SU1721089A1 - Strain of hybrid cultured mammalian mus musculus l - a producer of monoclonal antibody to glutamate receptors of human brain - Google Patents

Abstract

This invention relates to hybridoma technology and can be used to detect glutamate receptors in the human brain. The aim of the invention is to obtain a hybridoma strain producing monoclonal antibody (MonAt) to glutamate receptors in the human brain. The strain obtained on the basis of cells of the immune spleen of the mouse BALB / C and cells of the mouse myeloma line X-63-Ad-653. MonAt belongs to subclass 1d 61. The strain is deposited under VSKK (P) number 363 D. The cells of the strain are cultured under standard conditions on medium with 20% serum, as well as in the form of ascites tumor in syngeneic mice. Studying the interaction of MonAt with glutamate neuroreceptors by enzyme immunoassay revealed high specificity and the possibility of using this antibody for detecting immobilized glutamate receptors using the enzyme immunoassay. Binding of monat produced by strain A-HGR-7C5 with glutamate receptors on sections of post-mortem preparations of the human cerebral cortex showed that it was obtained and suitable for a comparative analysis of the distribution of glutamate neuroreceptors in norm and in the pathology of the central nervous system. AND

Description

The invention relates to hybridoma technology and can be used to detect the antigen, the glutamate receptors of the human brain.

The purpose of the invention is to produce a hybridoma strain producing monoclonal antibody to glutamate receptors in the human brain, suitable for detecting these receptors, studying their structure and functioning. The strain of hybridoma A-HGR-7C5 SCC (P) MZbZD prepared as follows,

BALB / C mice are immunized intraperitoneally with 50 µg of glutamatching membrane proteins isolated from post-mortem preparations of the human cerebral cortex and identified in experiments radioligand binding to H -1 glutamate as glutamate receptors, in complete Freydante's adjuvant. In parallel, the antigen is administered in an amount of 25 µg into the tail vein. After 7 days, the immunization is repeated without adjuvant. 7 days later, mice whose serum has a titer of 15000 (titer determined by ELISA) are immunized for 3 days, injecting 25 µg of antigen into the tail vein daily. After this time, 12x10 spleen cells of immune mice

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hybridized with 25x10 myeloma cells; X63.Ag8.653 mice in the presence of RPMI1640 medium containing 45% (v / v) polyethylene glycol (mol. 3350) and 15% dimethyl sulfoxide. After hybridization and washing with polyethylene glycol, cells are seeded into 96-well plates at 2x105 cells per well. For the cultivation and selection of hybrid cells, RPMI 1640 medium is used with the addition of 10% horse serum and 10% fetal bovine serum, .1–0 M Hyperxanthine,

 aminopterin 1.6 x 10 5Mthymidine. Producer hybrids are cloned by the finite dilution method, seeding 1 cell per well of the plate containing 4x104 cells of the spleen as a feeder layer. After the second cloning, almost 100% of the subclones produce antibodies to the glutamate receptors of the human brain. Antibody production is maintained for at least 28 passages in culture.

The strain is characterized by the following properties.

The culture medium is RPMI 1640 supplemented with 10% fetal bovine serum and 10% horse serum, 4 mM L-glutamine, 10 mM pyruvate Na. 100 μg / ml penicillin, 100 μg / ml streptomycin, amino acids and vitamins for the basal Eagle's medium, 0.05 mM mercaptoethanol. The cultivation is carried out at 37 ° C in an atmosphere of 5% carbon dioxide.

Sow dose Y5 cells / ml. After 3-4 days, the concentration of antibodies in the culture medium reaches 10-20 µg / ml. Contamination with bacteria, fungi and mycoplasma was not detected. For long-term storage, hybridoma cells are frozen in Fetal Bovine Serum supplemented with 10% dimethyl sulfoxide. Freezing mode: 4 ° C / min to 4 ° C, then 1 ° / min to -70 ° C, after which the cells are transferred to liquid nitrogen. The cells are thawed, transferring the ampoule from liquid nitrogen to a water bath at 37 ° C.

To grow a hybrid neurotromic strain in ascites form, the cells are administered intraperitoneally to BALB / c mice. processed piers, in the amount of 4x106 cells per mouse. Ascites ripens in 12–14 days.

Hybridomas are conventionally called A-HGR-7C5. The monoclonal antibody produced by the A-HGR-7C5 strain belongs to the IgG isotype, subclass G1, and binds to the supra-membrane region of the glutamate receptors of the human brain.

The specificity of the interaction of antibodies with neuroreceptors of L-glutamate is revealed by enzyme immunoassay and immunohistochemistry.

In order to obtain a preparation of monoclonal antibodies in an amount necessary for carrying out immunochemical studies, hybrid cells are placed in a plastic bottle with an area of 25 cm2 and 5x105 cells in 5 ml of RPMI 1640 medium with 10% fetal calf serum, 10% horse serum, 4 mM glutamine , 10 mM pyruvate Na, 100 µg / ml penicillin, 100 µg / ml streptomycin and 0.05 mM mercaptoethanol. Cells are cultured for 3-4 days at 37 ° C in an atmosphere containing 5% carbon dioxide. The culture medium is harvested by centrifugation for 10 min at 800d. The resulting supernatant is used to determine the specificity of the antibody to interact with the glutamate receptors of the human brain.

Determination of the specificity of the interaction of a monoclonal antibody with an antigen by ELISA.

A polystyrene 96-well plate is sorbed with antigen — 1 μg per well in 100 μl of carbonate buffer (100 mM, pH 9.6) at 4 ° С overnight. After the tablet was saturated with a 0.05% solution of Tween-20 in phosphate-buffered saline (5 mM NahtePCM. 140 mM NaCI, pH 7.4) to reduce the nonspecific binding of antibodies to plastic (1 h, 20 ° C), t 100 μl of culture medium strain A-HGR-7C5 (1 h, 20 ° C). The plate is then washed 4 times with phosphate-saline buffer containing 0.05% Tween-20, and rabbit antibodies against mouse immunoglobulins conjugated with horseradish peroxidase (2 µg / ml, 1 h, 20 ° C) are added to the rabbit. After the end of incubation, the incompatible reaction products are washed as described above, and the bound conjugate is determined by the cleavage of peroxidase substrate (HaCte) in the presence of the chromogen, orthophenylene diamine. The positive reaction is characterized by the development of a brown color, the intensity of which is measured by absorption at 492 nm. Glutamate utilizing enzymes, glutamate dehydrogenase and glutamate decarboxypase, are used as control antigens.

The results show that the antibodies effectively bind to the glutamate receptors of the human brain and do not interact with control antigens.

Determination of the specificity of monoclonal antigen by immunohistochemical analysis.

For this study, sections of postmortem preparations of the human cerebral cortex are used. Obtained at autopsy (no later than 1 h after death) bark samples are placed in liquid nitrogen and used to prepare sections (5 µm). A 10% formalin solution in phosphate-buffered saline is used to fix the sections. Fixation is carried out for 10 minutes at 20 ° C. Sections were washed with phosphate-saline buffer for 1 hour and monoclonal antibodies (culture medium) were added with 10% human serum (incubation for 30 minutes, 20 ° C). After washing with phosphate-saline buffer (10 min, 20 ° C), rabbit antibodies against mouse immunoglobulins with 10% human serum are applied. The sections are washed as described above and incubated with culture medium containing monoclonal antibodies against peroxidase and peroxidase (30 min, 20 ° C), washed again, the reaction is developed with the help of the substrate diamine benzidine in Tris-HCl buffer (50 mM Tris, 150 mM NaCI, pH 7.5) in the presence of H2U2. A monoclonal antibody detects intensely stained neuronal bodies, neuro saw, axial cylinders of myelinated and unmyelinated fibers using a light microscope. To determine the specificity of staining, sections were preincubated with L-glutamate (100 µm) in tris-citrate buffer (50 mM Tris, 10 mM CaCfc, pH 7.2) in

for 30 min at 37 ° C. Further processing of the slices is carried out as described above. Pretreatment of the slices with L-glutamate leads to a significant reduction and, in some cases, to the disappearance of the color.

As a control, monoclonal antibodies to endotoxin of gram-negative bacteria (Ig class M) and antibodies to collagen II (Ig class G) are used in these experiments. With these antibodies, no structures of the nervous tissue are detected.

These results indicate the high specificity of the monoclonal antibody -A-HGR-7C5, the possibility of using this antibody as a marker for detecting glutamate receptors and their distribution on sections of the human brain in normal conditions and in various pathologies of the central nervous system using the immunoenzyme method and, in perspective, the production of pure preparations of glutamate receptors on columns with immobilized antibodies.

Claims (1)

Claims of the invention The strain of hybrid cultured animal cells Mus musculus L. VSCK (P) N363D is a producer of monoclonal antibodies to glutamate receptors in the human brain.