RU2571208C1 - STRAIN OF HYBRID ANIMAL CELLS Mus musculus 1G7 - PRODUCER OF MONOCLONAL ANTIBODIES SPECIFIC FOR BOTULINUM TOXIN OF TYPE B - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of hybrid cells of animals Mus musculus 1G7 - producer of monoclonal antibodies to the botulinum toxin of type B. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures of the Federal State Institution of Science of State Research Centre of Applied Microbiology and Biotechnology (FSIS SRC AMB), the collection number is H-46.

EFFECT: invention enables to obtain highly specific monoclonal antibodies to botulinum neurotoxin of type B suitable for construction on their basis of test-systems for detection of the causative agent of botulism.

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Description

The invention relates to biotechnology and can be used to obtain monoclonal antibodies to type B botulinum toxin. Botulism is a severe foodborne neuropsychiatric disease caused by the action of botulinum neurotoxins (BoNTs) secreted by 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 botulism) 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. The existence of the complex protects botulinum toxins from the aggressive environment of the stomach during food intoxication and increases their life time in the bloodstream [Abbasova, S.G. 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), which differ in 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. This is confirmed by large outbreaks of botulism registered in 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 have been proposed. These Fab fragments can be used as immunosensors for detecting botulinum toxins in food and for other needs of the public health and defense [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, well-known foreign mus musculus hybrid cell strains - producers of MCAs, which can be used to detect botulinum neurotoxins, are not available for the production of MCAs and 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. Bodrykh, S.V. 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 both for medicine and for 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 examples in this area are 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 1G7 is proposed - the producer of highly specific monoclonal antibodies to type B botulinum toxin.

The strain of hybrid animal cells Mus musculus 1G7 was deposited in the state collection of pathogenic microorganisms and cell cultures of the Federal budget institution of science of the State scientific center of applied microbiology and biotechnology (FBUN SSC MPB), collection number - N-46, and is used as a producer of monoclonal antibodies to detect botulinum neurotoxin type B.

The pedigree of the strain.

Mus musculus 1G7 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 1G7.

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 animal hybrid cells of animals Mus musculus 1G7 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 1G7 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 1G7 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 // T 33. - M .: Higher. Shk., 1991. - S. 174-175].

The characteristic of a useful product.

The strain of animal hybrid cells Mus musculus 1G7 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 1G7.

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. Contamination of a strain of hybrid animal cells of Mus musculus 1G7 animals. 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.

Properties of a useful product. 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 1G7. Determination of specific activity against microorganisms of the genus Clostridium.

FIG. 2 Dot blot with ICA. 1G7. Determination of the sensitivity of MCA 1G7 in relation to strains of Clostridium botulinum containing botulinum neurotoxin type B.

FIG. 3 Dot blot with MCA 1G7. Determination of the sensitivity of MCA 1G7 in relation to recombinant botulinum neurotoxin type B.

FIG. 4 Definition of subclass affiliation of MCA 1G7.

FIG. 5. SDS-PAGE electrophoresis MCA 1G7.

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 1G7

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 HC50 B domain (amino acids N853 - E1291 protein botulinum toxin B precursor), 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 was produced and protein production was 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 p.].

A strain of Mus musculus hybrid cells is prepared as follows. Female BALB / c mice are immunized according to the scheme shown in the table 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. Ρ 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 × ⁸ 10 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.

Selection of hybrid cells is carried out 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, cultivation is carried out on RPMI-1640 medium with 20% fetal calf serum, 2 MM / L-glutamine, 100 μg / ml gentamicin.

Screening of hybrid clones.

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

To perform an indirect ELISA, 1 μg / well of type B recombinant bolulinic neurotoxin is added to the wells of a 96-well polystyrene immunoassay plate 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 substrate-indicator solution (10 μl of 30% H ₂ O ₂ and 8 mg of orthophenylenediamine per 10 ml of pH 5.0 phosphate-nitrate buffer) 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 1G7 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 1G7 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 1G7 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 under toxin production conditions

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).

table 2 Panel of microorganisms to determine the specific activity of MCA 3F11. Row A Row B Row C A1. Clostridium botulinum No. 311, type B B1. Clostridium perfringens ATCC 10543 C1. Salmonella paratyphi A 225 A2. Clostridium botulinum No. 312, type B B2. Clostridium difficile 2652K C2. Shigella flexneri 1a 8516 A3. Clostridium botulinum No. 364, type B B3. Escherichia coli O157: H7 5999 C3 Shigella sonnei 4385 A4. Clostridium botulinum ATCC 19697 Type A B4. Escherichia coli O86 Orskow C4. Yersinia enterocolitica 287 A5. Clostridium botulinum No. 345 type A B5. Escherichia coli 675 C5 Yersinia pseudotuberculosis III A6. Clostridium coccoides ATCC 29263 B6. Escherichia coli O6 25922 ATCC C6. Brucella abortus 544 A7. Clostridium histolyticum ATCC19501 B7. Salmonella typhi 65 C7 Escherichia coli 4879 A8. Clostridium novyi ATCC 19402 B8 Salmonella typhimurium 21 C8 Proteus vulgaris

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 1G7 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 Μ phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As the analysis showed, MCA 1G7 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 MICA 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 twice with a final concentration of 1 × 10 ⁵ μl / 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 Μ phosphate-saline buffer, 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 mcl / ml (Fig. 2).

Example 5. Determination of the sensitivity of MCA 1G7 in relation to recombinant botulinum neurotoxin type B

The specific activity of QOL 1G7 hybridomas against a botulinum neurotoxin type B was tested 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. Next, the membrane was blocked with an inert protein solution and incubated sequentially with 1G7 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 Μ 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 1G7 detect up to 0.5 ng / ml of botulinum toxin B (Fig. 3).

Example 6. Determination of the subclass affiliation of MCAs produced by 1G7 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 liquid of 500 μl to determine the isotypes of murine monoclonal antibodies no 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).

The subclass of the obtained MCAs showed that the strain of hybrid cells 1G7 produces monoclonal antibodies belonging to the IgG1 subclass of mouse immunoglobulins (Fig. 4).

Example 7. Isolation and purification of MCA 1G7

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 Μ Tris, 0.15 Μ 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 / h at room temperature. After applying the samples, the column is washed from unbound components and the immunoglobulins are eluted with 0.05 Μ glycine buffer, 0.15 Μ 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 1G7 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 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 3F11 (fraction No. 3-1.4 mg / ml) was obviously added in excess (1: 500 dilution) of 100 μl / well into the FSB, 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 4N 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 1G7, the detection limit of botulinum toxin B was 0.4 ng / ml (table 2, FIG. 6).

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 1G7, 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-46.

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