RU2566553C1 - STRAIN OF HYBRID CELLS OF ANIMALS Mus musculus 3F11 - PRODUCER OF MONOCLONAL ANTIBODIES SPECIFIC TO BOTULINUS TOXIN TYPE B - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: strain of hybrid cells of animals Mus. musculus 3F11 - producer of monoclonal antibodies specific to botulinus toxin type B is suggested. The strain is deposited to state collection of pathogens and cell cultures of Federal Budget Institution of Science of State Scientific Centre of Applied Microbiology and Bioengineering (FBIS SSC AMB),collection No. - H-45.

EFFECT: invention ensures production of the highly specific monoclonal antibodies to botulinic neurotoxin type B, suitable for construction of their base of the test-systems to identify botulinus.

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Description

The invention relates to biotechnology and can be used to obtain monoclonal antibodies to botulinum toxin type B.

Botulism is a severe foodborne neuropsychiatric disease caused by the action of botulinum neurotoxins (BoNTs) secreted by the anaerobic microorganisms Clostridium botulinum [Stanker, L.H. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food / L.H. Stanker, M.C. Scotcher, L. Cheng, K. Ching, J. McGarvey, D. Hodge, and R. Hnasko // Toxins (Basel). - 2013. - V. 5. - P. 2212-2226].

The widespread use of home canning, salting, smoking of fish and meat products without observing appropriate technologies affects the intensity of the epidemic manifestations of this infection. Infection of people occurs mainly as a result of eating foods containing botulinum toxins and the causative agents of Clostridium botulinum (food butulism), and through damaged skin (traumatic botulism) [Noskova, O.A. Clinical and epidemiological features and improvement of laboratory diagnosis of botulism in the Irkutsk region / O.A. Noskova, T.Yu. Zagoskina, E.N. Subycheva, E.Yu. Markov, S.E. Lekomtseva, O.B. Bodrykh, S.V. Balakhonov // Bulletin of the VSNS SB RAMS. - 2013. - No. 2 (90). Part 1. - S. 102-106]. In vivo, BoNTs exist as part of stable complexes with other proteins. Existence in the complex protects botulinum toxins from the aggressive environment of the stomach during food intoxication and increases their lifetime in the bloodstream [Abbasova SG Monoclonal antibodies to botulinum neurotoxins of types A, B, E and F / C.G. Abbasova, N.V. Rudenko, A.Yu. Gorokhovatsky, M.V. Kapralov, I.D. Vinogradova, Yu.V. Vertiev, V.A. Nesmeyanov, E.V. Grishin // Bioorganic chemistry. - 2011. - T. 37. - No. 3. - S. 344-353].

There are seven different serotypes of toxins (AH) that differ in the antigenic properties of the toxins they produce and some other characteristics. The greatest threat to humans is toxins of serotypes A, B, E and F. [Cheng, L.W. Detection of Botulinum Neurotoxin Serotypes A and B Using a Chemiluminescent versus Electrochemiluminescent Immunoassay in Food and Serum / L.W. Cheng, L.H. Stanker // Agric Food Chem. - 2013. - V 61. - P. 755-760].

In total, from 2001 to 2007, 139 cases of foodborne botulism were registered in the United States, of which the bulk of cases caused by intoxication BoNT / A and BoNT / E, and only 10 cases directly related to the consumption of food infected with BoNT / B. In the same time period, 387 cases of BoNT / B of childhood botulism were reported, due to the effect of BoNT / B. Although foodborne botulism associated with serotype B is less common, it nevertheless poses a serious threat to food safety. The evidence of this is the large outbreaks of botulism registered by the USA and Great Britain [Scotcher, M.S. Detection of botulinum neurotoxin serotype B at sub mouse LD50 levels by a sandwich immunoassay and its application to toxin detection in milk / M.C. Scotcher, L.W Cheng, L.H. Stanker // PLoS ONE. - 2010. - V 5. - P. 1-10].

Specific antibodies are widely used to diagnose botulinum infections. There is a Russian patent RU 2152036 (priority dated 05/12/1998, copyright of the NGO Biomed) that describes a method for determining botulinum toxins A and B based on specific F (ab) ₂ antibody fragments. Fab fragments of recombinant antibodies specific for types A and B botulinum toxins are proposed. These Fab fragments can be used as immunosensors for detecting botulinum toxins in food and for other health and defense ministries [US Pat. 5932449 USA, C07K 16/12, C12P 021/04. Detection of botulinum toxin / The Secretary of the Army, USA, No. 08792824, declared. 01/30/1997]. Most of the described botulinum toxin detection systems using antibodies are based on classical enzyme-linked immunosorbent assay.

Hybridomas are known that produce mouse MCAs for botulinum neurotoxin type B, which are characterized by the stability of antibody production, on the basis of which a number of highly sensitive test systems have been developed in the format of an enzyme immunoassay [Stanker, L.H. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food / L.H. Stanker, M.C. Scotcher, L. Cheng, K. Ching, J. McGarvey, D. Hodge, and R. Hnasko // Toxins (Basel). - 2013. - V. 5. - P. 2212-2226]. However, the famous foreign strains of hybrid cells of Mus museums - producers of MCA, which can be used to detect botulinum neurotoxins, are not available for MCA and the production of domestic diagnostic test systems. In Russia, currently there are no patented strains producing MCA for botulinum toxins.

Diagnosis of botulism is carried out on the basis of clinical, epidemiological and laboratory data. Currently used methods of laboratory diagnosis of botulism are laborious, costly and require the creation of special conditions for analysis or the use of special expensive equipment [Noskova, O.A. Clinical and epidemiological features and improvement of laboratory diagnosis of botulism in the Irkutsk region / O.A. Noskova, T.Yu. Zagoskina, E.N. Subycheva, E.Yu. Markov, S.E. Lekomtseva, O.B. Vigorous, SV. Balakhonov // Bulletin of the VSNS SB RAMS. - 2013. - No. 2 (90). Part 1. - S. 102-106].

The development of rapid detection methods for BoNTs remains an urgent problem for both medicine and the state sanitary and epidemiological control authorities due to their extreme toxicity and highest mortality in food poisoning. In addition, recently in the whole world attention to this category of toxins has increased due to the threat of bioterrorism [Abbasova SG Monoclonal antibodies to botulinum neurotoxins of types A, B, E and F / C.G. Abbasova, N.V. Rudenko, A.Yu. Gorokhovatsky, M.V. Kapralov, I.D. Vinogradova, Yu.V. Vertiev, V.A. Nesmeyanov, E.V. Grishin // Bioorganic chemistry. - 2011. - T. 37. - No. 3. - S. 344-353].

The most striking example in this area is the creation of hybridoma technology for producing monoclonal antibodies (MABs) with desired properties.

The objective of the invention is to obtain a strain of hybrid cultured cells producing monoclonal antibodies specific for type B botulinum toxin and suitable for constructing test systems based on them to detect the causative agent of botulism.

The problem is solved by the fact that a new strain of hybrid cultured animal cells of animals mus musculus 3F11 - a producer of highly specific monoclonal antibodies to botulinum toxin type B.

The strain of hybrid animal cells Mus musculus 3F11 was deposited in the state collection of pathogenic microorganisms and cell cultures of the Federal budget institution of science of the State scientific center for applied microbiology and biotechnology (FBUN SSC MPB), collection number - H-45, and is used as a producer of monoclonal antibodies to detect botulinum neurotoxin type B.

Pedigree strain

Mus musculus 3F11 animal hybrid cell strain is obtained by immunization of BALB / c mice. Mice are immunized with 4 times the introduction of recombinant botulinum toxin type B at a dose of 100 μg / mouse. On the third day after the last immunization, the splenocytes of the immune mice (1 × 10 ⁸ cells) are hybridized with the cells of mouse myeloma PZ-X63 Ag / 8-653 (1 × 10 ⁷ cells). A 50% solution of polyethylene glycol (Sigma, USA) was used as a merging agent. After hybridization, selection, screening, cloning, and cryopreservation of the hybridoma are performed.

Characterization of the strain of hybrid cells of animals Mus musculus 3F11.

Morphological characteristics

The hybrid cell culture consists of rounded cells loosely attached to the substrate the size of the original myeloma cell.

Cultural properties

The cultivation of the strain of hybrid animal cells of animals Mus musculus 3F11 is carried out at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide. The cultivation medium is RPMI - 1640 medium containing 20% fetal calf serum, 2 mM / L-glutamine, 100 μg / ml gentamicin. Cells are cultured as a stationary suspension in Costar plastic mattresses. Passage of 3F11 hybridoma cells is carried out 2 times a week with a dilution ratio of 1: 2 - 1: 3. The inoculum cell concentration is 1 × 10 ⁵ in 1 ml of medium. The maximum concentration of hybrid cells during cultivation is 1 × 10 ⁵ per 1 ml of medium. The hybrid clone does not lose the ability to synthesize antibodies at 10 passages in vitro (observation period).

Cultivation of hybridoma 3F11 in the animal

Female 20-week old BALB / c mice were pre-injected intraperitoneally with 0.3-0.5 ml pristan (Sigma, USA). After 2-4 weeks, the cultured cells of the strain located in the logarithmic phase of growth are sterile centrifuged for 5-10 minutes at 1000 rpm in an OPN-3 centrifuge. The supernatant is removed and the cell pellet is suspended in a sterile RPMI medium. Female BALB / c mice were intraperitoneally injected each with 1 ml of a cell suspension containing at least 10 million hybridoma cells. Hybridoma is vaccinated in 100% of cases. An ascitic tumor forms on days 7–10, animals are euthanized on days 10–21, and 3-5 ml of immuno-ascitic (IAG) fluid is removed from the abdominal cavity. Cells from the IAH are separated by centrifugation, and the titer of the MCA is determined in the supernatant by enzyme immunoassay [Egorov, A.M. Theory and practice of enzyme immunoassay / A.M. Egorov, A.P. Osipov, B.B. Dzantiev, E.M. Gavrilova // T33. - M .: Higher. school., 1991. - S. 174-175].

Product Feature

The mus musculus 3F11 animal hybrid cell strain produces specific monoclonal antibodies to botulinum toxin type B, the titer of which is in the culture fluid (QOL) 1: 640000, in the IAI 1: 1000000. Monoclonal antibodies from QOL and IAG were isolated by affinity chromatography on a column of protein G-Sepharose (Protein G Sepharose 4 Fast Flow). The purity of the obtained immunoglobulin fractions was evaluated in SDS-PAGE-electrophoresis under denaturing conditions. The concentration of immunoglobulins was determined spectrophotometrically at a wavelength of 280 nm (Smart Spec Plus spectrophotometer, Bio Rad, USA).

The productivity of the strain of hybrid cells of animals Mus musculus 3F11

MCA production in the cultivation medium is 20-50 μg / ml, in ascitic fluid - 5-10 mg / ml. Stable production of MCA is maintained for at least 10 passages in vitro with continuous cultivation.

The contamination of the strain of hybrid cells of animals Mus musculus 3F11. Hybrid line contaminants, including bacteria, yeast, fungi, were not detected. Mycoplasmas were not determined.

Cryopreservation and heating mode

The freezing medium contains fetal serum - 90%, dimethyl sulfoxide - 10%. 1-1.5 ml of the cell suspension is transferred into plastic cryovials and placed in a foam container with a wall thickness of 1 cm and left for 16-24 hours in a kelvinator at a temperature of -70 ° C and then immersed in liquid nitrogen.

Defrosting is carried out by lowering the tubes into water at a temperature of 37-41 ° C. Cells were diluted with RPMI 1640 medium (HyClone, USA) and centrifuged at 1000 rpm. The sediment is resuspended in a growth medium and the cells are transferred into culture bottles at a concentration of 200-300 thousand per milliliter. Cell viability after thawing is 60-80%. Each ampoule contains at least 10 million / ml cells.

Useful Product Properties

The hybridoma produces monoclonal antibodies belonging to the IgG1 subclass of mouse immunoglobulins.

The invention is illustrated by the following graphic materials.

FIG. 1. Dot blot with MCA 3F11. Determination of specific activity against microorganisms of the genus Clostridium.

FIG. 2. Dot blot with MCA 3F11. Determination of the sensitivity of MCA 3F11 in relation to strains of Clostridium botulinum containing botulinum neurotoxin type B.

FIG. 3. Dot blot with MCA 3F11. Determination of the sensitivity of MCA 3F11 in relation to recombinant botulinum neurotoxin type B.

FIG. 4. The definition of subclass affiliation MCA 3F11.

FIG. 5. SDS-PAGE electrophoresis of MCA 3F11.

FIG. 6. Enzyme-linked immunosorbent assay. Detection of botulinum toxin B by enzyme immunoassay.

For a better understanding of the invention, the following are examples of its specific implementation.

Example 1. Obtaining a strain of hybrid cells 3F11

Immunization

A recombinant protein consisting of botulinum toxin B heavy chain fragments was used as an antigen for immunization. To create superproducers of botulinum toxin B heavy chain fragments, DNA sequences encoding the C-terminal fragments of these toxins forming the HC50B domain (amino acids N853 - E1291 of the botulinum toxin precursor protein C), were obtained by PCR amplification with the genomic DNA of the pathogen, cloned into the expression vector pET22b (+) (Novagen, USA). The resulting expression constructs were transformed into E. coli cells of strain BL21 (DE3), bacterial biomass production and protein production were carried out at a temperature of + 300 ° C and 1 mm IPTG [Ryabko A.K. Development of a method for detecting botulinum neurotoxin based on immunodetection coupled with PCR amplification / A.K. Ryabko, A.E. Khlyntseva, A.V. Kolesnikov, O.N. Krasavtseva, I.G. Shemyakin, T.I. Kombarova, I.V. Zharnikova, A.V. Trump // Modern medicine and pharmaceuticals: analysis and development prospects: Materials of the VI International Scientific and Practical Conference (11/20/2012). - M .: Publishing house "Sputnik +". - 2012. - 70 s].

A strain of Mus musculus hybrid cells is prepared as follows. Female BALB / c mice are immunized according to the scheme shown in Table 1 below.

Blood from immune mice was taken on the third day after the last injection of antigen at a concentration of 100 μg according to Animal protocol No. P 02-19 p. 7.

The titers of specific antibodies in animal sera are determined using indirect solid-phase ELISA.

Hybridization

For fusion, spleen cells of mice and mouse myeloma cells PZ-X63 Ag / 8-653 in the amount of 1 × 10 ⁸ and 1 × 10 ^7, respectively, are used. The mixture of cells is centrifuged, the supernatant is thoroughly removed, and 1 ml of a 50% PEG solution is added to the cell pellet over 1 minute. The mixture is centrifuged for 15 minutes at 1000 rpm. Then the cells are dissolved in the growth medium. Cells dispense 96-well microplates (Costar, USA) onto a layer of peritoneal macrophages taken from BALB / c mice at 50 μl per well.

Selection

The hybrid cells are selected in GAT medium (PanEco, Russia), consisting of RPMI nutrient medium, in which 20% fetal bovine serum, 0.1 mM hypoxanthine, 0.04 mM thymidine, and 0.01 mM aminopterin are added. After 21 days, aminopterin is removed from the medium. Subsequently, the cultivation is carried out on RPMI - 1640 medium with 20% fetal calf serum, 2 mM / L-glutamine, 100 μg / ml gentamicin.

Hybrid Clone Screening

Specific hybrids are selected by enzyme-linked immunosorbent assay (ELISA).

To perform an indirect ELISA, 1 μg / well of recombinant bolulinic neurotoxin type B is added to the wells of a 96-well polystyrene plate for immunoassay and kept at 37 ° C for 2 hours or at 4 ° C for 18 hours. After incubation, the wells are washed 3-5 times with phosphate-buffered saline, pH 7.4, containing 0.05% tween-20 (Sigma). (FSB-T). Then, 160 μl of a 0.5% solution of bovine serum albumin (BSA), tested for the absence of peroxidase activity, is added to the wells and incubated at 37 ° C for 1 hour. FSB-T is washed three times. Then, 100 μl of the culture medium of the studied hybridomas are transferred to the wells and incubated for 1 h at 37 ° C. After that, the wells of the tablet are washed three times with a solution of PBS-T and the peroxidase conjugate is added to the wells to the whole mouse IgG molecule (Sigma, USA) in working dilution. The plate is incubated at 37 ° C for 1 hour, then the wells are washed 6 times with FSB-T solution. Next, the plates are washed and an enzymatic reaction is carried out: 100 μl of a substrate-indicator solution (10 μl of 30% H ₂ O ₂ and 8 mg of orthophenylenediamine per 10 ml of phosphate-nitrate buffer pH 5.0) are added to the wells. A positive reaction is evaluated by the appearance of a tan solution. The reaction is stopped by adding 50 μl of 4N sulfuric acid to the wells. The results are taken into account on a photometer (xMark, Microplate Spectrophotometr, Bio-Rad, USA) for ELISA at a wavelength of 492 nm.

Cloning of 3F11 hybrid cells was performed by limiting dilution in 96-well plates on a layer of peritoneal macrophages at the rate of 1 × 10 ⁴ cells per well. Clone screening is carried out by indirect solid-phase ELISA, as described above.

Cultivation

The cultivation of the strain of hybrid cells 3F11 is carried out at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide. The cultivation medium is RPMI - 1640 medium containing 20% fetal calf serum, 2 mM / L-glutamine, 100 μg / ml gentamicin.

Cryopreservation

Clones of 3F11 hybridoma are cryopreserved on a freezing medium consisting of fetal calf serum - 90%, dimethyl sulfoxide - 10%. Hybridoma clones are stored in Dewar vessels with liquid nitrogen.

Example 2. Cultivation of C. botulinum culture in the conditions of toxin production

Selected well-isolated, typical colonies of C. botulinum grown on solid nutrient medium (clostridial agar - 25.25 g, 1 g of yeast extract, 2 g of agar, 475 ml of distilled water), incubated under anaerobic conditions for 48 hours at 35 ° C, suspended in physiological saline (to an optical density of 4 according to Mac Farland) and heated for 10 minutes at 80 ° C to inactivate vegetative cells.

A warmed culture (1 ml) was added to a freshly prepared medium for the production of toxin (casein enzymic hydrolysate - 10 g / l, pepton aus Casein C / ESX Enzymatich for Tocingenwennung - 10 g / l, sodium thioglycollate - 0.25 g / l, yeast extract - 10 g / l, distilled water - 500 ml), tubes with 9 ml of medium, 1/10 ratio. Incubation was carried out at 35 ° C for 5 days under anaerobic conditions obtained in the anaerostat using a gas generating package (GasPak EZ, BD, USA).

Example 3. Determination of specific activity against microorganisms of the genus Clostridium

To conduct this study, a panel of 24 microorganisms was used, including microorganisms of the genus Clostridium and others (table 2).

Inactivated microbial cells were adsorbed at a concentration of 1 × 10 ⁶ cells / well using a BIO-DOT device (BIO RAD, USA) on a nitrocellulose membrane (GE Healthcare Life Sciences, Amersham, Germany). Microorganism strains were obtained from the microorganism collection FBSI SSC PMB. The membranes were then blocked with an inert protein solution and sequentially incubated with 3F11 MCA and a peroxidase conjugate to the whole mouse IgG molecule (Sigma, USA). The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, USA), 0.015% H ₂ O ₂ , 0.01 M phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As the analysis showed, MCA 3F11 specifically interacted with strains of Clostridium botulinum containing botulinum neurotoxin type B and did not give cross-reactions with other microorganisms (Fig. 1).

Example 4. Determination of the sensitivity of MCA 3F11 in relation to strains of Clostridium botulinum containing botulinum neurotoxin type B.

Sensitivity testing of QL with a 3F11 hybrid against botulinum toxin type B was performed by dot blot analysis. Clostridium botulinum type B strains with an initial concentration of 1 × 10 ⁸ μl / ml were sorbed onto a nitrocellulose filter using a Bio-Dot device (Bio-Rad, USA) and titrated in two steps to a final concentration of 1 × 10 ⁵ m. cells / ml (1st row - strain Clostridium botulinum No. 311, type B, 2nd row - Clostridium botulinum No. 312, type B, 3rd row - Clostridium botulinum No. 344, type B).

The membrane was then blocked with an inert protein solution and incubated sequentially with 3F11 MCA and peroxidase conjugate to the whole mouse IgG molecule (Sigma, USA). The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, USA), 0.015% H ₂ O ₂ , 0.01 M phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As a result of the analysis, it was found that MCA 3F11 reveal the ketochka of strains of Clostridium botulinum type B strains up to 1 × 10 ⁵ μl / ml (Fig. 2).

Example 5. Determination of the sensitivity of MCA 3F11 in relation to recombinant botulinum neurotoxin type B

The specific activity of QL hybridomas 3F11 was tested against botulinum neurotoxin type B by dot blot analysis. Botulinum toxin B with an initial concentration of 0.5 μg / ml was sorbed onto a nitrocellulose filter using a Bio-Dot device (Bio-Rad, USA) and titrated twice. The membrane was then blocked with an inert protein solution and incubated sequentially with 3F11 MCA and peroxidase conjugate to the whole mouse IgG molecule (Sigma, USA). The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, USA), 0.015% H ₂ O ₂ , 0.01 M phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As a result of the analysis, it was found that MCA 3F11 detect up to 1 ng / ml of botulinum toxin B (Fig. 3).

Example 6. Determination of the subclass affiliation of MCAs produced by 3F11 hybridoma

Subclasses of the obtained MCAs are determined using the isotype kit for murine monoclonal antibodies (Roche Diagnostic Corporation, USA). An immunochromatographic strip is immersed in a culture fluid of 500 μl to determine the isotypes of murine monoclonal antibodies, not deeper than 1.5 cm, for 5 minutes. After the development of the control band and the band corresponding to the tested Ig, the antibody subclass is determined using the control immunochromatographic strip (attached to the kit for determining isotypes of murine monoclonal antibodies).

Subclass determination of the obtained MCAs showed that the 3F11 hybrid cell strain produces monoclonal antibodies belonging to the IgG2b mouse immunoglobulin subclass (Fig. 4).

Example 7. Isolation and purification of MCA 3F11

The allocation of MCA from ascitic fluid is carried out by affinity chromatography on a column of protein G-sepharose (Protein G Sepharose 4 Fast Flow). Cellular components from the culture and ascitic fluids are removed by centrifugation (2000 g × 20 min), the pH of the culture fluid is adjusted to 8.6 using a 1 N NaOH solution, ascitic fluid is diluted 1: 1 with planting buffer (0.05 M Tris, 0.15 M NaCl, 0.02% NaN ₃ , pH 8.6). The column is balanced with landing buffer and samples are applied at a rate of 10 ml / hour at room temperature. After applying the samples, the column is washed from unbound components and the immunoglobulins are eluted with 0.05 M glycine buffer, 0.15 M NaCl, pH 2.3. The yield control of the immunoglobulin fraction is carried out using a UV detector (Pharmacia LKB, Sweden).

The isolated immunoglobulins are concentrated by precipitation with dry ammonium sulfate (up to 50% saturation) and transferred to a phosphate-buffered saline by gel filtration on a Sephadex G-25 column (PD-10, Pharmacia LKB, Sweden) with a capacity of 10 ml. The purity of the obtained immunoglobulin fractions was evaluated in SDS-PAGE-electrophoresis under denaturing conditions by the method of Laemmli [Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 / U.K. Laemmli // Nature. - 1970. - V. 227. - P. 680-685]. As a result, purified immunoglobulin preparations were obtained (Fig. 5).

Example 8. Determination by ELISA of the specific interaction of MCA produced by hybrid cells of Mus musculus strain with botulinum neurotoxin type B

MCA 3F11 in phosphate-buffered saline, pH 7.4 with an initial concentration of 5 μg / well, was titrated twice in vertical steps to a final concentration of 0.035 μg / well (100 μl / well in PBS). For titration, 96-well stripped ELISA plates were used. Positive (K +) and negative controls (K-) controls were added to the first vertical row of the tablet. Well A1 with negative control was used as a blank. The antibody plate was incubated for 1 h on a shaker at a temperature of 37 ° C, then washed with FSB-T 4 times. Non-specific binding sites were blocked by adding 160 μl of 0.5% fat milk to the wells, incubated for 1 h on a shaker at 37 ° C and washed 3 times with PBS-T. Recombinant botulinum toxin was titrated twice in horizontal direction with an initial concentration of 0.5 μg / ml in FSB to a final concentration of 0.0002 μg / ml in FSB (100 μl / well in FSB), incubated under the same conditions, and then the plate was washed 4 times in the FSB-T. Biotinylated MCA 1G7 (fraction No. 3-1.4 mg / ml) was obviously added in excess (1: 500 dilution) of 100 μl / well into the FSB, and the plate was incubated and washed four times as described above. Then, 100 μl horseradish streptavidin-peroxidase conjugate (Imtek, Russia) was added to the plate wells at a dilution of 1: 5000 (according to the instructions for use) in the FSB. After incubation and four times washing the plate, 50 μl of substrate (prepared solution of 3.3 ′, 5.5′-tetramethylbenzidine) was added to all wells. The tablet was placed for 3-5 minutes in a dark place, then the reaction was stopped with 4 N sulfuric acid (50 μl / well).

The tablet was placed for 3-5 minutes in a dark place at room temperature. The results were taken into account using an xMark plate spectrophotometer (Bio-Rad, USA) at a wavelength of 450 nm.

As a result of the analysis, it was found that using immobilized MCA 3F11, the detection limit of botulinum toxin B was 0.8 ng / ml (table 2, FIG. 6).

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 3F11, a producer of monoclonal antibodies to botulinum toxin type B, was deposited in the collection of microorganisms of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology (FBUN SSC PMB), collection number H-45.

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