RU2439148C1 - STRAIN OF HYBRID CULTIVATED CELLS OF ANIMAL Mus musculur 1E6-PRODUCER OF MONOCLONAL ANTIBODIES, SPECIFIC TO BACILLUS Anthracis spores - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: strain of hybridioma is obtained by immunization of mice of line BALB/c. Mice are immunised by conventional method by double with thirty day exposition subcutaneous introduction of inactivated spores of B. Anthracis strain "ÿ-1". On the third day after last buster-injection carried out is hybridisation of splenocytes of immune mice (1ù108 cells) with cells of mouse myeloma P3-X63 Ag/8-653 (1ù107 cells), using for fusion polyethylene glycol (Sigma, USA). After hybridisation carried out are selection, screening, cloning and cryopreservation of hybridoma. Obtained hybridoma is deposited in collection of microorganisms of FSSE SRC AMB under number H10. Invention relates to biotechnology and can be used for obtaining monoclonal antibodies (MCA) to spores Bacillus anthracis. Strain productivity is 20-50 mcg/ml of monoclonal antibodies (MCAla in ascitic liquid- 5-10 mg/ml. Hybridoma produces MCA, related to IgGl subclass of mouse immunoglobulins and spores specific to Bac.anthracis. ^ EFFECT: increase of strain productivity. ^ 7 dwg, 2 tbl, 9 ex

Description

The invention relates to biotechnology and can be used for specific detection of Bacillus anthracis spores.

The causative agent of anthrax has the ability to form spores that are highly stable in the external environment, which determines not only its further preservation in an anabiotic state in nature, but also the ability to multiply and accumulate in the soil. This feature of the spore form of B. anthracis contributes to the creation of long-term active soil foci and potentially dangerous territories, which leads to a constant threat of infection. Despite a significant number of methods for improving the laboratory diagnosis of anthrax, this problem has not yet been adequately solved.

To indicate biological pathogens, immunochemical methods of determination are successfully used. Including such modern highly specific tests as enzyme-linked immunosorbent assay (ELISA), latex agglutination, dot blot analysis, immunoblotting and immunomagnetic separation.

The basis for all immunoassay options are antibodies. The specificity of immunological reactions is directly dependent on the specificity of the antibody used to prepare the diagnostic drug. Diagnostics prepared on the basis of MCA are strictly specific, since they are aimed at identifying one antigen.

Therefore, one of the important approaches in the detection of anthrax spores in environmental objects is the use of MCAs produced by hybridomas.

Obtaining hybridomas, producers of highly specific MABs for B. anthracis spores is an extremely difficult task. The spores of B. anthracis have an extra layer - the exosporium, which is also present in some other closely related bacilli, such as Bacillus cereus and Bacillus thuringiensis. The exosporiums of these bacteria are similar in structure and have a high degree of homology [1]. In its composition, exosporium contains more than 130 different antigens, which are a complex mixture of proteins, lipids and carbohydrates. One of the main antigens of exosporium is the collagen-like glycoprotein BclA (Bacillus collagen-like protein of anthracis). According to the literature, BclA is an immunodominant antigen of the B. anthracis exosporium, i.e. most antibodies produced against spores interact with BclA [2].

Hybridoma strains are known (for example, EA-4-10-4 hybridoma) producing monoclonal antibodies obtained against spores of B. anthracis Sterne strain that interacted with purified recombinant BclA [2].

However, the sequences of BclA in closely related bacilli (B. anthracis, B. cereus, and B. thuringiensis) are homologous [3], which can lead to false-positive results of the detection of anthrax spores when using antibodies to this protein.

The objective of the invention is to obtain a strain of hybrid cultured cells that produce monoclonal antibodies specific for B. anthracis spores, but not interacting with BclA and suitable for constructing test systems on their basis for identifying the causative agent of anthrax in spore form.

The problem is solved by the fact that a new strain of hybrid cultured animal cells of Mus musculus 1E6 is proposed - a producer of highly specific monoclonal antibodies to B. anthracis spores that do not interact with recombinant BclA.

The technical result of the invention is the successful use of monoclonal antibodies produced by the proposed strain for the specific detection of B. anthracis spores by latex agglutination methods (example 8) and immunomagnetic separation followed by enzyme immunoassay (example 9) with a sensitivity of 2 × 10 ⁶ and 1 × 10 ³ spore in one ml, respectively.

The strain of hybrid cultured animal cells Mus musculus 1E6, a producer of highly specific monoclonal anti-spore antibodies B. anthracis, was deposited in the collection of microorganisms of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology (FGUN SSC BCH) collection number - H10.

The hybridoma strain was obtained by immunization of BALB / c mice. Mice were immunized according to the generally accepted method by double, with thirty-day exposure, subcutaneous administration of inactivated spores of B. anthracis strain STI-1. On the third day after the last booster injection, splenocytes of immune mice (1 × 10 ⁸ cells) were hybridized with mouse P3-X63 Ag / 8-653 mouse myeloma cells (1 × 10 ⁷ cells). Polyethylene glycol (Sigma, USA) was used as a fusion agent. After hybridization, selection, screening, cloning, and cryopreservation of the hybridoma were performed.

Characterization of hybridoma 1E6

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

Cultural properties. The cultivation of hybridoma strain 1E6 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. Passaging of 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).

The cultivation of hybridomas in the body of the animal. BALB / c mice of 20 weeks of age were treated with pristan (Sigma, United States) 0.5 ml intraperitoneally and after 2–3 weeks, 1 × 10 ¹⁰ hybrid cells were intraperitoneally injected. The appearance of immuno-ascites was recorded on days 7-15, and the selection of immuno-ascitic (IAG) fluids was performed on days 10-21. The synthesis of immunoglobulin produced by the hybridoma was determined by indirect ELISA [4].

The characteristic of a useful product. Hybridoma 1E6 produces specific monoclonal antibodies to B. anthracis spores, the titer of which is 1: 1000 in the culture fluid (QOL) and 1: 1,000,000 in the IAH. 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).

Strain productivity. MCA production in the cultivation medium is 20-50 μg / ml, in ascitic fluid - 5-10 mg / ml. MCA production in the culture fluid is maintained for 10 passages (observation period) and 5 passages during cultivation in the form of ascites tumors in mice (observation period).

Strain contamination. Hybrid line contaminants, including bacteria, yeast, fungi, were not detected. Mycoplasmas were not determined.

Cryopreservation. The freezing medium contains fetal calf serum - 90%, dimethyl sulfoxide - 10%.

Cryopreservation and heating mode. Hybrid cells are introduced into cryovials, placed in a foam container with a wall thickness of at least 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 quickly in a water bath at a temperature of 37 ° C.

Ampoules contain 1.0 ml of cryoprotective medium with a concentration of hybrid cells of 1 × 10 ⁶ . The viability of restored hybridomas after cryopreservation is 75-85%.

Properties of a useful product. The hybridoma produces monoclonal antibodies belonging to the IgG1 subclass of mouse immunoglobulins; affinity constant for MCA 1E6 is 3.9 × 10 ^-8 M. MCAs are specific for B. anthracis spores.

The invention is illustrated by the following graphic materials.

Figure 1 - Dot blot analysis of MCA 1E6 with cells of heterologous microorganisms.

Figure 2 - Dot blot with MKA 1E6. Determination of nonspecific activity against spore-forming representatives of the genus Bacillus.

Figure 3 - Dot blot analysis of MKA 1E6 with spores of B. anthracis strains. Determination of specific intraspecific (B. anthracis) activity.

Figure 4 - SDS-PAGE electrophoresis of MKA 1E6.

Figure 5 - Determination of subclass affiliation MCA 1E6.

6 - Immunoblotting MCA 1E6 with recombinant glycoprotein BclA exosporium strain B. anthracis STI-1.

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

Example 1. Obtaining hybridoma 1E6

Immunization

BALB / c mice are immunized with spores of B. anthracis STI-1 strain according to the following scheme:

- subcutaneous administration to mice of inactivated spores in a concentration of 1 × 10 ⁷ spores in complete Freund's adjuvant;

- after 30 days, reintroduction of mice with inactivated spores at a concentration of 1 × 10 ⁷ spores in incomplete Freund's adjuvant;

- after 30 days, an intravenous injection into mice of inactivated spores in a concentration of 1 × 10 ⁷ spores in physiological saline.

Blood was collected from immune mice on the third day after the last injection with inactivated spores at a concentration of 1 × 10 ⁷ spores 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

3 days after the last intravenous injection, the mouse spleen is removed and 1 × 10 ⁸ mouse splenocytes are hybridized with 1 × 10 ⁷ cells of the P3-X63 Ag / 8-653 myeloma line in the presence of 1 ml of polyethylene glycol (SIGMA, USA) for 1 minute.

Selection

After washing the polyethylene glycol, the cells were plated on 96-well plates on a layer of peritoneal macrophages taken from BALB / c mice. Selection of hybrid cells is carried out on a selective medium containing hypoxanthine-aminopterin-thymidine (HAT). After 21 days, aminopterin is removed from the medium. Subsequently, the cultivation is carried out on RPMI - 1640 Sigma medium with 20% fetal calf serum, 2 mM / L-glutamine, 100 μg / ml gentamicin.

Hybrid Clone Screening

Indirect solid-state ELISA is used to select positive hybrid clones producing MABs.

To conduct an indirect ELISA, 1 × 10 ⁶ spores / well of B. anthracis STI-1 strain are added to the wells of a 96-well polystyrene plate for ELISA and incubated at 65 ° C for 3 hours. Then, 25 μl are added to the wells 96 ° C ₂ H ₅ OH and incubated at a temperature of 65 ° C for 25 minutes. 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. Three times washed with phosphate-buffered saline pH 7.4 containing 0.5% tween-20 (PBS-T), the supernatant of the culture fluid was added to the wells in a volume of 100 μl and incubated at 37 ° C for 1 hour. After this, 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. After that, 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-citrate buffer pH 5.0) is 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 (Picon photometer, RF) for ELISA at a wavelength of 492 nm.

Cloning

Cloning of hybrid cells is carried out 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

Hybridoma clones are cultured 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

Hybridoma clones 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. Determination of nonspecific activity against microbial cells of other microorganisms by dot blot analysis

To conduct this study, a panel of 22 strains of microorganisms Y. pestis EV, Klebsiella pneumoniae 963, Y. enterocolitica 287, Fr. tularensis 15, Str. pneumonia 7465, Str. pyogenes 8198, Staphylococcus epidermidis 11047, Pseudomonas aeruginosa 10662, Legionella micadadeis 11371, Pasterella multocida 10322, M. tuberculosis H-37RV, C. jejuni ATCC 11168, Listeria monocytogenes, Brucella abortus vacc. Pcs. 19, Salm. paratyphi, Salm. typhimurium, Salm. Paratyphi A, Salm. enteritidis 204, Y. pseudotuberculosis, E. coli IM83, V. cholerae inab A25, V. cholerae Ogawa.

As a positive control, spores of B. anthracis STI-1 and M71 strains are used. On the nitrocellulose membrane (GE Water & Process Technologies, USA) using an BIO-DOT device (BIO RAD, USA), inactivated microbial cells are sorbed in concentrations from 1 × 10 ⁵ to 1 × 10 ⁷ spores / ml. Microorganism strains were obtained from the collection of microorganisms of the Federal State Institution of Science and Science of the Russian Academy of Medical Sciences. The membranes are then blocked with an inert protein solution and incubated sequentially with 1A6 mAb 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% diaminobsnzidine (Sigma, USA), 0.015% H ₂ O ₂ , 0.01 M phosphate-buffered saline, pH 7.4). The reaction is stopped by washing with distilled water.

As the analysis showed, MCA hybridomas 1E6 do not show cross activity with microbial cells of other microorganisms (lanes 1 to 22) and interact only with B. anthracis spores (lanes 23 and 24) (figure 1).

ICA specificity verification.

Example 3. Dot blot. Determination of nonspecific activity against spore-forming representatives of the genus Bacillus

For analysis on a nitrocellulose membrane (GE Water & Process Technologies, USA), inactivated bacillus spores in concentrations from 1 × 10 ⁵ to 1 × 10 ⁷ spores / ml are sorbed using a BIO-DOT device (BIO-RAD, USA). 21 strains of closely related spore saprophytes of the genus Bacillus (B. pantothenticus B-1398, B. larvae B-1400, B. polymyxa B-1559, B. thuringiensis B-214, B. natto B-193, B. alvei B -1555, B. brevis B-1567, B. megaterium B-1435, B. licheniformis B-1411, B. subtilis B-1405, B. antracoides B-217, B. pasteurii B-1962, B. cereus B- 160, B. sphaericus B-1152, B. maceraus B-1410, B. species B-1967, B. azotoformaus B-1956, B. pulvifaciens B-1402, B. firmus B-1994, B. laterosporus B-1404 , B. pumilis B-5007). Microorganism strains were obtained from the collection of microorganisms of the Federal State Institution of Science and Science of the Russian Academy of Medical Sciences. The membranes are then blocked with an inert protein solution and incubated sequentially with MKA 1E6 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).

As a positive control, spores of B. anthracis STI-1 and B. anthracis M-71 strains are used.

As a result of the analysis, it was found that 1A6 MCAs interact only with spores of B. anthracis at a concentration of 1 × 10 ⁵ spores / ml (lanes 22 and 23) and do not show nonspecific activity with respect to spores of other bacilli (lanes 1 through 21 ) (Fig.2).

Example 4. Determination of specific intraspecific (B. anthracis) activity

For analysis, inactivated bacillus spores in concentrations from 1 × 10 ⁵ to 1 × 10 ⁷ spores / ml are sorbed on a nitrocellulose membrane (GE Water & Process Technologies, USA) using a BIO-DOT device (BIO RAD, USA). The following are used in the work: 2 vaccines (B. anthracis STI-1, B. anthracis - 34F2), 4 attenuated strains (B. anthracis M-71, B. anthracis M-71/12, B. anthracis 228/8, B. anthracis 220). Microorganism strains were obtained from the collection of microorganisms of the Federal State Institution of Science and Science of the Russian Academy of Medical Sciences. The membranes are then blocked with an inert protein solution and incubated sequentially with 1A6 mAb 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)

Negative control - membranes treated with intact mouse serum in a 1/1000 dilution.

As a result of the analysis, it was found that MCA hybridomas 1E6 interact with all spores of the studied B. anthracis strains (lanes 2 through 7) (Fig. 3).

Example 5. Isolation and purification of MCA hybridoma 1E6

The allocation of MCA from ascitic fluid is carried out by affinity chromatography on a column of protein G-spharose (Protein G Sepharose 4 Fast Flow). Cellular components from the culture and ascitic fluids were removed by centrifugation (2000 g × 20 min), the pH of the culture fluid was adjusted to 8.6 using a 1 N NaOH solution, ascitic fluid was 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.

As a result, purified immunoglobulin preparations were obtained (FIG. 4).

Example 6. Determination of the subclass affiliation of the MCA hybridoma 1E6

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 determination of the subclass of MAB produced by hybridoma 1E6 showed that monoclonal antibodies belong to the IgG1 subclass of immunoglobulins (Fig. 5).

Example 7. Immunoblotting of MCA hybridomas 1E6 with recombinant glycoprotsin BclA exosporium strain B. anthracis STI-1

To verify the specificity of the MCA hybridoma 1E6 to the recombinant BclA glycoprotein of the exosporium strain B. anthracis STI-1, SDS-PAGE electrophores was performed on 10% polyacrylamide gel (PAG) for 1 hour at 220 V, 20 mA. Proteins from PAG are transferred to a nitrocellulose membrane (GE Water & Process Technologies, USA) for 1 hour at a current of 380 mA. After the transfer is complete, the membrane is blocked by immersing in a 1% BSA solution in FSB pH 7.4 for 1 hour at room temperature. For washing the membranes using FSB-T pH 7.4. Blocked and washed membranes are cut into strips and each stripe is incubated separately in solutions of MCA hybridomas 1E6, with Anti-His antibody (Sigma, USA) and intact mouse serum for 1 hour at 37 ° C. Further, after washing with a buffer, the strips are incubated with a peroxidase conjugate to the total mouse Ig fraction (Sigma, USA) in working dilution under the same conditions, washed with FSB-T. Visualization of the reaction is carried out 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-saline buffer - FSB, pH 7.4). The reaction is stopped by washing with distilled water.

Negative control - 1/1000 dilution of intact mouse serum. Positive control Anti-His antibody (Sigma, USA).

Monoclonal antibodies produced by hybridoma 1E6 do not interact in the immunoblot with the recombinant glycoprotein Bc1A of the exosporium strain B. anthracis STI-1 (Fig.6).

Example 8. Detection of Bacillus anthracis spores in latex agglutination reaction

For the formulation of the latex agglutination reaction (RLA), A-63-21 latex particles (Academician M.M.Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia) and 1A6 hybridoma are used. The synthesized monodisperse acrolein particles had an average diameter of 1180 nm. The immobilization of MCA on latex particles is carried out according to the manufacturer's protocol: to 50 μl of a 10% latex suspension add 100 μl of MCA hybridoma 1E6 with a concentration of 1 mg / ml, bring the volume to 0.5 ml and incubate for 2 hours at room temperature with stirring. To block unreacted groups add 4 ml of a 1% solution of ovalbumin (Sigma, USA) in 0.1 M borate buffer (BB), pH 8.2. Latex particles are washed from unbound MCA in the BB by three-fold 10-minute centrifugation at 1200 g. The pellet was resuspended in 5 ml BB with 1% ovalbumin.

The latex agglutination reaction is carried out in round-bottom 96-well plates with a U-shaped profile of the wells (company Pan Eco, Russia). As control strains, spores of the strains are used: B. anthracis 71/12, B. anthracis M-71, B. anthracis STI-1. For specificity control, B. licheniformis B-1411, B. subtilis B-1405, B. antracoides B-217, B. megaterium B-1435, B. cereus 160 strains were used. As a negative control, 0.1 M borate buffer was used, pH 8.2.

Dilution of spores of microorganism strains with step 2 is prepared in a volume of 25 μl in round-bottom 96-well plates in borate buffer. After adding 25 μl of latex particles with immobilized MCA hybridomas 1E6 to all wells, the plate is shaken and incubated for 1.5-2.0 hours at room temperature.

The results are taken into account visually using a four-cross system on a light background in good light.

4+ - all latex particles are agglutinated, uniformly cover the bottom of the hole, forming an "umbrella".

3+ - almost all latex particles are agglutinated, which is reflected in a decrease in the diameter of the "umbrella".

2+ - half of the latex particles are agglutinated.

1+ - most particles are not agglutinated and settled on the bottom of the hole, but the diameter of the “button” is still different from the “point”.

“-” - there are no signs of agglutination, latex particles settled on the bottom of the hole in the form of a “dot” of bright blue in the center of the hole.

A positive reaction is considered to be rated at least 3+.

As a result of the latex-agglutination reaction, it was found that latex particles with immobilized MCA 1E6 detect spores of anthrax strains at a concentration of 4.5 × 10 ⁵ -1.8 × 10 ⁶ spores / ml and do not reveal heterologous cultures of the genus Bacillus at a concentration of 2 × 10 ⁸ spores / ml and below (Fig.7).

Example 9. Identification of spores of Bacillus anthracis by ELISA using magnetic particles with immobilized MCA hybridoma 1E6.

Magnetic immunosorbents (MIS) developed using the method described in the invention (Pat. RF. No. 2138813, G-01, No. 33/543, dated September 27, 1999, Bull. No. 27) are used for ELISA using magnetic particles. The activation of magnetic particles is carried out by oxidation using sodium perchlorate (ChemMed, RF) as follows: 3 ml of distilled water containing 0.20 g of sodium perchlorate is poured into 0.5 g of magnetic particles. Incubated at a temperature of (22 ± 4) ° C for 1 hour in the dark. Then the magnetic particles are washed with buffered saline solution (PBS) until the extinction value is absent on a spectrophotometer at a wavelength of 280 nm. The immobilization of MCA hybridoma 1E6 on magnetic particles is carried out as follows: 2 ml of MCA hybridoma 1E6 with a concentration of 2 mg / ml is added to 0.4 g of activated magnetic particles. Incubated for 2 hours at a temperature of (22 ± 4) ° C. For ELISA, immunoproxidase conjugates are prepared by periodic oxidation according to PKNakane, A. Kawaoi [5]: 1 ml of 0.3 M sodium bicarbonate solution and 0.025 ml of 32% formalin are added to 5 mg of horseradish peroxidase, incubated mixture on a shaker for 30 minutes. Next, 1 ml of sodium periodate was added to the reaction mixture and incubated on a shaker for 30 minutes. Then, 1 ml of 0.16 M ethylene glycol was added to the reaction mixture and incubated for 1 hour on a shaker. Next, the reaction mixture was dialyzed overnight at + 4 ° C against 0.01 M carbonate-bicarbonate buffer, pH 9.6. After dialysis, 1 ml of MCA 1E6 at a concentration of 5 mg / ml is added to activated horseradish peroxidase and incubated on a shaker for 2 hours, 5 mg of sodium borohydride are added and incubated in the dark for 2 hours at + 4 ° C. Then dialyzed night against 0.1 M PBS. After dialysis, BSA (10 mg BSA per 1 ml conjugate) is added.

ELISA using magnetic particles with immobilized MCA 1E6 (MIS). 50 μl of 10% suspension of MIS is added to eppendorfs. Then, 200 μl of FSB with albumin-tween (USB, USA) (FSB-AT) is added to eppendorf with a negative control, 200 μl of suspensions of killed cultures of anthrax microbe and strains of heterologous microorganisms are added to other eppendorfs. All suspension eppendorfs are incubated at a temperature of (37 + 1) ° C for 40 minutes. After incubation, the liquid is removed by holding the eppendorfs with a permanent magnet (Invitrogen, Norway), and the MIS is washed twice with phosphate-buffered saline with tween (FSB-T). Then, 200 μl of working dilution of the conjugate are added to each eppendorf. The eppendorfs with the conjugate are incubated at a temperature of (37 + 1) ° C for 15 min and washed with FSB-T 6 times. After that, 200 μl of substrate-indicator solution is added to the eppendorfs. Take into account the color change of the solutions in eppendorfs for 1-3 minutes. The supernatant is transferred 100 μl into the wells of a polystyrene plate for enzyme immunoassay (Table 1), holding eppendorf with a permanent magnet, and the reaction is stopped by adding 50 μl of a 4 N sulfuric acid solution (ChemMed, RF). The results are taken into account on a photometer (Picon photometer, RF) for ELISA at a wavelength of 492 nm. Positive are considered the results if the optical density of the samples was 2 or more times higher than the optical density of the negative control.

As control strains, spores of strains are used: B. anthracis 71/12, B. anthracis 228/8, B. anthracis STI-1 in concentrations from 1.0 × 10 ⁷ spores / ml to 1.0 × 10 ² spores / ml . To control specificity, spores of strains are used: B. cereus 160, B. megaterium 2, B. cereus 19, B. cereus 16, B. cereus 8035 B. subtilis 37, B. subtilis 83 B. megaterium 5, B. megaterium 89, B. cereus 104, B. cereus 250, in concentrations from 1.0 × 10 ⁵ spores / ml to 1.0 × 10 ⁴ spores / ml.

As a result of the tests, it was found that magnetic particles with immobilized MCA 1E6 reveal spores of anthrax strains at concentrations of 1 × 10 ² -1 × 10 ³ spores / ml and do not reveal control heterologous cultures of the genus Bacillus in spore form at a concentration of 1.0 10 ⁵ m.k. / ml and below when using the conjugate in a working dilution of 1: 600 (Table 2).

Information sources

1. Quinlant J. J. and Foegeding P. M. Monoclonal Antibodies for Use in Detection of Bacillus and Clostridium Spores. // Appl. Environ. Microbiol. - 1997. - V.63. - p. 482-87.

2. Christopher Steichen, Ping Chen, John F. Kearney, and Charles L. Turnbough, Jr. Identification of the Immunodominant Protein and Other Proteins of the Bacillus anthracis Exosporium. // Bacteriology, March 2003. - V.185, No. 6. - p. 1903-1910.

3. Tomas A. Lcski, Clayton C. Caswell, Marcin Pawlowski, David J. Klinke, Janusz M. Bujnicki, Sean J. Hart, and Slawomir Lukomski. Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting. // Applied and Environmental Microbiology, Nov. 2009, p.7163-7172.

4. Egorov A.M., Osipov A.P., Dzantiev B.B., Gavrilova E.M. Theory and practice of enzyme immunoassay. / T 33 - M .: Higher. Shk., 1991 .-- S.174-175.

5. Nakane P.K., Kawaoi A. Peroxidase-labelled antibody-a new method of conjugation. // J. Histochem. Cytochem. - 1974. - V.22, N 4. - P.506-508-1084-1091.

Table 1 The scheme of introduction of the test material (suspension, positive and negative controls) Hole number Suspend B. anthracis STI-1, spore / ml Suspend B. anthracis 71/12, spore / ml Suspend B. anthracis 228/8, spore / ml Suspend heterologous spore strains / ml one 2 3 four 5 6 BUT form - - 1.0 × 10 ⁵ B. subtilis 37 1.0 × 10 ⁵ B. cereus 104 1.0 × 10 ⁵ B. cereus 19 AT 1.0 × 10 ² 1.0 × 10 ² 1.0 × 10 ² 1.0 × 10 ⁴ B. subtilis 37 1.0 × 10 ⁴ B. cereus 104 1.0 × 10 ⁴ B. cereus 19 FROM 1,0 × 10 ³ 1,0 × 10 ³ 1,0 × 10 ³ 1.0 × 10 ⁵ B. subtilis 83 1.0 × 10 ⁵ B. cereus 250 1.0 × 10 ⁵ B. cereus 16 D 1.0 × 10 ⁴ 1.0 × 10 ⁴ 1.0 × 10 ⁴ 1.0 × 10 ⁴ B. subtilis 83 1.0 × 10 ⁴ B. cereus 250 1.0 × 10 ⁴ B. cereus 106 E 1,0 × 10 ⁵ 1,0 × 10 ⁵ 1,0 × 10 ⁵ 1.0 × 10 ⁵ B. megaterium 5 1.0 × 10 ⁵ B. cereus 160 1.0 × 10 ⁵ B. megaterium 2 F 1.0 × 10 ⁶ 1.0 × 10 ⁶ 1.0 × 10 ⁶ 1.0 × 10 ⁴ B. megaterium 5 1.0 × 10 ⁴ B. cereus 160 1.0 × 10 ⁴ B. megaterium 2 G 1.0 × 10 ⁷ 1.0 × 10 ⁷ 1.0 × 10 ⁷ 1.0 × 10 ⁵ B. megaterium 89 1.0 × 10 ⁵ B. cereus 8035 - N Denied. the control Denied. the control Denied. the control 1.0 × 10 ⁴ B. megaterium 89 1.0 × 10 ⁴ B. cereus 8035 -

table 2 The results of spectrometric processing of ELISA one 2 3 four 5 6 7 8 9 10 eleven 12 BUT 0,000 - - 0.145 0.132 0.181 AT 0.385 0.378 0.401 0.136 0.129 0.169 FROM 0.527 0.590 0.562 0.158 0.199 0,111 D 0.589 0.601 0.628 0.154 0.187 0.142 E 0.610 0.627 0.679 0.133 0.132 0.164 F 0.715 0.694 0.764 0.128 0.146 0.170 G 0.801 0.842 0.879 0.175 0.161 - N 0.160 0.127 0.149 0.150 0.153 -

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 1E6, a producer of monoclonal antibodies specific for Bacillus anthracis spores, was deposited in the microorganism collection of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology (FGUN SSC BCH), collection number H10.

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