RU2401303C1 - Animal mus musculus hybrid cell g10b6 clone l-producer of monoclonal diphtheria toxin antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is presented a new G10B6 hybrid cell clone producing monoclonal diphtheria toxin antibody in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of diphtheria toxin (manufactured by State Institution Gamaleya's Epidemiology and Microbiology Research Institure) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesizes monoclonal antibodies specifically reacting with diphtheria toxin. The antibody titre is 1:10000 1:20000 in the culture fluid, and 1:1000000 in ascitic. Antibodies can be used for designing immunobiological systems of diphtheria toxin detection exceeding available analogues in sensitivity, specificity and reproducibility.

EFFECT: invention can be used for creation of the diagnostic test-systems exceeding available analogues in sensitivity, specificity and reproducibility.

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Description

The invention relates to the field of microbiology and biotechnology, specifically to hybridoma technology, and is a clone of G10B6 hybrid cells of Mus Musculus L animals producing monoclonal antibodies (hereinafter: MKAT) to corynebacter toxin causative agent of diphtheria in cell cultures and the abdominal cavity of syngeneic animals (hereinafter: DT). Hybridoma can be used in diagnostic test systems for the specific indication of DT in clinical samples.

The causative agent of diphtheria (Corynebacterium diphteriae) and the toxin produced by it are the cause of acute respiratory disease in people, often fatal. The toxin Corynebacterium diphteriae completely determines the symptoms of diphtheria and is one of the central objects for monitoring bacterial toxins in the clinic. Currently, the analysis of toxins is of great importance in connection with the threat of epidemics. In some cases, including with diphtheria, it is not possible to accurately diagnose based on symptoms [Murray PR, Rosenthal KS, Pfaller MA // Medical Microbiology, 6th Edition, 2009. Elsevier], and therefore the task of laboratory diagnosis of toxins consists in developing tests with the highest possible sensitivity and specificity. DT must be detected at a concentration not exceeding 10 ng / ml [Pappenheimer A.M. Diphtheria toxin // Annu. Rev. Biochem. 1977, 46: 69-94].

Currently, systems based on enzyme-linked immunosorbent assay (ELISA) using antibodies against toxins are the most used and reliable for the rapid and sensitive diagnosis of toxins [The EFSA Journal. 2007, 130: 4-352].

The sensitivity and specificity of ELISA is determined to a large extent by the quality of the antibodies. Monoclonal antibodies have advantages over polyclonal antibodies, because based on them receive standardized drugs.

To determine the DT, MKAT is used in sandwich ELISA variants. The toxin from the solution is bound by polyclonal antibodies adsorbed on a solid support, then (the second layer) the detecting MKAT is conjugated to the toxin conjugated with a label (for example, peroxidase, phosphatase or biotin). The concentration of the tested toxin is determined by the calibration curve obtained in the sandwich version of the ELISA with a toxin of known concentration. The sensitivity of the determination of DT in the ELISA sandwich version reaches 0.1-4 ng / ml [Sviridov VV, Zaitsev EM, Delvig AA, Mazurova IK, Semenov BF. The use of monoclonal antibodies to determine diphtheria toxin and its structural components // Molecular Genetics, Microbiology and Virology. 1986, 1: 30-35; Engler KH, Efstratiou AJ. Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria // J Clin Microbiol. 2000, 38 (4): 1385-1389]. The use of polyclonal antibodies can reduce the reproducibility of the results due to the non-standardity of these drugs.

A known method for the quantitative detection of biological toxins based on immunoassay using biological microarrays. A biological microchip is an ordered array of individual microcells on a solid substrate, in which MKAT is immobilized to various biotoxins of bacterial and plant origin. The use of microchips allows you to simultaneously analyze the sample for the presence of several biotoxins. The microchip uses a competitive variant and a sandwich variant of immunoassay. In a competitive variant, DT and five other toxins are simultaneously analyzed, namely the staphylococcal toxin SeB, the B subunit of cholera toxin, the lethal factor and the protective antigen of anthrax toxin and the tetanus toxin C fragment. A competitive immunofluorescence assay is used in the chip, in which MKAT to DT and five other toxins (staphylococcal toxin SeB, cholera toxin B subunit, lethal factor and protective antigen of anthrax toxin and tetanus toxin C fragment) are locally immobilized on the surface of epoxy slides. For detection of DT and other toxins, Alexa Fluor 532 labeled with fluorescent dye is used, the toxins indicated above are in constant concentration. Also, native (unlabeled) toxins are introduced into the analyzed sample, which compete for binding sites on MKAT with highly purified, dye-conjugated toxins. The concentration of the tested toxin is determined by the calibration curve obtained in a competitive ELISA with a toxin of known concentration. Fluorescence signals are recorded using a microarray scanner equipped with a diode laser with an excitation wavelength of 532 nm. The sensitivity of the determination of DT in the biological chip reaches 306 ng / ml [Rucker VC, Havenstrite KL, Herr AE. Antibody microarrays for native toxin detection // Anal Biochem. 2005, 339 (2): 262-270].

Known hydrogel microchip, which is an ordered array of three-dimensional hydrogel cells on a solid substrate, obtained by photo- or chemically modified polymerization and containing immobilized MKAT to various biotoxins of bacterial and plant origin [RF Patent No. 2216547, С07К 17/08, publ. 2003]. The hydrogel microchip is designed to screen a wide range of hazardous toxins. However, the chip currently detects a limited number of toxins and, in particular, does not detect DT, which poses a serious threat to human health and life.

Known closest to the claimed clone of hybrid cultured animal cells Mus musculus L MM DT 12 is a producer of monoclonal antibodies to DT class IgG1 [Zaitsev EM, Sviridov BB, Titova NG, Lebedev VS, Garipova MI, Volgareva GM, Semenov BF. Antitoxic properties of monoclonal antibodies to diphtheria toxin // Molecular Genetics, Microbiology and Virology. 1985, 2: 37-41]. The clone was obtained as a result of hybridization of splenocytes of mice immunized with a commercial diphtheria toxoid preparation (manufactured by TsNIIVS named after Mechnikov) with X63-Ag8.6 myeloma cells. Monoclonal antibodies MM DT 12 bind to the B-fragment of DT. The main disadvantage of the aforementioned clone producing MKAT to DT is that, when it is involved in the ELISA sandwich, paired with another MKAT, a rather high optical density is observed in negative control samples (without toxin), apparently due to the nonspecific binding interaction and those showing MKAT [Sviridov VV, Zaitsev EM, Delvig AA, Mazurova IK, Semenov BF. The use of monoclonal antibodies to determine diphtheria toxin and its structural components // Molecular Genetics, Microbiology and Virology. 1986, 1: 30-35].

The objective of the invention is to obtain a clone of hybrid cells producing highly specific MKAT against DT that do not have binding to other bacterial toxins, with a detection limit of the toxin in ELISA below 10 ng / ml.

The problem is solved by the clone of hybrid cultured animal cells Mus musculus L G10B6 - a producer of monoclonal antibodies to DT.

The specified clone of hybrid cultured animal cells of Mus musculus L G10B6 animals is obtained by fusion of SP-2/0 line myeloma cells and popliteal lymph node cells of BALB / c mice immunized with DT (manufactured by the N.F. Gamalei State Research Institute of Epidemiology and Microbiology), using a polyethylene glycol with a molecular weight of 4000 with a subsequent selection of hybrid cells as a merging agent on a selective medium: on an Eagle medium modified with Dulbecco with 20% cow fetal serum, 4 mM L-glutamine, 5 mM mercaptoethanol and GAT (0.1 mM hypoxanthine, 4 × 1 0 ^-7 M aminopterin, 1.6 × 10 ^-5 M thymidine). By the method of limiting dilutions, stable clones producing antibodies are selected, which specifically bind DT in indirect ELISA. At the next stage, clones that determine the DT in a concentration below 10 ng / ml in the sandwich ELISA are selected from clones producing MKAT specific for DT. Next, clones are selected that do not cross-interact with other bacterial toxins in the microchip, namely ricin, the lethal factor of the toxin Bacillus anthracis, the protective antigen of the toxin Bacillus anthracis, cholera toxin, staphylococcal toxins SeA, SeB, SeI, SeG. The selected clone G10B6 produces antibodies that, upon detection of DT in the ELISA sandwich, can detect DT at a concentration not exceeding 10 ng / ml. In particular, the sensitivity of the ELISA sandwich variant, where MKAT G10B6 is used as binding antibodies, and MKAT, which recognize another antigenic determinant of DT, are used as developing antibodies, is 1.3 ng / ml. This sensitivity is preserved when determining DT in swabs of the nasopharynx of a person.

MKAT can be used for the quantitative detection of DT in simultaneous and parallel analysis with a number of other biological toxins in the format of a biological microchip. The sensitivity of the determination in the microchip format reaches 2 ng / ml.

Hybridoma G10B6 is characterized by the following properties.

1. Karyological characteristics.

The karyotype of G10B6 clone cells by species is murine, by the modal number of chromosomes 88.

2. Morphological characteristics.

The G10B6 hybridoma clone is represented by large rounded cells, morphologically similar to the cells of the original SP-2/0 myeloma line.

3. Standard in vitro growing conditions.

The inoculum concentration when grown in vitro is (1.0-2.0) × 10 ⁵ cells / cm ³ . The culture medium is Wednesday Needle in the modification of Dulbecco with 10% serum of the fetal cow. Cultivation temperature (37 ° C). The content of CO ₂ in the atmosphere of cultivation is 5%.

4. Cultural properties of the clone.

Hybridoma G10B6 is a suspension-monolayer, about 60% of the cells are in suspension, not attaching to the surface of the culture vessel, the frequency of passage at a seeding dose (1.0-2.0) × 10 ⁵ cells / cm ³ - 3-4 days. The hybrid clone does not lose its ability to synthesize antibodies when passaged in vitro for 6 months (observation period). The productivity of the clone for antibodies in vitro is in the magnitude of the titer of the supernatant in ELISA 1: 10000-1: 20,000.

5. The cultivation of hybridomas in the body of the animal.

Animal species - inbred mice of the BALB / c line. The dose of cells is 1-2 × 10 ⁶ per mouse. 10-14 days before the introduction of the hybridoma, 0.5 ml of 2, 6, 10, 14-tetramethylpentadecane (pristan) is injected into the abdominal cavity of mice. The tumor grows in a mixed ascites-solid manner. Ascites is formed on days 7-12 in a volume of 3 to 10 ml in the mouse. Digestibility 100%. The hybrid clone retains the ability to produce antibodies at a constant level (control by antibody titer) when cells are transplanted into animals for 6 passages (observation period). The titer of MKAT in ascites liquids in ELISA is 1: 2 × 10 ⁶ -1: 4 × 10 ⁶ .

6. Clone contamination.

G10B6 contamination by bacteria, yeast and fungi was not detected.

7. Biotechnological characteristics of a useful product.

Antibodies produced by cells of clone G10B6 belong to the class of IgG1 immunoglobulins according to ELISA with typing monospecific antisera against individual classes of mouse immunoglobulins. MKAT CT-F5 / H3 light chains belong to the κ type.

By immunoblotting, it was shown that MKAT G10B6 binds to the B fragment of DT.

Affinity constant calculated by the Beatty method [Beatty JD, Beatty BG and Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay // J Immunol Methods, 1987; 100: 173-179], for MKAT G10B6 it is 0.8 × 10 ⁹ M ^-1 .

8. The method of cryopreservation.

The cryopreservation medium contains 90% of the fetal serum of the cow and 10% of the cryoprotectant - dimethyl sulfoxide (DMSO).

Freezing and warming mode

Freezing: the cell pellet obtained after low-speed centrifugation is suspended in cryopreservation medium at a rate of 1-3 × 10 ⁶ cells per ml of medium. The cell suspension is mixed and poured into 1 ml cryovials (Nunc). Labeled tubes are placed in a low-temperature refrigerator (-70 ° C) for a day, and then in a Dewar vessel with liquid nitrogen (for long-term storage). Cell recovery after thawing: rapid thawing in a water bath at 37 ° C until completely thawed. Then the cell suspension is transferred to a sterile centrifuge tube with 10 ml of culture medium without serum. Cells are pelleted by centrifugation (10 min at 150 g), then the cell pellet is suspended in Eagle's Dulbecco modification containing 10% serum of the fetal cow and transferred to a culture vessel. Cell viability after cryopreservation is (80 ± 10)%.

The invention is illustrated by graphic materials.

Figure 1. IFA DT sandwich in FSB and nasopharyngeal swabs on a tablet using MKAT G10B6 as binding antibodies.

When analyzing the obtained data, the optical absorption (A 492 nm) is calculated as the median of the optical absorption of three identical microplate cells. The analytical sensitivity of the analysis, i.e. the smallest tested concentration of diesel fuel is determined as the concentration corresponding to the value of optical absorption exceeding, by at least two standard deviations, the optical absorption of a multiple measured zero point.

The sensitivity value is determined experimentally by the obtained calibration curves and calculated as follows:

IF ₀ + 2 SD

where IF ₀ is the value of the fluorescence intensity for each measurement of the zero sample; SD is the standard deviation from the arithmetic mean value IF ₀ .

SD is calculated by the following formula

,

,

- среднее арифметическое значение интенсивности флуоресценции при измерении нулевой пробы; n - число измерений.Where

- the arithmetic mean value of the fluorescence intensity when measuring a zero sample; n is the number of measurements.

For the presented curve, the sensitivity is 1.3 ng / ml.

Figure 2. Calibration curves for determining the concentration of DT in a sandwich ELISA on microarrays using MKAT G10B6 as binding antibodies.

Calculation of the intensity of the fluorescent signal from each cell of the biochip and determination of the concentrations of toxins are carried out using special software ImagelAssay (IMB, Moscow). When analyzing the data obtained, the fluorescence intensity is calculated as the median signal from four identical gel cells.

The sensitivity value is determined experimentally from the obtained calibration curves and calculated, as indicated in the signature to figure 1. For the presented curve, the sensitivity is 2 ng / ml.

The invention is illustrated by examples.

Example 1. Obtaining clone G10B6

A hybridoma is obtained by fusion of mouse SP-2/0 mouse myeloma with the BALB / c mouse lymph node cells of the conventional category immunized with whole DT (manufactured by the N.F. Gamalei State Research Institute of Epidemiology and Microbiology). On the first day of immunization, the toxin is introduced into the pads of the hind legs of mice at 15 μg / mouse DT diluted in 0.1 ml of physiological saline, with the addition of 0.1 ml of incomplete Freund's adjuvant. On days 14 and 28, DT is administered at a dose of 15 μg / mouse with incomplete Freund's adjuvant and in physiological saline, respectively. 6 days after the last immunization, popliteal lymph nodes are removed and 5 × 10 ⁶ mouse lymphocytes are hybridized with 1 × 10 ⁶ cells of the SP-2/0 myeloma line in 1 ml of polyethylene glycol 4000 for 1 min of joint incubation.

After removal of polyethylene glycol by centrifugation, the cells are plated on 96-well plates on a layer of feeder cells - macrophages isolated from the peritoneal cavity of the mouse inbred line. Selection of hybrid cells is carried out on the Eagle medium in the modification of Dulbecco, with 20% serum of the fetal cow, 4 mm L-glutamine, 5 mm mercaptoethanol and GAT. After 21 days - the time of complete death of myeloma cells, hybridoma cells are cultured on a medium without aminopterin. Further cultivation of hybridomas is carried out on a medium that does not contain selective components of GAT. 3-fold cloning of hybridomas is carried out by the method of limiting dilutions in 96-well plates on a layer of feeder cells. In the last two clonings, the percentage of positive clones is 100%.

Indirect ELISA with DT is used to screen for clones producing MKAT specifically binding to DT. DT is adsorbed into the wells of the ELISA plate at a protein concentration of 1 μg / ml in 0.1 M bicarbonate buffer pH 9.0. 100 μl of antigen solution was added to each well and incubated overnight at 4 ° C. After sorption, free binding sites are blocked with a 1% solution of bovine serum albumin at 37 ° C for 1 h. Wells of the plate are washed with FSB with 0.1% tween-20, then 100 μl / well of the analyzed supernatants taken from the wells of the tablet are added to which hybridoma antibodies grow and produce. Incubated for 1 h at 37 ° C. Wash off, as described above, add 100 μl / well of peroxidase conjugate of rabbit antibodies against mouse immunoglobulins, incubate for 40 min at 37 ° C, wash and introduce 100 μl / well of peroxidase substrate: a solution of orthophenyl diamine at a concentration of 1 mg / ml in 50 mM citrate buffer pH 4.5 containing 0.015% hydrogen peroxide. After color development, the reaction is stopped by adding 100 μl / well of 10% sulfuric acid. The color intensity is recorded spectrophotometrically, determining the optical absorption at a wavelength of 492 nm. Selected clones producing MKAT specifically binding DT.

Example 2. The use of MKAT G10B6 in the sandwich variant of ELISA for diphtheria DT

In the ELISA sandwich variant, MKAT G10B6 is used as binding antibodies and as detecting antibodies, MKATs are directed against epitopes of the DT molecule with which MKAT G10B6 does not interact. MKAT G10B6 is sorbed with the first layer at the bottom of the ELISA microplate at 100 μl / well at a concentration of 10 μg / ml. Free plastic binding centers are blocked with a 1% solution of milk powder for 1 h at 37 ° C. Wash the FSB with 0.1% tween-20. Then, the analyzed samples are introduced in a volume of 100 μl / well containing DT in various dilutions, incubated for 45 min at 37 ° C, and the FSB is washed with 0.1% tween-20. A conjugate of detecting MKAT with biotin is introduced in a volume of 100 μl / well at an antibody concentration of 10 μg / ml. Incubated for 45 min at 37 ° C, washed, conjugated streptavidin with horseradish peroxidase, incubated (45 min at 37 ° C) and washed. Then, a peroxidase substrate is added: a solution of orthophenyldiamine at a concentration of 1 mg / ml in 50 mM citrate buffer pH 4.5 containing 0.015% hydrogen peroxide. After 15 minutes, the reaction was stopped by the addition of 10% sulfuric acid. The color intensity is recorded spectrophotometrically at a wavelength of 492 nm. Analysis of solutions with a known concentration of DT shows that the sensitivity of the determination of DT reaches 1.3 ng / ml. When incubating DT with MKAT G10B6 not in the FSB, but in the presence of swabs of the human nasopharynx, the sensitivity of the determination of DT does not change and amounts to 1.3 ng / ml (Fig. 1).

Example 3. Quantitative analysis of diesel fuel using a biological microchip

MKAT G10B6 and MKAT are immobilized on hydrogel chips against 8 other toxins (ricin, lethal factor of the toxin Bacillus anthracis, protective antigen of the toxin Bacillus anthracis, cholera toxin, staphylococcal toxins SeA, SeB, SeI, SeG). The concentration of each of the antibodies in the polymerization mixture is 0.8 ± 0.2 mg / ml, the volume of the gel element is 0.1 nl. Each gel element contains one of the nine aforementioned MKAT; in addition, control gel elements that do not contain antibodies are included in the chip.

Add 30 μl of the analyzed sample and 30 μl of a solution showing biotinylated monoclonal antibodies specific for DT (as in Example 2), and incubate on the biochip for 17 h at 37 ° C. After the washing procedure (20 min, PBS with 0.1% Tween-20), fluorescently labeled streptavidin was added, incubated for 10 min at 37 ° C, washed again and the fluorescent signals were recorded (FIG. 2).

Hydrogel chips are prepared as follows [RF Patent No. 2216547, MKI S07K 17/08, publ. 2003]: glass slides for the manufacture of microchips treated with solutions of 1 N. NaOH, H ₂ SO ₄ , washed with water, then immersed in a solution of 1% 3-methacryloxypropyltrimethoxysilane in ethanol, washed in ethanol, water and dried. A polymerization mixture containing gelling monomers based on methacrylamide and N-substituted amino sugars, as well as MKAT to be immobilized, is applied using a QArray robot (Genetix, UK) in the form of microdrops with a volume of 0.1 nl onto the surface of the activated substrate. Gel cells are polymerized under an ultraviolet lamp with a maximum radiation of 350 nm (Sylvania GTE lamp, F15T8 / 350B1, UK) at a distance of 8 cm from the lamp, for 50 min at 20 ° C in a stream of nitrogen. When applied with a pin of 150 microns, gel elements of a hemispherical shape with a diameter of 120 microns are obtained. After polymerization, biochips are washed for 40 minutes with FSB with 0.1% tween-20. To reduce the nonspecific interaction of the reagents with the surface of the biochip, the surface is treated with blocking buffer for 1 h, then washed with distilled water.

The quality of the obtained biochips is checked in transmitted light using a biochip analyzer (IMB RAS) equipped with special software TestChip and QualityControl (IMB RAS). As a result of quality control, biochips are rejected for which deviations of the radii of gel elements exceed 5% inside each biochip and 8% between all biochips of a given batch.

Example 4. Simultaneous analysis of several biotoxins, including DT, on one microchip

The analysis is carried out as in example 3, but biotinylated MKAT, detecting DT, are mixed with biotinylated antibodies to other toxins (ricin, lethal factor of the toxin Bacillus anthracis, protective antigen of the toxin Bacillus anthracis, cholera toxin, staphylococcal toxins SeI, Se, SeA, Se, SeA, Se, Se ) The detection limits for biotoxins for parallel analysis are the same as for each individual toxin. For DT, the minimum detectable dose is 2 ng / ml.

The proposed assays can be used to test for the presence of toxin in clinical specimens.

Claims (1)

A clone of hybrid cultured animal cells Mus musculus L G10B6 is a producer of monoclonal antibodies to diphtheria toxin.

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