RU2059721C1 - Strain of hybrid cultured murine cells mus musculus l used for preparing monoclonal antibodies to the human thyrotropic hormone - Google Patents

Abstract

FIELD: medicinal biotechnology. SUBSTANCE: method involves the use of the strain of hybrid cultured murine cells producing high-affinitive monoclonal antibodies to the human thyrotropic hormone. Strain is obtained by hybridization of murine splenocytes (strain Balb/c) with myeloma cells X63 Ag.653. Monoclonal antibodies belong to the isotype Ig G1, their concentration in cultural fluid and ascitic fluid is 15-20 mcg/ml and 5-10 mg/ml, respectively. Crossed reactivity of antibody obtained with relative human thyrotropic hormones is 0.06%, not above. Method is used for procedure development used for diagnosis in endocrinology. EFFECT: improved method of hormone assay. 2 dwg, 2 tbl

Description

 The invention relates to biotechnology and can be used to obtain monoclonal antibodies (MKAT) to human thyroid stimulating hormone (TSH).

 For the correct and timely diagnosis of diseases associated with impaired functions of the pituitary and thyroid glands, as well as for monitoring the effectiveness of treatment, an assessment of the level of TSH in human blood is necessary. Modern methods of immunoassay (enzyme immunoassay, immunofluorescence, immunoradiometric) are based on the use of monoclonal antibodies.

Monoclonal antibodies to TSH were obtained in a number of laboratories abroad, the binding constants of which ranged from 10 ⁸ to 10 ¹¹ M ^-1 (M.M. Benkirane, D. Bon, S. Costagliola, F. Paolucci, B. Darbouret, P. Prince, P. Carayon // Endocrinology. 1987. V. 121, N 3. 1171-1177; JMBraun, G. Charreire // J. Immunol. Meth. 1990.132, N 2. 197-203; M. Soos, K.Siddle // J. Immmunol. Matt. 1982. V. 51, N 1. 57-64).

 It is advisable to consider the well-known domestic strain of hybrid cultured T-32 cells (A.S. USSR N 1742324, class C 12 N 5/00, 1992) as the closest analogue of our technical solution according to a number of signs.

 Our proposed strain is similar to the prototype according to the production method (hybridization of splenocytes of Balb / c mice with cells of mouse myeloma X63 Ag 653). Also a common quality is the high specificity for human thyroid stimulating hormone (TSH), the absence of cross-reactions with related glycoprotein hormones of the pituitary and placenta (LH, FSH, hCG).

 The affinity of antibodies produced by strain T-32 is sufficient to create an enzyme-linked immunosorbent assay system that allows you to determine the level of TSH in human biological fluids in the concentration range from 2.4 to 50.0 µI / ml. However, normal serum TSH concentrations range from 0.30 to 5.00 μMU / ml. Consequently, such a test system will allow only coarse screening and identification of patients with elevated TSH values (consistent with hypothyroidism). Moreover, most of the normal values and low TSH concentrations cannot be determined due to insufficient sensitivity.

 The aim of the proposed technological solution was to obtain stable hybrid cultured mouse cells of the BALB / c line producing high-affinity McAb for human TSH, which do not give cross-reactions with structurally similar glycoprotein hormones (luteinizing, follicle-stimulating, and chorionic gonadotropic).

 The affinity of the proposed monoclonal antibodies makes them suitable for the development of immunometric test systems for the determination of TSH in biological fluids with a sensitivity of not more than 0.15 μMU / ml. Such sensitivity allows differential diagnosis of normal, low and high concentrations of TSH in biological fluids (corresponds to eugiper and gyrotyroidism).

 To obtain hybridoma cells producing McAb, cells of the mouse myeloma line X63.Ag8.653 were hybridized using 50% polyethylene glycol 4000 with splenocytes from the mouse line BALB / c immunized with TSH.

 Female BALB / c mice were immunized for 4 months with human thyroid stimulating hormone. A hormone with immunological activity of at least 6 IU / mg was used for immunization.

 After hybridization, the cells were resuspended in RPMI 1640 medium containing components of a selective medium (hypoxanthine, aminopterin, thymidine). Supernatants were analyzed by immunometric method. The specificity of monoclonal antibodies was evaluated in competitive ELISA. Positive clones were recloned and expanded as an ascites tumor in BALB / c mice, which were injected with pristan 10-12 days before the introduction of the cells. Antibodies from ascites fluids were isolated by precipitation with ammonium sulfate, followed by ion exchange chromatography on DEAE cellulose. The obtained McAt were used in the enzyme-linked immunosorbent two-site method for determining TSH.

Getting strain:
1. Immunization.

 The BALB / c line itself was immunized intraperitoneally for 4 months, injecting every 2 weeks with 30 μg of TSH preparation in 0.5 ml of PBS emulsion with complete (first injection) or incomplete (subsequent injections) Freund adjuvant. 4 days before hybridization, 20 μg of TSH in PBS was intravenously administered. For immunization, a TSH preparation was used, which, according to an analysis carried out using a Bio-Merieux test system, had an immunological activity of at least 6 IU / mg.

 2. Obtaining hybridomas.

 Hybridization of the splenocytes of the immunized animals was carried out with X63.Ag8.653 myeloma cells in a cell ratio of 2: 1 according to the Köhler-Milstein method using PEG 4000. After hybridization, the cells were resuspended in RPMI 1640 medium with a double concentration of components of the selective medium (hypoxanthine, aminopterin, thymidine ), and 100 μl were scattered into 96-well plates containing 100 μl of the macrophage feeder layer. The culture medium at all stages of growing hybridomas contained 20% fetal calf serum, 4 mm glutamine. Hybridomas were cloned at least 2 times by limiting dilution method. The presence of positive clones was determined by enzyme immunoassay.

 3. Scanning of primary clones.

 Testing the culture fluid for the presence of antibodies to TSH was carried out by the immunometric two-site method. As a solid phase, polystyrene tablets with the maximum sorbing ability of Nunk were used.

The tablets were sensitized with an immunoglobulin fraction of rabbit antiserum to TSH (Hormon Cooperative). Antiteta sorbed from solution with a concentration of 10 ug / ml in PBS overnight at ⁴ ° C or 1.5 hours at 37 ° ^C. All subsequent incubations were performed for 1 h at 37 ° ^C. Between incubations unbound components were washed with PBS 0.05 % tween-20.

 After blocking the remaining free binding sites on the plastic with a 0.5% gelatin solution, TSH at a concentration of 10 ng / ml was introduced into PBS. Then, culture supernatants were added. Antigen-bound antibodies were determined using a rabbit anti-mouse IgG peroxidase conjugate. Orthophenylenediamine was used as a chromogen. Optical absorption was measured on a Multiscan spot spectrophotometer.

 4. Production of monoclonal antibodies in the form of ascites.

 Positive monoclones were grown as an ascites tumor in BALB / c mice, which were injected with 0.5 ml pristan 7-10 days before the introduction of cells. After the development of an ascites tumor in mice, animals were sacrificed by cervical dislocation, ascites fluid was collected, cells were removed by centrifugation.

The ascites fluid containing 5.10 mg / ml antibody, was frozen at -20 ° ^C.

 5. Purification of monoclonal antibodies.

 Antibodies from ascites fluid were isolated by precipitation of an immunoglobulin fraction of 50% of saturation with ammonium sulfate, followed by desalination on a Sephadex G-25 column and ion-exchange chromatography on DEAE-cellulose. Antibodies were bound to cellulose equilibrated with 5 mM sodium phosphate buffer, pH 8.0; elution was carried out in a gradient of ionic strength from 0.01 to 0.30 M created by sodium chloride.

The purity was checked by polyacrylamide gel electrophoresis under denaturing conditions. Impurities were not more than 5%
A subclass of the obtained antibodies in the culture supernatant was determined using monospecific antibodies to murine immunoglobulin isotypes according to the recommendations of the supplier. Antibodies synthesized by clone 624D belong to the IgG1 subclass.

 6. Determination of the specificity of monoclonal antibodies.

Specificity was evaluated by competitive ELISA. For this purpose, the wells of polystyrene plates TTG solution was added at a concentration of 400 ng / ml (2 mIU / ml) in PBS and incubated overnight at ⁴ ° C or 1.5 hours at 37 ° ^C. To block nonspecific binding, 0.5 % solution of gelatin in PBS. Then, the studied antibodies were incubated at an excessive concentration (5 μg / ml) and hormones: LH, FSH, hCG, TSH beta-subunit and TSH in the concentration range from 10 to 1000 ng / ml, which corresponds to 0.07-7.00 IU / ml for LH and FSH; 0.14-14.00 IU / ml for hCG and 0.07-7.00 μMU / ml for TSH. Monoclonal antibodies bound to immobilized thyrotropin exhibited a rabbit anti-mouse IgG antibody peroxidase conjugate. The percentage inhibition of the binding of monoclonal antibodies to TSH immobilized in phase, when competing hormones are added to the solution, was determined by the formula
(VO / B) x 100% where Va is the concentration of TSH immobilized in phase,
In the concentration of LH, FSH, hCG and beta subunits of TSH in solution, at which 50% inhibition of the binding of TSH, immobilized in phase, with these antibodies is achieved.

The obtained B2T antibodies interact with the antigenic determinant exposed on the beta subunit as part of intact TSH. Moreover, the percentage of cross-reactions with LH, FSH and hCG was not more than 0.06% and with a free beta subunit of TSH 1.60%
The resulting strain of hybrid cultured cells was deposited in the Specialized collection of transplantable somatic vertebrate cells of the All-Union collection of cell cultures under the number 624D.

The strain is characterized by the following features:
Standard growing conditions: 37 ^° C, 5% CO ₂ , RPMI 1640 medium containing 20% ETS.

Cultural properties: cells are cultured in a monolayer at a concentration of 5 · 10 ⁵ 1 · 10 ⁶ cells / ml, subculture time is about 48 hours.

Characterization of hybridoma cultivation in the animal organism: the hybridoma is cultured in the organism of BALB / c mice, a dose of 5 · 10 ⁶ cells per mouse is used for inoculation, the animals are sensitized for 7-10 days by intravenous injection of 0.5 ml pristan, ascites are formed in 7-14 days.

 Characterization of the biosynthesis of monoclonal antibodies: the yield of monoclonal antibodies in the culture fluid is 15-20 μg / ml, in ascites fluid 5-10 mg / ml.

 The stability of antibody production is maintained for at least 20 massages in culture and 3 passages in animals.

 Characterization of monoclonal antibodies produced by 624D hybridoma: the hybridoma produces monoclonal antibodies of the IgG1 class against human thyrotropin. The activity of monoclonal antibodies is determined by the method of heterogeneous enzyme-linked immunosorbent assay. Thyrotropin is used as an antigen. The specificity of antibodies was evaluated in a competitive enzyme-linked immunosorbent assay using luteinizing, follicle-stimulating hormones and chorionic gonadotropin as competing ones. The percentage of cross-reactivity of the obtained antibodies with these hormones was not more than 0.06% in molar ratio.

Hybridoma cryopreservation method: 624D hybridoma cryopreservation is carried out in an ETS containing 15% DMSO. The first three days of the ampoule with the cells stored at ^-70 ° C, then transferred to liquid nitrogen. Hybridoma viability was evaluated using a 0.5% trypan blue solution. Viability is about 80%
The use of the strain is illustrated by the following example:
Solid-phase immunometric analysis of thyrotropin.

In the wells the plates for immunoassay sorbed monoclonal antibody 624D in a concentration of 10 ug / ml in 100 ul PBS for 1.5 hours at 37 ° ^C. Subsequent incubation was carried out for 1 hour at 37 ° ^C. Between incubations unbound components washed with PBS, containing 0.05% tween-20.

 Nonspecific binding was blocked with a 0.5% solution of bovine serum albumin in PBS. Then, 100 μl of a sample with an unknown concentration of TSH or a standard TSH solution was added. After incubation and washing, 100 μl of the peroxidase conjugate of the second antibodies to thyrotropin was added.

 Orthophenylenediamine was used as the chromogen (to prepare 10 ml of the substrate solution, 6 mg of orthophenylenediamine and 5 μl of 30% hydrogen peroxide in 0.15 M citrate-phosphate buffer, pH 5.0) were taken. After color development, the reaction was stopped by adding 100 μl of 4.9% sulfuric acid to the plates and the optical absorption was measured at 492 nm.

 The concentration of thyrotropin in the sample was calculated from the calibration curve. To construct this curve according to the scheme given by us in the example, 100 μl of standard TSH samples with concentration values: 0.0, 0.5, 1.0, 2.5, 5.0, were added to the wells of the plates blocked by bovine serum albumin. 7.5, 15.0 μMU / ml in parallel. The optical density at 492 nm resulting from the stoppage of the enzymatic reaction by sulfuric acid was determined using a Titertec Multiscan MCC / 340 plate spectrophotometer.

 In FIG. Figure 1 shows a sample of a typical calibration curve of the dependence of the optical density at 492 nm on the concentration of TSH in the sample obtained as a result of analysis using 3 different series of reagents. Digital material for each series of reagents indicating standard deviations calculated on the basis of 4 parallel determinations is shown in table 1.

 The standard samples used to construct the calibration curve were standardized relative to the II International standard preparation of human thyrotropin 80/558. The sensitivity of the analysis was defined as (x ± 2s), where x is the average concentration obtained from the analysis of the control sample not containing thyrotropin, s standard deviation.

 Our method was also tested on the sera of healthy (euthyroid) donors and patients with thyroid diseases (with hyper- and hypothyroidism). The results obtained by our method were correlated with the values obtained by testing the same samples in the TSH-EIA (TSH-ELISA) enzyme-linked immunosorbent assay (manufactured by Hoffman la Roche) (numerical data are given in Table 2). Moreover, the correlation coefficient calculated on the basis of a comparison of 25 samples amounted to 0.995 (Fig. 2).

 Thus, the proposed technical solution can be used to create a solid-phase enzyme-linked immunosorbent assay system for the quantitative determination of thyroid-stimulating hormone in human serum in the range of 0.30-15.00 μMU / ml with a sensitivity of not more than 0.15 μMU / ml. It can be used in clinical analysis of patients to differentiate hypo-, hyper- and euthyroid states.

Claims (1)

 The strain of hybrid cultured mouse cells of mus musculus L. HSCC (P) N 624D used to obtain monoclonal antibodies to human thyroid stimulating hormone.

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