RU2525663C1 - STRAIN OF HYBRID CULTURED ANIMAL CELLS Mus musculus 2F9-PRODUCER OF MONOCLONAL ANTIBODIES SPECIFIC FOR LYSOSTAPHIN AND INHIBITING ITS LYTIC ACTIVITY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: new strain of hybrid cultured animal cells Mus musculus 2F9 - producer of monoclonal antibodies specific for lysostaphin inhibiting its lytic activity and suitable for its quantitation is proposed. High-affinity monoclonal antibodies (mAb) of hybridoma 2F9 enables to determine lysostaphin in the samples to the concentrations of 0.8 ng/ml. The mAb of hybridoma 2F9 can be used to study the structural and functional properties of lysostaphin and quality control of preparations based on lysostaphin.

EFFECT: improved properties.

3 dwg, 1 tbl, 5 ex

Description

The invention relates to biotechnology and can be used to obtain monoclonal antibodies (MCAs) to lysostaphin.

Lysostaphin, a zinc-dependent glycyl-glycine endopeptidase produced by Staphylococcus simulans cells, is specific for bacteria of the Staphylococcus species. aureus. The molecular weight of lysostaphin is 27 kD. Lysostaphin destroys the cell wall of S. aureus bacteria. Lysostaphin is non-toxic and weakly immunogenic for animals and is considered as a promising component of antibacterial drugs for the treatment of infections caused by staphylococci, in particular for the treatment of endocarditis caused by antibiotic-resistant S. aureus strains. Lysostaphin is the only drug that can destroy the biofilms of bacteria S. aureus and S. epidermidis. Lysostaphin is active against all antibiotic-resistant strains of pathogenic staphylococci, including MRSA strains, and is superior in effectiveness to other anti-staphylococcal biological preparations - bacteriophages, bacteriocins and phage endolysins.

Recombinant lysostaphin practically does not differ in properties from the active form of lysostaphin from bacteria S. simulans. All clinical strains of S. aureus are sensitive to recombinant lysostaphin with a MIC from 0.03 to 2 μg / ml (Bastos M C F, Coutinho B G and Coelho MLV Lysostaphin: A Staphylococcal Bacteriolysin with Potential Clinical Applications Pharmaceuticals 2010, 3, 1139-1161 )

There is evidence of obtaining hybridomas (A1G5A1, A2C1D10 and L1F3), the monoclonal antibodies of which are specific for lysostaphin and were used to purify it using affinity chromatography. A hybridoma LTN1 is also known that produces MAB to a lysostaphin fragment between 3 and 13 amino acids: a peptide (THEHSAQWLN). MCAs LTN1 specifically bind to a 17 kDa lysostaphin hydrolysis product (Huang J., Wu H., Zhang J., Huang O. Purification of recombinant lysostaphin by monoclonal antibody affinity chromatography. [Chinese article] Sheng Wu Gong Cheng Xue Bao 2009 Jan; 25 (1): 147-51).

There is no information on hybridoma-producing MCAs that inhibit the lytic activity of lysostaphin.

The objective of the invention is to obtain a strain of hybrid cultured cells producing monoclonal antibodies specific for lysostaphin, inhibiting its antistaphylococcal activity and suitable for the quantitative determination of lysostaphin.

The problem is solved by the fact that a new strain of hybrid cultured animal cells of Mus musculus 2F9 animals is proposed - a producer of monoclonal antibodies specific for lysostaphin, inhibiting its lytic activity and suitable for its quantitative determination.

The strain is deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM - OBOLENSK", collection number - H-29. Depositor: Federal State Institution of Science "State Scientific Center for Applied Microbiology and Biotechnology" of the Federal Service for Supervision of Consumer Rights Protection and Human Well-being (FBUN SSC PMB).

The hybridoma strain is obtained by immunizing BALB / c mice with recombinant lysostaphin for 56 days. On the third day after the last booster injection, splenocytes of immune mice (10 ⁸ cells) are hybridized with cells of mouse myeloma PZ-X63 Ag / 8-653 (10 ⁷ cells). Polyethylene glycol with a molecular weight of 4000 (Merk, Germany) (Nowinski RC, Lostrom ME, Tam MR, Stone MR and Burnette WN The isolation of hybrid lines producing monoclonal antibodies against the p 15 (E) protein of ceotropic murine is used as a fusion agent Leukemia viruses. Virology, 1979, 93, 111-126.). After hybridization, selection, screening, cloning, and cryopreservation of the hybridoma are performed.

Characterization of a strain of hybrid cultured animal cells of Mus musculus 2F9 animals (hybridomas 2F9).

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

Cultural properties. The cultivation of the hybridoma strain 2F9 is carried out at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide. The cultivation medium is DMEM or RPMI - 1640 medium containing 10% fetal calf serum, 2 mM / L-glutamine, 50 μg / ml gentamicin. Cells are cultured as a stationary suspension in Costar plastic mattresses. Passage of hybridoma cells is carried out 2 times a week with a dilution ratio of 1: 2 - 1: 3. The inoculum concentration of cells is 10 ⁵ in 1 ml of medium. The maximum concentration of hybrid cells during cultivation is 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. Animal species - inbred mice of the BALB / c line. The dose of cells during vaccination is 10 ⁶ per mouse during the initial administration and 10 ⁵ per mouse during the secondary administration. 14 days before the introduction of hybridoma cells, mice were injected intraperitoneally with 0.5 ml of 2,6,10,14-tetramethylpentodecane (pristan). Immunoascitic fluid is formed on the 14-20th day in the volume of 3-5 ml. The synthesis of MCA produced by the hybridoma is determined by indirect enzyme-linked immunosorbent assay (ELISA).

The characteristic of a useful product. Hybridoma 2F9 produces specific monoclonal antibodies to lysostaphin, the titer of which is 1: 1000 in culture fluid (QOL), and 1: 100000 in immuno-ascitic fluid (IAG). Monoclonal antibodies from QOL and PAH are isolated by precipitation with ammonium sulfate followed by affinity chromatography on protein A or G sepharose. The homogeneity of the isolated antibodies is checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Strain productivity. MCA production in the cultivation medium is 20-50 μg / ml, in ascitic fluid - 2-4 mg / ml. MCA production in the culture fluid is maintained for 10 passages (observation period) and 5 passages when cultured in the form of ascites tumors in mice (observation period)

Strain contamination. Contaminants, including bacteria, yeast, fungi, were not detected in the hybrid line. The presence of microplasmas and viruses in the hybridoma was 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 10 ⁶ . The viability of restored hybridomas after cryopreservation is 65-75%.

Properties of a useful product. Hybridoma 2F9 produces monoclonal antibodies belonging to the IgG1 subclass of mouse immunoglobulins. MCAs are specific for native and recombinant lysostaphin and have an affinity constant of 1.72 × 10 ¹⁰ M ^-1 (Beatty JD, Beatty BG, Vlahos WG Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J. Immunol. Methods, 1987, 100, 1987, 100 , 173-179.), Inhibit the antistaphylococcal activity of lysostaphin and are suitable for its quantitative determination.

Evidence of the properties of MCA hybridomas 2F9 are presented in the following figures:

Figure 1. Enzyme-linked immunosorbent assay for the determination of lysostaphin in the analyzed sample using monoclonal antibodies hybridoma 2F9.

Figure 2. Inhibition of S. aureus culture growth on solid nutrient medium with lysostaphin in the presence of 1F5, 2F9 and 2G10 hybridomas.

Figure 3. Immunoblot of monoclonal antibodies hybridomas with lysostaphin preparations.

Example 1. Obtaining hybridoma 2F9.

Immunization

BALB / c mice are immunized with a recombinant lysostaphin preparation according to the scheme:

- subcutaneous administration to mice of 200 μg of lysostaphin at 4 points in complete Freund's adjuvant;

- after 28 days, reintroduction to mice of 100 μg of lysostaphin at 4 points in incomplete Freund's adjuvant;

- after 28 days, an intravenous injection in mice of 20 μg of lysostaphin in physiological saline.

Blood from immune mice is taken on the third day after lysostaphin injection. The titers of specific antibodies in animal sera are determined using solid-phase ELISA.

Hybridization.

After 3 days, the mouse spleen was removed and 10 ⁸ mouse splenocytes were hybridized with 10 ⁷ cells of the PZ-X63-Ag8.653 myeloma line in the presence of 1 ml of a 50% solution of polyethylene glycol with a molecular weight of 4000 (Merk, Germany) 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 DMEM "Sigma" with 15% fetal calf serum, 2 mm / L-glutamine, 50 μg / ml gentamicin.

Screening of hybrid clones.

For the selection of positive hybrid clones producing MAB, use the enzyme-linked immunosorbent assay.

For ELISA, 500 ng of lysostaphin, prepared in 0.01 M carbonate buffer pH 9.6 in a volume of 100 μl, is added to the wells of a 96-well polystyrene plate for enzyme-linked immunosorbent assay and then kept at 6 ° C for 18 hours. washed with a buffered saline solution containing 0.05% Tween-20 (PBS-Tw). Pipette 100 μl of a 0.5% solution of bovine serum albumin (BSA), tested for the absence of peroxidase activity, and incubated at 37 ° C for 45 minutes. Wash PBS-Tv three times and add supernatant of culture fluid to the wells in a volume of 100 μl. The panel is incubated at 37 ° C for 1 hour. After this, the wells of the tablet are washed three times with PBS-Tv solution and antibodies against mouse immunoglobulins labeled with horseradish peroxidase in working dilution are added to the wells. The plate is incubated at 37 ° C for 40 minutes, then the wells are washed 5 times with PBS-TV solution and 100 μl of tetramethylbenzidine are added to the wells. The reaction is stopped by adding 50 μl of 2 M sulfuric acid to the wells. The tablets are scanned on a Picon tablet reader (Russia), measuring absorbance at 450 nm.

Cloning.

Cloning of hybrid cells is carried out by the method of limiting dilutions in 96-well plates on a layer of mouse peritoneal macrophages at the rate of 10 ⁴ cells per well. Clone screening is performed by 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 DMEM or RPMI - 1640 medium containing 15% fetal calf serum, 2 mM / L-glutamine, 50 μg / ml gentamicin.

Cryopreservation.

Hybridoma clones are cryopreserved on a freezing medium consisting of calf embryonic serum - 90% and dimethyl sulfoxide - 10%. Hybridoma clones are stored in Dewar vessels with liquid nitrogen.

Example 2. Determination of the amount of lysostaphin in enzyme-linked immunosorbent assay.

To conduct a solid-phase ELISA, lysostaphin at a concentration of 4 μg, 800 ng, 160 ng, 32 ng, 6.4 ng, 3.2 ng, 1.6 ng and 0.8 ng are added to the wells of a 96-well polystyrene plate for enzyme-linked immunosorbent assay ml of 0.01 M carbonate buffer (pH 9.6) in a volume of 100 μl and incubated at a temperature of 6 ° C for 16 hours. The subsequent steps of the analysis are carried out similarly to the steps described for screening hybrid clones. Monoclonal antibodies are added to the wells in a volume of 100 μl with a concentration of 5 μg / ml. Negative control - 1/1000 dilution of intact mouse serum.

In ELISA, monoclonal antibodies of hybridoma 2F9 specifically interact with lysostaphin and determine up to 0.80 ng / ml of protein in the analyzed sample (figure 1).

Example 3. Inhibition of the lytic activity of lysostaphin in a suspension of bacteria staphylococcus MKA hybridoma 2F9.

The S. aureus overnight agar culture was suspended in PBS. Bacteria inactivate by heating the suspension at a temperature of 80 ° C for 1 hour. The heated suspension is diluted to a turbidity equal to an optical density of 0.32 at a wavelength of 590 nm.

90 μl of culture fluid hybrid 1F5 and 2F9 are mixed with 10 μl of lysostaphin at a concentration of 300 μg / ml. The mixture is incubated for 1 hour at a temperature of 37 ° C. After incubation, the mixture is added to 1 ml of an inactivated suspension of S. aureus with an optical density of 0.32. The hydrolysis of staphylococcus cells is carried out for 1 hour at a temperature of 37 ° C. After the reaction, the suspension was diluted with PBS twice and the absorbance was measured (table 1).

Monoclonal antibodies produced by 2F9 hybridoma, in contrast to 1F5 mAb, inhibit the lytic effect of lysostaphin in an inactivated suspension of S. aureus bacteria (table 1).

Example 4. Inhibition of growth inhibition of staphylococcus culture on a solid nutrient medium with lysostaphin MCA hybridoma 2F9.

The overnight agar culture of S. aureus was suspended in PBS until a turbidity equal to the optical density standard of 10 units. The resulting suspension was diluted 10 times with PBS and 0.2 ml of the diluted suspension was uniformly applied to nutrient agar LA, poured into a Petri dish. The thickness of the agar is 15 mm. 200 μl of culture fluid hybridomas 1F5, 2F9 and 2G10 are mixed with 20 μl of lysostaphin at a concentration of 300 μg / ml. The resulting mixture was successively diluted twice with hybridoma culture fluid to a concentration of lysostaphin 15 and 7.5 μg / ml. The mixtures are incubated for 1 hour at a temperature of 37 ° C. After incubation, paper filters are added to the lysostaphin titration tubes and, after wetting, they are applied to LA agar with seeded staphylococcus culture. The plates are incubated for 18 hours at 37 ° C.

Monoclonal antibodies produced by 2F9 hybridoma, in contrast to the 1A5 and 2G10 hybridomas, inhibit the bactericidal effect of lysostaphin when growing S. aureus bacteria on a solid nutrient medium (Figure 2).

Example 5. Immunoblot.

To test the specificity of the 2F9 MCA hybridoma to recombinant lysostaphin and lysostaphin produced by the bacteria S. Simulans, SDS protein electrophoresis was performed on 10% polyacrylamide gel (PAG) for 2 hours at 220 V, 20 mA. PAAG proteins are transferred onto a nitrocellulose membrane (Hybond-P) 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 20 mM Tris buffer, pH 7.4 for 1 hour at room temperature. For washing the membranes and preparing solutions of MCA and the conjugate of antibodies against immunoglobulins G mice with horseradish peroxidase, 20 mM Tris buffer, pH 7.4, containing 150 mM NaCl and 0.5% Tween 20. Blocked and washed membranes are cut into strips containing adsorbed fractions of lysostaphin preparations, and each lane is incubated separately in MCA solutions with a concentration of 1 μg / ml for 1 hour at a temperature of 37 ° C. Further, after washing with a buffer, the strips are treated with a solution of anti-mouse antibodies conjugated with horseradish peroxidase (Sigma) in working dilution under the same conditions, washed with a buffer and stained with diaminobenzidine using 0.5 mg / ml 3.3′-diaminobenzidine with 0.1 mg / ml NiCl ₂ , 0.1 mg / ml CoCl ₂ , 0.03% H ₂ O ₂ in PBS pH 7.2. The reaction is stopped by washing with distilled water.

Monoclonal antibodies produced by 2F9 hybridoma, according to the immunoblot, interact with native and recombinant lysostaphin (Fig. 3). Non-inhibiting lytic activity of lysostaphin MCA (hybridomas 1F5 and 2G10), in addition to the main band of lysostaphin (27 kD), three additional bands (23, 18 and 16 kD) in the recombinant protein and the 23 kD band in the natural protein are detected, and MCA 2F9 - three additional bands (21, 19 and 18 kDa) in both recombinant and natural proteins.

Thus, based on the presented examples, MCA 2F9 hybridomas are specific for lysostaphin and inhibit its lytic activity. High affinity 2F9 mAb hybridomas allow the determination of lysostaphin in samples to concentrations of 0.8 ng / ml. MCA hybridomas 2F9 can be used to study the structural and functional properties of lysostaphin and quality control of drugs based on lysostaphin.

Table 1 Inhibition of the lytic activity of lysostaphin MCA in a suspension of bacteria S. aureus. Mixture composition Optical density hybridoma culture fluid 2F9, suspension of bacteria S. aureus 0.16 hybridoma culture fluid 2F9, lysostaphin, S. aureus bacterial suspension 0.15 hybridoma culture fluid 1F5, S. aureus bacterial suspension 0.16 hybridoma culture fluid 1F5, lysostaphin, suspension of bacteria S. aureus 0.12 Hybridoma culture medium, S. aureus bacterial suspension 0.16 Hybridoma culture medium, lysostaphin, S. aureus bacterial suspension 0.12

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 2F9, a producer of monoclonal antibodies specific for lysostaphin, inhibiting its lytic activity and suitable for the quantitative determination of lysostaphin, was deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM - OBOLENSK", collection number H-29.

Images