SU1693045A1 - Strain of hybridous cultured mammalian cells, mus musculus l - a producer of monoclonal antibodies to human transferrine - Google Patents

Abstract

This invention relates to hybridoma technology and can be used to determine transferrin in serum and urine. The strain is obtained by fusion of cells of the murine myeloma line Sp 2 / 0- Ag 14-1 with the spleen cells of BaLb / c mice immunized with a highly purified human transformer. Hybrid clones are selected on selective medium containing hypoxanthine, aminopterin and thymidine, and cloned in semi-solid agar. The strain ICIOSp2 / 0 is stored in the specialized collection of transplanted somatic cells of the vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR under No. 345D. The population doubling time is 48 hours, the passaging frequency is 2-3 days, the multiplicity of sowing is 1/2 - 1/3. In BaLb / c mice, when inoculated with 2-10x10 cells of the strain, ascitic tumors form after 10-16 days. The titer of monoclonal antibodies is 3 for cultured liquids and 6 for authentic liquids, the concentration of monoclonal antibodies in culture is 100 µg / ml, in ascites fluid 10 mg / ml. The antibodies belong to the IgG 1 class. The sensitivity of the determination of human transferrin is 10 µg / ml. The specificity of monat is shown by immunoblotting. with s

Description

This invention relates to hybridoma technology and can be used to determine transferrin in serum and urine.

Strain of hybridoma ICIO S 2/0 was prepared as follows.

BaLb / c mice are immunized 3-fold intraperitoneally with 10 μg of highly purified human transferrin: the first time with Freund's complete adjuvant, the second time with Freund's incomplete adjuvant, and the third time without an adjuvant at 1 month intervals. On the third day after the last immunization, spleen cells of the immunized mice were hybridized with Sp2 / OAg 14 mouse myeloma cells.

ratio of 108 spleen cells to 107 myeloma cells. Polyethylene glycol is used as a hybridizing agent for gas chromatography m.v. 4000. Hybrid clones are seeded on selective medium containing hypoxanthine (10 ML aminopterin (4x10 M), thymidine (1.6x10 M) at the rate of 10 myeloma cells per 1 well of a 96-well plate. After 2 weeks, the production of specific antibodies synthesized by the hybrid by ELISA using conjugates of rabbit-anti-mouse antibodies with peroxidiza and highly purified transferrin as antigen.

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com agar. After recloning cells from ascitic tumors, the clone that actively generates monoclonal antibodies is converted into a mass culture and is designated ICIOSp2 / 0. The percentage of positive clones in the second stage is 70%. The strain ICIOSp2 / 0 is deposited in the specialized collection of digestible somatic cells of the Institute of Cytology of the Academy of Sciences of the USSR VSKK (P) under No. 345 D.

At the time of certification, the passage number of the ICIOSp2 / 0 strain is 60 in culture and 25 in animals.

The strain is characterized by the following properties.

Cultivation medium: Dulbecco's medium with 15% bovine fetal serum, 1 mm sodium pyruvate, 50 µm mercaptoethanol and 50 µg / ml gentamicin. Cells are cultured in air with 7.5% COa at 37 ° C. The culture consists of cells adhering weakly to the substrate. The growth pattern is a stationary suspension. The doubling time of the population is 48 hours, the passivation frequency is after 2-3 days, the multiplicity of sowing is 1 / 2-1 / 3, and the cell concentration (1-2) x105 per 1 ml is sown.

The introduction of strain cells into the abdominal cavity of BaLb / c mice causes the formation of ascites tumors in them. The dose of cells for administration (2-10) x10. 1-2 weeks before the cells are introduced, mice are sensitized by intraperitoneal injection of 0.5 ml of pristan (2,4,10,14-tetramethylpentadecane) with 0.5 ml of mineral oil. The time of the formation of ascitic tumors is 10-16 days. Ascites is 100-digestible.

Marker symptom: the production of specific monAT to human transferrin. Class lylgGL

The motility of glucose-6-phosphate dehydrogenase and lactate dehydrogenase isoenzymes is characteristic of mouse cells.

Contaminants of strain ICIOSp2 / 0, including bacteria, fungi, yeast and mycoplasma, were not detected. Characteristic monat, Strain MCIOSp2 / 0 hybridoma produces mouse monoclonal immunoglobulins G, subclass G1. The affiliation of immunoglobulins to this class and subclass is determined by enzyme immunoassay using conjugates of antibodies to mouse immunoglobulins of different classes and subclasses.

Antibody specificity is assessed by immunoblotting. For this, wire electrophoresis of human serum proteins with sodium dodecyl sulfate polyacrylamide gel (10% PAAG, 20 mg protein per well), then transfer the proteins to nitrocellulose strips and incubate with a monat solution at a concentration of 10 µg / ml. Antibodies bound to the antigen are detected using a conjugate of rabbit antibodies against mouse immunoglobulins labeled with peroxidase. In this case, the binding of monAT is revealed with only one protein zone corresponding to a protein with m.v. 78 kilodalton, which corresponds to m.v. transferrin. The interaction of monoclonal antibodies with any other human serum proteins was not detected.

Thus, it was found that antibodies produced by the strain react specifically with human transferrin.

The hybridoma strain stably produces monAT to human transferrin without changing affinity during long-term cultivation (60 passages in culture and 25 in animals).

The titer of monoclonal antibodies is determined by an indirect enzyme immunoassay. It is 1000 for culture liquids and 106 for ascites fluids. The concentration of monAt in a culture is 100 µg / ml, in ascites fluid 5-6 mg / ml. To determine the binding constants, monAt were incubated with several different concentrations of transferrin for 2 hours, and then the concentration of unbound antibodies was determined by enzyme immunoassay. The calculation of the constant was carried out according to the Scatchard equation. It is equal to (1.35 ± 0.35) x10 10 M.

The method of cryoconservation.

Cryoprotective environment: culture medium with 30% fetal bovine serum and 10% dimethyl sulfoxide (cryoprotector). Cells (10 cells in 1 ml) are suspended in a cold cryoprotective medium (+ 4 ° C). The suspension is poured into sterile ampoules at the rate of (1-2) x107 cells per vial. The vials are cooled to -70 ° C at a rate of 1 ° C / min and transferred to a container with liquid nitrogen.

To defrost the cells, the ampoules are removed from nitrogen and quickly thawed to 37 ° C in a water bath. Cells are washed twice in phosphate buffer. Over 60% of viable cells are obtained by trypan blue staining.

MonAT from ascitic fluids is obtained by salivating ammonium sulfate 3 times at a concentration of 40% of saturation, followed by dialysis against potassium phosphate buffer pH 7.5 with the addition of 0.1 M NaCl, followed by

affinity chromatography on protein A. The degree of purification of immunoglobulins by electrophoresis is about 90%.

Example. Highly purified human transferrin is sorbed on a polystyrene 96-well plate, making 100 μl of transferrin solution with a concentration of 10 μg / ml in phosphate buffer with a pH of 7.4 with 0.1 M Nad and incubating for 1 hour at 37 ° C. Then the plates are washed 3 times with the same buffer with the addition of 0.05% tween-20 and the perioidase conjugate of monAt of the ICIO Sp2 / 0 clone, obtained by the periodate method, in a concentration of 1 µg / ml is added to the sample containing 5-100 µg

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Claims (1)

transferrin (in the same phosphate buffer). They are incubated for 30 minutes, then washed 6 times with the same buffer with the addition of tween, and 100 µl of substrate each are added — orthophenylenediamine 0.25 mg / ml solution and 0.03% hydrogen peroxide in acetate buffer pH 5.5. After 15 min, the absorbance at 492 nm was measured. The results show that the minimum detectable concentration of transferrin is 78 mcc per 1 ml. Claims of the Invention Hybrid cultured cell strain of animals Mus musculus L No. BCKK (II) 345 D. Produces monoclonal antibodies to human transferrin.