RU2401300C1 - Animal mus musculus hybrid cell clone l-producer of monoclonal cholera toxin antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is produced a new CT-E6/E10 hybrid cell clone producing monoclonal cholera 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 cholera toxin (SIGMA) 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 synthesises monoclonal antibodies specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:10000 in the cultural fluid, 1:1000000 in ascitic.

EFFECT: produced antibodies exceeds the analogues available in sensitivity.

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Description

The invention relates to the field of microbiology and biotechnology, specifically to hybridoma technology, and is a clone of CT-E6 / E10 hybrid cells of Mus Musculus L animals producing monoclonal antibodies (hereinafter, MKAT) to cholera vibrio toxin in cell cultures and the abdominal cavity of syngeneic animals ( XT). Hybridoma can be used in diagnostic test systems for specific indication of cholera toxin in the food industry, in environmental protection, in medicine.

Cholera vibrio (Vibrio cholerae) and the toxin produced by it are the cause of acute intestinal disease in people, often fatal. The cholera vibrio toxin fully determines the symptoms of cholera and is one of the central objects for monitoring bacterial toxins in the environment and food. Currently, the analysis of toxins is of great importance due to the threat of bioterrorism, as many natural toxins, including cholera toxin, can be used as components of biological weapons. The dose of the toxin causing the disease is usually extremely small [Scarlatos A., Welt B., Cooper B., et al. // J. Food. Sci. 2005.V.70 (8). P.121-124], in connection with which the task of laboratory diagnosis of toxin is to develop tests with the highest possible sensitivity. XT must be tested at a concentration not exceeding 1 ng / ml [Ramamurthy T., Bhattacharya SK, Uesaka Y., Horigome K., Paul M., Sen D., Pal S.C., Takeda T., Takeda Y., Nair GB // J. Clin. Microbiol. 1992. V.30 (7). P.1783-1786].

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, V.130. P.4-352].

The sensitivity and specificity of ELISA is determined to a large extent by the quality of the antibodies. Antigenic similarity between the representatives of the cholera-like group of toxins, primarily this refers to the similarity between XT and the heat-labile toxin E. coli (LT), dictates the feasibility of using MKAT, which have strict specificity for XT.

For the determination of toxins, MKAT are standardly used in competitive and sandwich ELISA variants. In a competitive embodiment, MKAT is adsorbed onto a solid support, then a solution of highly purified toxin conjugated with peroxidase, biotin or a fluorescent label and a sample containing the toxin are added. The toxin contained in the sample competes for binding sites on MKAT with a highly purified label conjugated toxin. The concentration of the tested toxin is determined by the calibration curve obtained in a competitive ELISA with a toxin of known concentration. The sensitivity of the determination of XT in competitive ELISA is 26 ng / ml [Rucker V.S., Havenstrite K.L., Herr A.E. Antibody microarray for native toxin detection. // Anal. Biochem. 2005. V.339. P.262-270]. In the test used MKAT (firm "Biodesign International").

In the sandwich embodiment, the toxin from the solution is bound by MKAT adsorbed on a solid support, then (the second layer) other detectable MKAT are conjugated to the toxin conjugated with a label (for example, peroxidase, biotin or fluorescein). 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 XT in the sandwich ELISA reaches 1 ng / ml [N. Koike, K. Okada, Y. Yabushita, D.Y. Zhang, K. Yamamoto, T. Miwatani, T. Honda. Rapid and differential detection of two analogous enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli by a modified enzyme-linked immunosorbent assay. // FEMS Immunol. Med. Microbiol. 1997 17 (1): 21-25].

When using MKAT (Biodesign International) in a sandwich variant with GM1 ganglioside immobilized on the liposomes, the XT and LT cell receptor, the detection sensitivity reaches 10 fg / ml [Ann-Yoon S., DeCory TR, Baeumner AJ, Durst R. / Anal. Chem. Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin. 2003.75.2256-2261]. The use of GM1 ganglioside limits the specificity of the assay because GM1 binds both XT and LT.

A known method for the quantitative detection of biological toxins based on ELISA 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 microarrays allows you to simultaneously analyze the sample for the presence of several biotoxins with a sensitivity not inferior to the sensitivity of standard immunological tests. The microchip uses a competitive version and a sandwich variant of ELISA. Sensitivity

XT determination in a biological chip reaches 1.6 ng / ml [F.S. Ligler, C. Rowe Taitt, L.C. Shriver-Lake, K.E. Sapsford, Ya Shubin and J.P. Golden Array biosensor for detection of toxins. 2003. Anal. Bioanal. Chem. 377 (3): 469-477]. This sensitivity is achieved using a biosensor chip. At the same time, XT and four other toxins were analyzed, namely ricin, staphylococcal toxin SeB, botulinum toxin A and toxin Bacillus globigii. An ELISA sandwich is used in the chip, in which MKAT binding to XT and four other toxins (ricin, staphylococcal toxin SeB, botulinum toxin A and toxin Bacillus globigii) are locally immobilized on the glass surface by forming an avidin-biotinylated antibody complex. Fluorescein-labeled MKAT is used to detect XT and other toxins. Fluorescence signals are recorded using a confocal microscope. For the detection of XT, MKAT clone of the hybridoma 3D 11 (Biodesign International) is used, which do not have strict specificity and bind to both XT and LT.

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 N 2216547, publ. 2003]. The hydrogel microchip is designed to screen a wide range of dangerous toxins that can be used as components of biological weapons. However, the chip currently detects a limited number of toxins and, in particular, does not detect XT, which poses a serious threat to human health and life.

Known closest to the claimed clone of hybrid cultured animal cells Mus musculus L HSCV (P) N 469D producer of monoclonal antibodies to cholera enterotoxin [RF patent N1835848, MKI C12N 5/00, publ. 1995], producing IgM class monoclonal antibodies (N 469), obtained against chromatographically purified cholera toxin, by hybridization of splenocytes with X63.Ag8.653 myeloma cells. MKAT N 469 binds to the B-subunit of cholera toxin. The main disadvantage of the clone producing MKAT No. 469 is that when it participates in the ELISA sandwich variant, cholera toxin with an insufficiently high sensitivity of 1 ng / ml is detected. In addition, quite low productivity is characteristic of clone N 469 - the antibody titer in the culture fluid is 1: 2501: 500, and in ascites 1: 1,000,000. Clone N 469 produces IgM antibodies, which makes it difficult to obtain pure antibody preparations because to obtain a preparation of pure IgM antibodies, the standard technique is not suitable - protein A Sepharose chromatography.

The objective of the invention is to obtain a clone of hybrid cells that produce highly specific MKAT against cholera toxin, do not have binding to the thermolabile toxin E. coli and other bacterial toxins, with a detection limit of the toxin in ELISA below 1 ng / ml.

The problem is solved by the clone of hybrid cultured animal cells Mus musculus L CT-E6 / E10 - producer of monoclonal antibodies to cholera toxin.

The specified clone of hybrid cultured animal cells of Mus musculus L CT-E6 / E10 animals is obtained by fusion of SP-2/0 line myeloma cells and lymph node cells of BALB / c mice immunized with cholera toxin (Sigma), using molecular weight polyethylene glycol as a merging agent 4000 and subsequent selection of hybrid cells in selective medium: on Eagle’s medium in the Dulbecco modification with 20% fetal serum of a cow, 4 mM L-glutamine, 50 μM mercaptoethanol and GAT (0.1 mM hypoxanthine, 4 × 10 ^-7 M aminopterin, 1 , 6 × 10 ⁵ M thymidine). Stable antibody-producing clones in the indirect ELISA specifically bind XT and non-bind LT are selected by the limiting dilution method. In the next step, clones are selected from clones producing MKAT specific for XT, which, in the ELISA sandwich variant, determine XT at a concentration below 1 ng / ml. Then, 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, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG. Selected clone CT-E6 / E10 Produces antibodies that, upon detection of XT in the ELISA sandwich variant, can detect XT at a concentration not exceeding 1 ng / ml. In particular, the sensitivity of the ELISA sandwich variant, where MKAT CT-E6 / E10 is used as the developing antibody, and MKAT, which recognize the other antigenic determinant of XT, are used as binding antibodies, is 0.2 ng / ml. This sensitivity is maintained when determining cholera toxin in water from an open reservoir, broth and milk.

MKAT can be used for the quantitative detection of cholera toxin 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 0.50 ng / ml.

The hybridoma ST-E6 / E10 is characterized by the following properties.

1. Karyological characteristic

The karyotype of clone cells CT-E6 / E10 by species is murine, by the modal number of chromosomes 88.

2. Morphological characteristics.

The CT-E6 / E10 hybridoma clone is represented by large rounded cells that are similar in morphology to the cells of the original myeloma line SP-2/0.

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 CT-E6 / E10 is a suspension-monolayer, about 60% of the cells are in suspension, not attached 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 in vitro antibody clone productivity is 1: 10,000 in the titer of the supernatant in ELISA.

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: 1 × 10 ⁶ .

6. Clone contamination.

No contamination with CT-E6 / E10 by bacteria, yeast and fungi was detected.

7. Biotechnological characteristics of a useful product. Antibodies produced by CT-T6 / E10 clone cells belong to the IgG class of immunoglobulins according to ELISA with typing monospecific antisera against individual classes of mouse immunoglobulins. MKAT light chains CT-E6 / T10 belong to the "to" type.

By immunoblotting, it was shown that MKAT CT-E6 / E10 binds to the B subunit of cholera toxin.

Affinity constants calculated using the Beatty method [Beatty JD, Beatty BG, and Vlahos WG: Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J. Immnol. Methods., 1987; 100: 173-179], for MKAT CT-E6 / E10 is 0.52 × 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).

The mode of freezing and heating.

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 the Needle medium in the Dulbecco modification containing 10% of the fetal bovine serum and transferred to a culture vessel. Cell viability after cryopreservation is (80 ± 10)%.

The invention is illustrated by graphic materials.

Figure 1. Sandwich analysis of ELISA of cholera toxin in PBS, milk, meat broth and water on a tablet using MKAT CT-E6 / E10 as developing 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 lowest test CT concentration is defined as the concentration corresponding to the value of optical absorption exceeding 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 _o + 2 * SD,

where IF _o 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 _o .

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 0.20 ng / ml.

Figure 2. Calibration curves for determining the concentration of CT in the ELISA sandwich assay on microarrays using MKAT CT-E6 / E10 as developing 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 0.50 ng / ml.

The invention is illustrated by examples.

Example 1. Obtaining a clone CT-T6 / E10

A hybridoma is obtained by fusion of mouse SP-2/0 mouse myeloma with conventional BALB / c mouse lymph node cells immunized with whole cholera toxin (Sigma). On the first day of immunization, the toxin is introduced into the pads of the hind legs of mice, 2.5 μg / mouse of cholera toxin 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, cholera toxin is administered with incomplete Freund's adjuvant at a dose of 5 and 20 μg / mouse, 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, 50 μM 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 XT is used to screen for antibody producing clones producing MKAT specifically binding XT. Cholera toxin 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 is 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 PBS 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 add 100 μl / well of peroxidase substrate: 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 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 XT.

For screening antibody-producing clones producing MKAT that do not interact with the heat-labile E. coli toxin, indirect ELISA with the heat-labile E. coli toxin is used. The analysis is carried out as with XT, but the thermolabile E. coli toxin 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 is added to each well and incubated overnight at 4 ° C. Clones producing MKAT that do not interact with the thermolabile toxin E coli are selected.

Example 2. The use of MKAT CT-E6 / E10 in the sandwich variant of ELISA for cholera toxin

In the ELISA sandwich variant, MKAT CT-E6 / E10 is used as detecting antibodies, and as binding antibodies, MKATs are used against epitopes of the cholera toxin molecule with which MKAT CT-E6 / E10 does not interact. Binding MKAT adsorb the first layer at the bottom of the ELISA microplate at 100 μl / well at a concentration of 10 μg / ml. Free plastic binding sites are blocked with a 1% milk powder solution for 1 hour at 37 ° C. Wash the FSB with 0.1% tween-20. Then, the analyzed samples were introduced in a volume of 100 μl / well containing cholera toxin in various dilutions, incubated for 45 min at 37 ° C, and the FSB was washed with 0.1% tween-20. Contribute CT-E6 / E10 conjugate with biotin 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 ± 5 min, 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 cholera toxin shows that the sensitivity of XT determination reaches 0.2 ng / ml. Upon incubation of cholera toxin with MKAT CT-E6 / E10 not in FSB, but in 10% milk, broth, and water from an open reservoir, the sensitivity of XT determination does not change and amounts to 0.2 ng / ml.

Example 3. Quantitative analysis of cholera toxin using a biological microchip

Binding MKAT (as in Example 2) is immobilized onto hydrogel chips against cholera toxin and MKAT against 8 other toxins (ricin, lethal toxin factor Bacillus anthracis, protective antigen of the toxin Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, 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 of CT-E6 / E10 developing biotinylated monoclonal antibodies and incubate on a biochip for 17 h at 37 ° C. After the washing procedure (20 min, PBS with 0.1% Tween-20), fluorescently labeled streptavidin is added, incubated for 10 min at 37 ° C, washed again and the fluorescent signals are recorded. The detection limit for XT is 0.50 ng / ml.

Hydrogel chips are obtained as described, for example, in [RF patent N 2216547, MKI C07K 17/08, publ. 2003]. Glass slides for the manufacture of microchips are 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, F15T 8 / 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 nonspecific interaction, biochips are 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 cholera toxin on one microchip. The analysis procedure, as in example 3, but the biotinylated MKAT CT-E6 / E10, detecting XT, is mixed with biotinylated antibodies to other toxins (ricin, lethal toxin factor Bacillus anthracis, protective antigen of the toxin Bacillus anthracis, diphtheria toxin A, staphylococcus aureus , SeB, Sel, SeG). The detection limits for biotoxins for parallel analysis are the same as for each individual toxin. For XT, the minimum detectable dose is 0.50 ng / ml.

The proposed analyzes can be used to test for the presence of toxin in food, water and biomaterials.

Claims (1)

A clone of hybrid cultured animal cells Mus musculus L CT-E6 / E10 is a producer of monoclonal antibodies to cholera toxin.

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