the optical density is measured at a wavelength of 492 nm.

EFFECT: technical result is to reduce the duration of the analysis of the content of tetrodotoxin with the expansion of the range of detectable analogues, simplify the process of preparing the conjugate for immunization, and eliminate the cross-reaction between the obtained antibodies and the components of the studied extracts.

6 cl, 3 ex, 1 dwg

Description

The invention relates to the field of biochemistry, veterinary medicine and food industry, namely to laboratory diagnostics, and can be used to determine the content of tetrodotoxin (TTX) and its analogs in marine organisms.

Recently, invasive TTX-containing species are increasingly found in Europe, America and Oceania, and their spread in these regions leads to an increase in cases of TTX poisoning. Along with TTX, its analogs are often found in biological objects, which have different toxicity due to their different affinity for voltage-dependent sodium channels (NaV). Most of the studied TTX analogs have a lower affinity for NaV channels compared to TTX; however, the affinity of some TTX analogs is comparable to or greater than TTX, and the content of these analogs may exceed the content of TTX in some organisms. Thus, TTX analogs can make a significant contribution to the overall toxicity of the organism. Therefore, it is extremely important to measure the total content of TTX and its analogues when monitoring toxins in seafood. Today, TTX is commonly detected using a mouse bioassay, liquid chromatography in combination with fluorescence or mass spectrometric detection, and gas chromatography with mass spectrometry. However, chromatographic methods are expensive and laborious, and bioassays show low accuracy. The fastest, easiest to use and most sensitive method for detecting low molecular weight toxins is the enzyme-linked immunosorbent assay (ELISA). Most ELISA assays designed for the determination of TTX use TTX-specific monoclonal antibodies, which excludes the possibility of detecting TTX analogs.

There is a known method of indirect competitive ELISA for determining the content of TTX in extracts of marine organisms, which consists in the fact that the plate with the surface covered with maleimide is activated, for this the plate is washed three times with washing buffer containing 0.1 M phosphate-buffered saline (PBS) and 0.05% Tween 20, pH 7.2, add a solution of dithiol carboxylate in ethanol, incubate for 3 hours at room temperature, wash three times with washing buffer, add a solution of N-hydroxysuccinimide, a solution of 1-ethyl-3- (3 -dimethylaminopropyl) carbodiimide, incubated for 30 minutes at room temperature, washed three times with washing buffer, added ethylenediamine solution, incubated for 30 minutes at room temperature, washed three times with washing buffer, added ethanolamine solution, incubated for 30 minutes at room temperature temperature, washed three times with washing buffer, poured a solution of TTX and 37% formaldehyde, incubated for 15 hours at room temperature t and TTX and washed three times with wash buffer. After activation, the test extract is added to the plate, mixed with a solution of monoclonal antibodies, incubated for 30 minutes at room temperature, washed three times with washing buffer, the plate is incubated in blocking buffer containing 1% BSA for 30 minutes at room temperature, washed three times with washing buffer, add conjugate of secondary antibodies with horseradish peroxidase, incubate for 30 minutes at room temperature, wash three times with washing buffer, then add the substrate tetramethylbenzidine, incubate for 10 minutes at room temperature and measure the optical density at a wavelength of 620 nm (Reverté, L., de la Iglesia, P., del Río, V., Campbell, K., Elliott, CT, Kawatsu, K., Campàs, M. Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer- Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS / MS, and Mouse Bioassay // Analytical Chemistry, 2015, v. 87 (21), pp. 10839-10847).

The disadvantages of the method include:

- the specificity of monoclonal antibodies exclusively for TTX, which means their inability to bind TTX analogues, and as a consequence, the impossibility of using the method to calculate the total concentration of TTX and its analogues;

- high cost of tablets with maleimide-coated surface;

- a long multi-stage process of activating the tablet.

There is a known ELISA method for determining the content of toxins in extracts of toxic organisms, which consists in the fact that the plate is sensitized with a solution of polyclonal antibodies to TTX obtained by immunizing rabbits with a conjugate of snail lymph hemocyanin with TTX (KLH-TTX) and isolated using affinity chromatography on EAH-Sepharose sorbent 4B, covalently linked to TTX, incubation at 5 ° C overnight, washed with PBS, add a blocking solution containing 4% Block Ace reagent, incubate at 5 ° C overnight and wash twice with washing buffer containing PBS and 0 , 05% Tween 20. After that, the test extract and the biotin-TTX conjugate are added, incubated at 37 ° C for 15 minutes, washed with washing buffer and streptavidin-horseradish peroxidase conjugate is added, incubated at 37 ° C for 15 minutes, washed three times with wash buffer. Then add the substrate - orthophenienediamine, incubate at 37 ° C for 5 minutes, stop the reaction by adding 2 M hydrochloric acid solution and measure the optical density at 490 nm (Sato S., Takaishi S., Yasumoto K., & Watabe S. Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin // Toxins, 2019, v. 11 (10), p. 551.).

The disadvantages of the method include:

- The duration of the analysis - it takes about 2 days;

- Limited determination of tetrodotoxin analogs - 4-epiTTC, 11-oxoTTC, 5,6,11-trideoxyTTC;

- Laborious process of obtaining a conjugate for immunization;

- As a carrier protein, the conjugate for immunization contains snail lymph hemocyanin, an animal protein. Antibodies generated against such a conjugate may cross-react with components of marine extracts.

The closest technical solution in terms of the number of essential features is the method of indirect competitive ELISA for determining the content of TTX in extracts of marine organisms, which consists in the fact that the plate is sensitized with a BSA-TTX conjugate solution in 0.05 M carbonate buffer by incubation at 4 ° C overnight , washed three times with wash buffer containing 0.01 M PBS and 0.05% Tween 20, pH 7.2-7.4, after which the plate is incubated in blocking buffer containing 1% BSA (0.01 M PBS, pH 7, 2-7.4 at room temperature for 2 hours, washed three times with washing buffer In the plate, the test extract is mixed with a solution of monoclonal antibodies, incubated at 37 ° C for 1.5 hours, washed three times with washing buffer. secondary antibodies with horseradish peroxidase, incubated at 37 ° C for 1 hour, washed five times with washing buffer. temperature of 37 ° C for 15 minutes, stop the reaction by adding 2N sulfuric acid solution and measure the optical density at a wavelength of 450 nm (Tao, J., Wei, WJ, Nan, L., Lei, LH, Hui, HC, Fen , GX, Rong, J. Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China // Food Additives & Contaminants: Part A, 2010, v. 27 (11), pp. 1589-1597).

The disadvantages of the method include:

- the specificity of monoclonal antibodies exclusively for TTX, which means their inability to bind TTX analogues, and as a consequence, the impossibility of using the method to calculate the total concentration of TTX and its analogues;

- high cost of the substrate - tetramethylbenzidine;

- the duration of the process, since it takes about a day to conduct the analysis.

The technical problem posed to the invention is the reduction of the duration of the analysis of the TTX content with a simultaneous expansion of the range of identified analogs, simplification of the preparation of the conjugate for immunization, the elimination of cross-reaction between the obtained antibodies and the components of the investigated extracts.

The technical problem posed is solved by the fact that in the known method of indirect competitive enzyme-linked immunosorbent assay for determining the content of toxins in extracts of marine organisms, including sensitization of the plate by incubation with a solution of bovine serum albumin conjugate with TTX (BSA-TTX), washing the sensitized plate with a buffer solution, incubation in buffer, washing the plate from the blocking buffer with washing buffer, adding extracts of marine organisms and a solution of specific antibodies to the wells of the plate, followed by incubating the plate, washing the plate with washing buffer, adding conjugate of secondary antibodies with horseradish peroxidase, incubation followed by washing with washing buffer, adding at a temperature of 37 ° C for 15 minutes, stopping the reaction by adding a solution of sulfuric acid and measuring the optical density, according to the invention, polyclonal antibodies are used as specific antibodies. Received by immunizing mice with phytohemagglutinin-TTX conjugate (PHA-TTX) and isolated by affinity chromatography on Affigel-10 sorbent covalently linked to TTX; Orthophenyleneamine is taken as a substrate, and the optical density is measured at a wavelength of 492 nm.

The use of polyclonal antibodies obtained by immunization of mice with a PHA-TTX conjugate and isolated by affinity chromatography on an Affigel-10 sorbent covalently bound to TTX makes it possible to expand the spectrum of detected toxins in extracts of marine organisms, and provides the detection of TTX and its five analogs - 4-epiTTC , 4,9-anhydroTTC, 5-deoxy TTX, 11-deoxy TTX, and 5,6,11-trideoxy TTX. Due to the fact that the composition of the conjugate for immunization as a carrier protein includes a plant protein - PHA, a cross-reaction of the obtained antibodies with the components of the investigated extracts is excluded.

The use of cheaper orthophenylenediamine as a substrate instead of the expensive tetramethylbenzidine used in the prototype makes it possible to reduce the cost of analysis.

The developed method makes it possible to determine the toxins contained in marine organisms, in particular in nemertines, polychaetes, crabs, and octopuses.

As a blocking buffer, TBS-Triton-X-100 buffer is used, containing 0.05 M Tris-HCl, 0.15 M NaCl, 0.005% Triton X-100, 1 mg / ml skim milk pH 7.4.

To reduce the time of the assay, incubation for sensitizing the plate, incubation in a blocking buffer, incubation of the plate with a solution of specific antibodies and incubation with a conjugate of secondary antibodies with horseradish peroxidase are carried out at a temperature of 20-37 ° C for 30-100 minutes. Shortening the incubation time for plate sensitization and incubation in blocking buffer does not lead to a decrease in sensitivity, while speeding up the process of obtaining the result.

Carrying out the above incubations in the stated parameters provides optimal conditions for both the preparation of the plates themselves and for subsequent analysis, which ultimately allows obtaining reliable data on the content of TTX and its analogs in extracts of marine organisms. A decrease in the incubation temperature when the plate is sensitized leads to an increase in the incubation time, as a result of which the total analysis time increases. An increase in temperature above 37 ° C does not increase the degree of tablet sensitization. In addition, incubation of the plate with polyclonolic antibodies and with secondary antibodies conjugated with horseradish peroxidase at temperatures below 20 ° С leads to an increase in the total time of the sample, an increase in temperature above 37 ° С can lead to a change in the affinity of the components of the antigen-antibody complex, which may affect the accuracy of the determination of the content of toxins. Incubation for 30-100 minutes ensures sufficient antibody-antigen binding.

Washing with a washing buffer after incubation with a BSA-TTX conjugate solution, incubation in a blocking buffer, incubation of the plate with a solution of specific antibodies and incubation with a conjugate of secondary antibodies with horseradish peroxidase is carried out no more than four times. This ensures the removal of unbound components from the system, while at the same time not decreasing the fraction of bound molecules, which makes it possible to increase the accuracy of determining the quantitative content of toxins in marine organisms.

The plate is sensitized with a BSA-TTX conjugate solution in 0.05 M carbonate buffer containing 0.15 M NaCl, pH 9.5, which provides better solubility of the conjugate, and, as a consequence, a more uniform coating of the surface of the plate wells with the conjugate layer.

To carry out the method, it was previously prepared according to known methods with some modifications. Commercial TTX citrate (Alomone Labs, Israel) was used for antigen preparation according to the method of Wang et al. (Wang, R., Huang, A., Liu, L., Xiang, S., Li, X., Ling, S., Wang, S. Construction of a single chain variable fragment antibody (scFv) against tetrodotoxin (TTX ) and its interaction with TTX // Toxicon, 2014, v. 83, pp. 22–34.) with some changes. Briefly, 0.2 ml of a TTX citrate solution (1 mg / ml) was mixed with 1 ml of a PHA solution in 0.01 M PBS with 0.15 M NaCl, pH 6.0 (2 mg / ml) and 0.21 ml 8 % formalin in FSB, pH 7.8. The mixture was incubated with constant stirring at 37 ° C for 72 hours. The resulting conjugate was purified from the residue of unbound TTX and formalin by dialysis with a molecular weight cutoff of 10 kDa in four changes of 50 ml PBS, pH 7.2. The conjugate was diluted using PBS, pH 6.0 to a final concentration of 0.5 mg / ml, stored at -20 ° C until further use.

Ten male BALB / c mice were used for immunization. For the first immunization, the conjugate solution was mixed with Freund's complete adjuvant (Sigma, USA) in a 1: 1 ratio. A 0.2 ml aliquot of the suspension was injected into the hind leg thigh muscle of the animal. One month after the first immunization, the mice were injected with a suspension of the conjugate with incomplete Freund's adjuvant (Sigma, USA) in a 1: 1 ratio. Three weeks later, a third immunization was given by injecting antigen without adjuvant intraperitoneally. The injections were repeated every even week. Blood volume of 0.2 ml was collected from each mouse from the tail vein every odd week. 50 μl of heparin solution (5000 U / ml) was added as an anticoagulant. Serum was separated by centrifugation (1000 × g, 10 min, 4 ° C), combined, diluted with glycerol in a 1: 1 ratio, and stored at -20 ° C until further use. To isolate TTX-specific immunoglobulins, a column with TTX covalently bound to an Affigel-10 sorbent (Bio-Rad, USA) was used. The sorbent was synthesized according to the protocol of Takati et al. (Takati, N., Mountassif, D., Taleb, H., Lee, K., Blaghen, M. Purification and partial characterization of paralytic shellfish poison-binding protein from Acanthocardia tuberculatum // Toxicon, 2007, v. 50 (3 ), pp. 311–321) with some modifications. Briefly, 5 ml of Affigel-10 in isopropanol was treated with 50 μl of ethylenediamine and 250 μl of dimethylformamide at 20 ° C. for 30 minutes. Then it was washed with 50 mM phosphate buffer (PB, pH 7.4), 5% glutaraldehyde (Sigma, USA) was added, and the mixture was incubated at 20 ° C for 30 minutes. The sorbent was washed with PB, 20 μg of TTX citrate was added and incubated at 20 ° C for 4 hours. Unbound toxin was washed out using PB. Then the sorbent was incubated in 5 ml of PB with 0.1 M NaBH ₄ at 20 ° C for 2 hours. The serum was applied to the column, washed with 0.05 M Tris-HCl with 0.15 M NaCl, pH 7.5. Antibodies bound to TTX were eluted using glycine buffer containing 0.15 M NaCl, pH 3.0, followed by elution with glycine buffer containing 0.15 M NaCl, pH 2.2. To prevent irreversible denaturation of immunoglobulins, the pH of the eluates was adjusted to 7.5 using 1.5 M Tris-HCl, pH 7.5. Antibodies were concentrated using Vivaspin centricones with a molecular weight cutoff of 30 kDa (Sartorius, Germany), mixed with glycerol in a 1: 1 ratio to a final concentration of 0.5 mg / ml, and stored at -20 ° C until further use.

For competitive ELISA, a 96-well plate (Nunc Maxisorp, Thermo Fisher Scientific, USA) was sensitized with BSA-TTX conjugate in 0.1 M carbonate buffer containing 0.15 M NaCl, pH 9.5. The BSA-TTX conjugate was prepared in the same manner as for the PHA-TTX conjugate (see above). 100 μl BSA-TTX conjugate (4 μg / ml) was added to each well and incubated at 20-37 ° C for 30-100 minutes. Then it was washed with four changes of TBS-Triton-X-100 buffer containing 0.05 M Tris-HCl, 0.15 M NaCl and 0.005% Triton X-100, pH 7.4. After that, 120 μl of blocking solution containing 1 mg / ml skim milk in TBS-Triton-X-100 buffer was added to each well, the plate was incubated at 20-37 ° C for 30-100 minutes and washed four times with TBS-Triton buffer. -X-100. 50 μl of test extract or TTX calibration solution was added to the first well in each row, and each row (except for the last well) was filled with two-fold serial dilutions of the respective solutions. After that, 50 μl of a solution of polyclonal antibodies to TTX (dilution 1: 150) in TBS-Triton-X-100 buffer was added to each well. Antibody solution without TTX was added to the last wells of the plate as a positive control. The plate was incubated at 20-37 ° C for 30-100 minutes. It was then washed four times with TBS-Triton-X-100 buffer; 100 μl of goat anti-mouse antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific, USA) in a solution of TBS-Triton-X-100 (dilution 1: 1600) was added to each well and incubated at 20-37 ° C for 30 100 minutes. The plate was washed as described above. Then, 100 μl of a solution of orthophenyleneamine in 0.1 M phosphate-citrate buffer, pH 5.0, was added to each well and incubated for 15 minutes. Thereafter, 50 μl of 5% H ₂ SO _{4 was} added to each well to stop the reaction. The absorbance of the samples was measured at 492 nm using an ELx800uv plate reader (Bio-Tek Instruments Inc., USA). The TTX content in all wells was calculated using a calibration curve (Fig. 1B).

The limits of detection (LoD), limits of quantification (LoQ), working range, mean, standard deviation (SD), recovery, and coefficient of variation (CV) of the ELISA method were calculated. LoD and LoQ were calculated using the method of the international conference on harmonization (Shrivastava, A., Gupta, V. Methods for the determination of limit of detection and limit of quantitation of the analytical methods // Chronicles of Young Scientists, 2011, v. 2 (1), p. 21) as follows: LoD = 3.3 × SD / s, LoQ = 10 × SD / s, where s is the slope of the standard curve. All statistical procedures, including the calculation of the mean, SD, and CV, were performed using the Microsoft Excel 2010 Analysis Toolkit (Microsoft Corp., Redmond, Washington, USA). The degree of extraction was calculated based on the analysis of extracts of TTX-free nemertines of the species Cerebratulus marginatus, with TTX applied at a concentration of 70, 200, 350, 1000 ng / ml. LoD and LoQ were 25.54 and 77.34 ng / ml, respectively. The working range was 25.54–2500 ng / ml (Fig. 1A, B). In the linear range, the mean intraplate CV for repeats was 2.02-4.67% (FIG. 1B), while the mean interplate CV was 5.66% (FIG. 1C, D). The method recovery was 101.6% for 1000 ng / ml and 70% for the range 70-350 ng / ml. The CV for data obtained by ELISA and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was 14.64%. Thus, the data obtained using HPLC-MS / MS and ELISA are consistent, and the proposed modification of ELISA is a reproducible, sensitive and accurate method for measuring the total content of TTX. The association constant Ka of the antigen-antibody interaction was determined by the linearization method (Orosz, F., Ovadi, J. A simple method for the determination of dissociation constants by displacement ELISA // Journal of Immunological Methods, 2002, v. 270 (2), pp. 155-162, its value was 6 * 10 ⁸ M ^-1 .

To determine the quantitative and qualitative content of TTX and its analogs, a calibration curve was preliminarily constructed using calibration solutions.

The invention is illustrated in figure 1, where:

Figure 1A shows a standard ELISA curve for quantifying the total concentration of TTX and its analogs;

Fig. 1B shows the linear part of the standard curve. The number of repeated measurements for each concentration (n) is 8. The curve is in good agreement with the experimental data (R² = 0.994, y = -0.1938x + 1.2743);

in fig. 1B shows the standard curves for four replicates of ELISA on different plates;

in fig. 1D - shows the linear part of the standard curves for four ELISA conducted on different plates.

Example 1

An extract of nemertean species Cephalothrix simula was prepared as described earlier (Vlasenko, AE, Velansky, PV, Chernyshev, AV, Kuznetsov, VG, Magarlamov, TY Tetrodotoxin and its analogs profile in nemertean species from the sea of Japan // Toxicon, 2018, v . 156, pp. 48-51.) Using 100 mg tissue samples. The pH was adjusted to ~ 7.5 with 0.5 M Tris-HCl, 1.5 M NaCl, pH 7.5. The final extracts were stored at -20 ° C until further use.

For competitive ELISA, a 96-well plate (Nunc Maxisorp, Thermo Fisher Scientific, USA) was sensitized with BSA-TTX conjugate in 0.1 M carbonate buffer containing 0.15 M NaCl, pH 9.5. 100 μl BSA-TTX conjugate (4 μg / ml) was added to each well and incubated at 37 ° C for 30 minutes. Then it was washed with four changes of TBS-Triton-X-100 buffer containing 0.05 M Tris-HCl, 0.15 M NaCl and 0.005% Triton X-100, pH 7.4. Thereafter, 120 μl of blocking solution containing 1 mg / ml skim milk in TBS-Triton-X-100 was added to each well, and the plate was incubated at 37 ° C for 30 minutes and washed with TBS-Triton-X-100 buffer. 50 μl of test extract or TTX calibration solution was added to the first well in each row, and each row (except for the last well) was filled with two-fold serial dilutions of the respective solutions. After that, 50 μl of a solution of polyclonal antibodies to TTX (dilution 1: 150) in TBS-Triton-X-100 buffer was added to each well. Antibody solution without TTX was added to the last wells of the plate as a positive control. The plate was incubated at 37 ° C for 30 minutes. Then it was washed with TBS-Triton-X-100 buffer; 100 μl of goat anti-mouse antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific, USA) in a solution of TBS-Triton-X-100 (dilution 1: 1600) was added to each well and incubated at 37 ° C for 30 minutes. The plate was washed as described above. Then, 100 μl of a solution of orthophenyleneamine in 0.1 M phosphate-citrate buffer, pH 5.0, was added to each well and incubated for 15 minutes. Thereafter, 50 μl of 5% H ₂ SO _{4 was} added to each well to stop the reaction. The absorbance of the samples was measured at 492 nm using an ELx800uv plate reader (Bio-Tek Instruments Inc., USA). The TTX content in all wells was calculated using a calibration curve (Fig. 1B). According to HPLC-MS / MS, the C. simula extract contained 23,616 ng of TTX and its analogs (including 4-epitTTC, 4,9-anhydroTTC, 5-deoxyTTC, 11-deoxyTTC, and 5,6,11-trideoxyTTX) per 1 g of body weight. The content of TTX and its analogs, revealed by ELISA, was 29,069 ± 4,874 ng / g of body weight.

Example 2

The extract of nemertean species Cerebratulus marginatus was prepared as described earlier (Vlasenko, AE, Velansky, PV, Chernyshev, AV, Kuznetsov, VG, Magarlamov, TY Tetrodotoxin and its analogs profile in nemertean species from the sea of Japan // Toxicon, 2018, v 156, pp. 48-51), using 100 mg tissue samples, 200 ng of TTX was added. The pH was adjusted to ~ 7.5 with 0.5 M Tris-HCl, 1.5 M NaCl, pH 7.5. The final extracts were stored at -20 ° C until further use.

For competitive ELISA, a 96-well plate (Nunc Maxisorp, Thermo Fisher Scientific, USA) was sensitized with BSA-TTX conjugate in 0.1 M carbonate buffer containing 0.15 M NaCl, pH 9.5. 100 μl BSA-TTX conjugate (4 μg / ml) was added to each well and incubated at 30 ° C for 45 minutes. Then it was washed with four changes of TBS-Triton-X-100 buffer containing 0.05 M Tris-HCl, 0.15 M NaCl and 0.005% Triton X-100, pH 7.4. Thereafter, 120 μl of blocking solution containing 1 mg / ml skim milk in TBS-Triton-X-100 buffer was added to each well, and the plate was incubated at 30 ° C for 45 minutes and washed with TBS-Triton-X-100 buffer. 50 μl of test extract or TTX calibration solution was added to the first well in each row, and each row (except for the last well) was filled with two-fold serial dilutions of the respective solutions. After that, 50 μl of a solution of polyclonal antibodies to TTX (dilution 1: 150) in TBS-Triton-X-100 buffer was added to each well. Antibody solution without TTX was added to the last wells of the plate as a positive control. The plate was incubated at 30 ° C for 45 minutes. Then it was washed with TBS-Triton-X-100 buffer; 100 μl of goat anti-mouse antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific, USA) in TBS-Triton-X-100 solution (dilution 1: 1600) was added to each well and incubated at 30 ° C for 45 minutes. The plate was washed as described above. Then, 100 μl of a solution of orthophenyleneamine in 0.1 M phosphate-citrate buffer, pH 5.0, was added to each well and incubated for 15 minutes. Thereafter, 50 μl of 5% H ₂ SO _{4 was} added to each well to stop the reaction. The absorbance of the samples was measured at 492 nm using an ELx800uv plate reader (Bio-Tek Instruments Inc., USA). The content of TTX in all wells was calculated using the calibration curve (Fig. 1B) and amounted to 142 ± 8 ng / ml.

Example 3

Polychaete extract Nereis sp. (prepared according to the procedure described earlier for nemerteans (Vlasenko, AE, Velansky, PV, Chernyshev, AV, Kuznetsov, VG, Magarlamov, TY Tetrodotoxin and its analogs profile in nemertean species from the sea of Japan // Toxicon, 2018, v. 156, pp. 48-51), 1000 ng of TTX was added using tissue samples of 100 mg, pH was adjusted to ~ 7.5 with 0.5 M Tris-HCl, 1.5 M NaCl, pH 7.5. The final extracts were stored at -20 ° C until further use.

For competitive ELISA, a 96-well plate (Nunc Maxisorp, Thermo Fisher Scientific, USA) was sensitized with BSA-TTX conjugate in 0.1 M carbonate buffer containing 0.15 M NaCl, pH 9.5. 100 μl BSA-TTX conjugate (4 μg / ml) was added to each well and incubated at 20 ° C for 100 minutes. Then it was washed with four changes of TBS-Triton-X-100 buffer containing 0.05 M Tris-HCl, 0.15 M NaCl and 0.005% Triton X-100, pH 7.4. Thereafter, 120 μl of blocking solution containing 1 mg / ml skim milk in TBS-Triton-X-100 buffer was added to each well, and the plate was incubated at 20 ° C for 100 minutes and washed with TBS-Triton-X-100 buffer. 50 μl of test extract or TTX calibration solution was added to the first well in each row, and each row (except for the last well) was filled with two-fold serial dilutions of the respective solutions. After that, 50 μl of a solution of polyclonal antibodies to TTX (dilution 1: 150) in TBS-Triton-X-100 buffer was added to each well. Antibody solution without TTX was added to the last wells of the plate as a positive control. The plate was incubated at 20 ° C for 100 minutes. Then it was washed with TBS-Triton-X-100 buffer; 100 μl of goat anti-mouse antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific, USA) in a solution of TBS-Triton-X-100 (dilution 1: 1600) was added to each well and incubated at 20 ° C for 100 minutes. The plate was washed as described above. Then, 100 μl of a solution of orthophenyleneamine in 0.1 M phosphate-citrate buffer, pH 5.0, was added to each well and incubated for 15 minutes. Thereafter, 50 μl of 5% H ₂ SO _{4 was} added to each well to stop the reaction. The absorbance of the samples was measured at 492 nm using an ELx800uv plate reader (Bio-Tek Instruments Inc., USA). The TTX content in all wells was calculated using the calibration curve (Fig. 1B) and amounted to 1010 ± 10 ng / ml.

The above examples confirm that the claimed method makes it possible to determine the total concentration of TTX and its analogs in extracts of marine organisms, in particular, 4-epi TTX, 4,9-anhydro TTX, 5-deoxy TTX, 11-deoxy TTX and 5,6,11-trideoxy TTX, 11 -oxoTTC, 5,6,11-trideoxyTTC. The analysis time is less than that of the prototype, and is 5 hours. The method excludes the possibility of cross-reaction between the obtained antibodies and the components of the investigated extracts, which makes it possible to simplify the procedure for preparing extracts and to reduce the total time of the analysis and its cost.

This work was supported by the Russian Foundation for Basic Research (RFBR), grant no. 18-04-00808 A.

Claims (6)

1. A method of performing an indirect competitive enzyme-linked immunosorbent assay for determining the content of toxins in extracts of marine organisms selected from tetrodotoxin (TTX), 4-epiTTC, 4,9-anhydroTTC, 5-deoxyTTC, 11-deoxyTTC and 5,6,11-trideoxyTTC, according to which the plate is sensitized by incubation with a solution of bovine serum albumin with tetrodotoxin (BSA-TTX) conjugate in 0.05 M carbonate buffer, the sensitized plate is washed with a buffer solution, incubated in a blocking buffer, the plate is washed from the blocking buffer with washing buffer, extracts are added to the wells of the plate marine organisms and a solution of specific antibodies, followed by incubation of the plate, wash the plate with washing buffer, add conjugate of secondary antibodies with horseradish peroxidase, incubate followed by washing with washing buffer, add a substrate, incubate at 37 ° C for 15 minutes, stop the reaction by adding a solution of sulfuric acid acid and measure the optical p density, and polyclonal antibodies are used as specific antibodies, which are obtained by immunizing mice with phytohemagglutinin-TTX conjugate (PHA-TTX) and isolated by affinity chromatography on Affigel-10 sorbent covalently linked to TTX; Orthophenylenediamine is taken as a substrate, and the optical density is measured at a wavelength of 492 nm. 2. The method according to claim 1, according to which the blocking buffer is a buffer containing 0.05 M Tris-HCl, 0.15 M NaCl, 0.005% Triton X-100, with the addition of 1 mg / ml skim milk. 3. The method according to claim 1, according to which the incubation for sensitizing the plate, incubation in a blocking buffer, incubation of the plate with extracts of marine organisms and a solution of specific antibodies and incubation with a conjugate of secondary antibodies with horseradish peroxidase is carried out at a temperature of 20-37 ° C for 30 -100 minutes. 4. The method according to claim 1, according to which a buffer containing 0.05 M Tris-HCl, 0.15 M NaCl with 0.005% Triton X-100, pH 7.4 is used as a wash buffer. 5. The method according to claim 1, according to which the wells of the plate are washed with washing buffer no more than four times. 6. The method according to claim 1, according to which a 0.15 M NaCl solution is additionally introduced into the 0.05 M carbonate buffer.

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