RU2460787C1 - Hybrid cell strain of mus musculus animals 10g4 - producer of monoclonal antibodies specific to capsular f1 antigen yersinia pestis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a hybrid cell strain of Mus musculus 10G4 deposited in the collection of microorganisms Federal State Research Institution State Research Centre for Applied Microbiology and Biotechnology, No. N-13.

EFFECT: strain may be used for producing monoclonal antibodies to capsular F1 antigen Yersinia pestis and is applicable for constructing the based plague agent test systems.

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Description

The invention relates to biotechnology and can be used to obtain monoclonal antibodies (MCAs) to the capsular F1 antigen of Yersinia pestis.

The causative agent of plague (Y. pestis) is one of the most virulent microorganisms for humans and the only representative of bacteria related to pathogens of the first (highest) hazard class.

Having an unlimited natural reservoir in rodents, the plague still remains an undefeated disease, outbreaks of which are regularly recorded in different parts of the globe.

About 2,000 cases of plague are reported each year, and the World Health Organization has recently recognized plague as a newly circulating disease [1].

The regularity of the epidemiological process is determined, on the one hand, by its nature as a natural focal transmissible infection with peculiar features of the course of epizootics among various species of rodents, and on the other, by factors contributing to the manifestation of the anthroponous transmission mechanism. In natural foci, the plague manifests itself in the form of epizootics among more than 200 species of rodents and rabbit-like world fauna. Plague is transmitted between them in a transmissible and probably alimentary way [2].

The plague microbe is unstable outside the body of warm-blooded animals and ectoparasites and is sensitive to the effects of physical, chemical and biological factors. The plague pathogen tolerates low temperature much better, withstanding three times freezing and thawing.

Under favorable temperature conditions, humidity and the absence of competing microbes, the plague microbe remains viable in water for a month, milk for up to 3 months, in the ground for up to 2 months. In the dried sputum of the plague sick, the microbes survive for a week, in the wet - more than a month (at low temperatures). In infected and starving fleas - up to 396 days [3].

One of the most labile properties of the plague pathogen is the synthesis of capsular antigen F1. This symptom is directly dependent on the nature of the epizootic process: as the epizootic fades, an increase in the number of cultures with a reduced ability to synthesize capsular antigen, up to its complete loss. This correlates with an increase in the number of seropositive animals with respect to F1 [4].

The Y. pestis EV strain of the NIIEG line has an increased substrate specificity and, at the same time, a low growth rate at an optimum temperature for synthesis of capsular antigen of -37 ° C [5].

Classical plague immunodiagnostics is based on the detection of capsular F1 antigen or anti-F1 antibodies.

Practical healthcare urgently needs certified modern immunodiagnostic test systems for indicating plague pathogens. In connection with the development of immunochips and multiparameter diagnostic systems, great importance is attached to the production of specific immunoreagents, primarily monoclonal or monoreceptor polyclonal antibodies.

The specificity of immunological reactions is directly dependent on the specificity of the antibody used to prepare the diagnostic drug. Diagnostics based on monoclonal antibodies (MCAs) are strictly specific because they are aimed at identifying a single antigen.

The objective of the invention is to obtain a strain of hybrid cultured cells that produce monoclonal antibodies specific for the capsular F1 antigen of Y. pestis and suitable for constructing test systems based on them to detect the plague pathogen.

The problem is solved by the fact that a new strain of hybrid cultured animal cells of animals Mus musculus 10G4 is proposed - a producer of highly specific monoclonal antibodies to the capsular F1 antigen Y. pestis.

The strain of hybrid animal cells Mus musculus 10G4 was deposited in the state collection of pathogenic microorganisms and cell cultures of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology (FGUN SSC BCH) collection number - H13.

Mus musculus 10G4 animal hybrid cell strain is obtained by immunization of BALB / c mice. Mice are immunized with 4 times the introduction of a recombinant antigenic drug F1 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). Polyethylene glycol (Sigma, USA) is used as a fusion agent. After hybridization, selection, screening, cloning, and cryopreservation of the hybridoma are performed.

Characterization of the strain of hybrid cells of animals Mus musculus 10G4

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 10G4 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 10G4 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 10G4 hybridoma in an animal.

20-week-old BALB / c mice were treated with a pieran (Sigma, USA) 0.5 ml intraperitoneally and after 2–3 weeks 1 × 10 ¹⁰ hybrid cells were intraperitoneally injected. The appearance of immunoascites is recorded on the 7-15th day, the selection of the immunoascitic (IAJ) fluid is performed on the 10-21th day. The synthesis of immunoglobulin produced by a hybridoma is determined by indirect enzyme-linked immunosorbent assay (ELISA) [6].

The characteristic of a useful product. The mus musculus 10G4 animal hybrid cell strain produces specific monoclonal antibodies to the capsular F1 antigen Y. pestis, the titer of which is 1: 640000 in the culture fluid (QL) and 1: 1,000,000 in the IAG. 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 10G4. 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).

Contamination of the strain of hybrid cells of animals Mus musculus 10G4. 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.

The invention is illustrated by the following graphic materials.

Figure 1 Dot blot with MCA 10G4. Determination of nonspecific activity against microbial cells of other microorganisms.

Figure 2 Dot blot with MCA 10G4. Determination of specific activity for the capsular F1 antigen of Y. Pestis.

Figure 3 SDS-PAGE electrophoresis of MCA 10G4.

Figure 4 Definition of subclass affiliation MCA 10G4.

Figure 5 Immunoblotting MCA 10G4 with capsular F1 antigen Y. pestis.

6 Detection of capsular F1 Y. pestis in latex agglutination reaction.

Fig. 7 Detection of capsular F1 antigen of Y. pestis by ELISA using 10K4 MCA.

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

Example 1. Obtaining a strain of hybrid cells 10G4

Immunization

For immunization, BALB / c mice aged 3-4 months are used. Immunization is carried out according to the following scheme:

0 day - 100 μg of antigen in PBS emulsified in PAF (0.2 ml) subcutaneously;

21 days - 100 μg of antigen in PBS emulsified in NAF (0.2 ml) subcutaneously;

51 days - 100 μg of antigen in PBS intravenously;

58 day - 100 μg of antigen in PBS intravenously;

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

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 are 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 μg / well of capsular F1 Y. pestis antigen is added to the wells of a 96-well polystyrene plate for ELISA and incubated at 37 ° C for 1 hour or at 4 ° C for 18 hours. Three times washed with phosphate-buffered saline pH 7.4, containing 0.5% tween-20 (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. Wash FSB-T three times, add the supernatant of the culture fluid to the wells in a volume of 100 μl and incubate 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 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 of 10G4 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 10G4 hybrid cells 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 10G4 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. Determination of nonspecific activity against microbial cells of other microorganisms by dot blot analysis

To conduct this study, a panel of 37 microorganisms of various species was used. As a positive control, the capsular F1 antigen of Y. pestis was used. Inactivated microbial cells were adsorbed at a concentration of 1 × 10 ⁶ cells / L on a nitrocellulose membrane (GE Water & Process Technologies, USA) using a BIO-DOT device (BIO RAD, USA). 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 were then blocked with an inert protein solution and incubated sequentially with 10G4 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, United States), 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 10G4 specifically interacted with the F1 antigen of Y. pestis and did not give cross-reactions with other closely related yersinia (Fig. 1).

Checking the specificity of MCA 10G4

Example 3. Determination of specific activity to F1 antigen of Y. pestis

The specific activity of QOL 10G4 hybridomas against a capsular F1 antigen of Y. pestis was tested by dot blot analysis. An F1 antigen with an initial concentration of 0.025 μg / well was sorbed onto a nitrocellulose filter using a Bio-Dot device (Bio-Rad, USA) and titrated in two steps to a final concentration of 0.0002 μg / well. Next, the membrane was blocked with an inert protein solution and incubated sequentially with 10G4 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, United States), 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 10G4 detect up to 7 ng / ml F1 antigen (figure 2).

Example 4. Isolation and purification of MCA 10G4

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 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. 3).

Example 5. The definition of subclass affiliation MCA 10G4

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 10G4 produces monoclonal antibodies belonging to the IgG1 subclass of mouse immunoglobulins (figure 4).

Example 6. Immunoblotting MCA 10G4 with capsular F1 antigen Y. pestis

To test the specificity of MCA 10G4 with the Y. pestis capsular F1 antigen, SDS-PAGE electrophoresis was performed on 10% polyacrylamide gel (PAGE) for 1 hour at 220 V, 20 mA. F1 from PAGE is 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. The blocked and washed membrane is incubated in a solution of the 10G4 hybridoma MCA for one hour at a temperature of 37 ° C. Further, after washing with a buffer, the membrane is incubated with a peroxidase conjugate to the total mouse Ig fraction (Sigma, USA) in working dilution under the same conditions, washed with PBS-T. The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, United States), 0.015% H ₂ O ₂ , 0.01 M phosphate-buffered saline - FSB, pH 7.4). The reaction is stopped by washing with distilled water.

As a result of the analysis, it was found that MCA 10G4 interact with the capsular F1 antigen Y. pestis (Fig. 5).

Example 7. Detection of capsular F1 Y. pestis in latex agglutination reaction

For the formulation of the latex-agglutination reaction (RLA), polyacrolein latex particles with a diameter of 1 and 1.2 μm are used (Institute of Bioorganic Chemistry named after Academician M.M.Shemyakin and Yu.A. Ovchinnikov RAS, Moscow) and ICA 10G4. 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 10G4 with a concentration of 1 mg / ml, the volume is adjusted to 0.5 ml and incubated 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 96-well plates with a V-shaped profile of the wells (company Pan Eco, Russia). As a control, capsular F1 antigen and cells of the Y. pestis EV strain NIIEG grown at 37 ° C are used. To control specificity, the following strains were used: Y. pestis EV NIIEG (grown at 28 ° C), Y. enterocolitica 287, Y. pseudotuberculosis III, S. enteritidis 1127, S. typhi H-901, Sal. paratyphi A-225, Sh. disenteriae 1362, E. coli ATCC 25922, E. coli 1282 GISC, L. pneumophila ATCC-33152, P. alcalifaciens 1068-50, P. aeruginosa ATCC27853.

As a negative control using 0.1 M borate buffer, pH 8.2.

Dilutions of cells of microorganism strains with step 2 are prepared in a volume of 25 μl in 96-well plates in borate buffer. After adding 25 μl of latex particles with immobilized MCA 10G4 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 covering the bottom of the well, 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 color 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 10G4 reveal F1 antigen at a concentration of 0.0038 μg / ml and cells of Y. pestis EV strain NIIEG (at 37 ° C) at a concentration of 3.6 × 10 ⁵ cells / ml and do not reveal heterologous cultures of microorganisms at a concentration of 5 × 10 ⁸ cells / ml and below (Fig.6, Fig.7, table 1, table 2, table 3).

Example 7. Detection of capsular F1 antigen of Y. pestis by ELISA using MCA 10G4

MCA 10G4 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. F1 antigen was titrated twice in a horizontal step with an initial concentration of 0.1 μg / well in FSB to a final concentration of 0.000097 μg / well in FSB (100 μl / well in FSB), incubated under the same conditions, and then washed plate 4 times in the FSB-T. Biotinylated MCA 13F8 (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, 100 μl of the substrate-indicator mixture were added to the wells (0.008 mg of orthophenylenediamine in 10 ml of phosphate-citrate buffer, pH 5.0 with 1 μl / ml 33% H ₂ O ₂ ). The plate was placed for 3-5 min in a dark place at room temperature, then the reaction was stopped with 4N sulfuric acid (50 μl / well). The results were taken into account using an xMark plate spectrophotometer (Bio-Rad, USA) at a wavelength of 492 nm.

As a result of the analysis, it was found that for immobilized MCA 10G4, the most optimal concentration is 5 μg / well, which allows you to confidently determine the minimum amount of F1 (4 ng / ml) (table 4).

Table 1 Latex-agglutination reaction results one 2 3 four 5 6 7 8 9 10 eleven 12 BUT 4+ 4+ 4+ 4+ 4+ 4+ 3+ 3+ 3+ 3+ 3+ -

table 2 Latex-agglutination reaction results one 2 3 four 5 6 7 8 9 10 eleven 12 BUT 1+ 1+ 1+ - - - - - - - - - AT 4+ 4+ 4+ 4+ 4+ 3+ 3+ 2+ 2+ - - -

Table 3 Latex-agglutination reaction results one 2 3 four 5 6 7 8 9 10 eleven 12 BUT - - - - - - - - - - - - AT - - - - - - - - - - - - FROM - - - - - - - - - - - -

Information sources

1. Bleves, S, Cornelis, G.R. (2000) How to survive in the host: the Yersinia lesson. Microbes Infect 2.

2. Domaradsky I.V. Plague, Moscow, "Medicine", 1998

3. Sanitary rules 3.4./4.2. 19-30 - 2005 2005 “Prevention of people with plague. Laboratory diagnosis of plague. "

4. Punsky E.E., Adamenko N.I. // Medical and geographical problems of the arid zone. - Ashgabat, 1982.- P.36-38.

5. Guidelines for the prevention of plague. / Ed. Naumova A.V., Samoilova L.V. - Saratov, 1992 .-- 278 p.

6. Egorov A.M., Osipov AP, Dzantiev BB, Gavrilova EM Theory and practice of enzyme immunoassay. / T. 33 - M .: Higher. Shk., 1991 .-- P.174 - 175.

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 10G4 is a producer of monoclonal antibodies specific for the capsular F1 antigen Yersinia pestis, deposited in the microorganism collection of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology under the number H-13.

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