SU1527257A1 - Strain of hybrid cultivable mus musculus cells as producer of monoclonal antibodies to inducer of amiotrophic leukospongiosis - Google Patents

Abstract

The invention relates to hybridoma technology and can be used in the preparation of diagnostic products. Strain ALMA-7 deposited under the number VSKK (P) N 223D. A strain is obtained by immunization of BALB / C mice with the PRP protein with a mol.m. of 27-32 kDa according to the scheme. Activated lymphocytes are fused with myeloma cells X63-AG8 / 653 in a 4: 1 ratio using PEG 4000. The hybridoma produces monoclonal antibodies of class JGG 2a. Stable production of antibodies for 29 passages. The strain in mice is not intertwined. 1 tab.

Description

The invention relates to hybridoma technology and may be called upon in the preparation of diagnostic preparations.

Strain was prepared as follows.

Mice of the BALB / C line with a mass of 18-20 g are immunized with Prg protein with mol. 27-32 kDa, obtained by sequential treatment of the cerebral suspension with nucleases, proteases, detergents in combination with gradient centrifugation, according to the scheme: 4 times every 7 days, intragastric injection of ipr protein with complete Freund adjuvant . The protein concentration of pgp is 1.0 mg / ml. 7 days after the last immunization, a booster dose of protein was introduced into the spleen in a volume of 0.1 ml.

On day 3, after a booster dose of the protein, spleens were removed from the mice and a suspension of activated lymphocytes was prepared, used for fusion with X63-Ag 8/653 digestible myeloma cells in a 4: 1 ratio. Cell fusion is carried out using a 50% polyethylene glycol 4000 solution. Hybrid cells are cultured on GAT selective medium (hypoxaitin – aminopterin – thymidine). Out of 125 clones formed during fusion with the X63-Ag 8/653 parent, 9 hybrid clones were detected that gave specific binding to the pgp protein in the ELISA assay. By serial passage of the cells of the most productive clone ALMA-7, they entered into mass

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culture When inoculated with linear mice BALB / C ALMA-7 cells at a dose of 6-12-10, ascites tumors are induced.

The obtained strain ALMA-7 is deposited under the VSKK number (P) No. 223D and is characterized by the following features.

Morphological signs.

The culture consists of rounded cells of varying size, weakly attached to the substrate, with dramas occupying most of the cell. The nucleoli are large, there are two nuclear cells. The cytoplasm has the appearance of a thin rim, or the nucleus is shifted to one side of the cell.

Cultural features.

The culture medium is Dulbecco's medium with 15% fetal caloric effusion, sodium pyruvate, 4 r / ji glucose, 2 1 glutamine, 50 µg / ml of the antibiotic gentamicin. The growth pattern is a stationary suspension. Shooting method - shaking. Passing frequency - in 2-3 days. A seeding dose of 150,000 cells in 1 ml. The multiplicity of sieving (1: 3) - (1: 5). Stable production of antibodies for 29 passages.

Cultivation in vivo.

When inoculated with linear BALB / c mice, ALMA-7 cells at a dose of 6-12-10 induce the formation of ascitic tumors after an average of 18 days. The lush pretreatment (at least 5-7 days prior to the introduction of the cells) is treated with a pristan at a dose of 0.4-0.5 ml per mouse to improve ascito formation. On average, 3.0 ml of ascitic fluid is obtained from one mouse. The ascitic fluid is clarified by centrifuging at 3000 rpm for 10 minutes. igG is precipitated with ammonium sulfate (30% saturation) and is purified by affinity chromatography. The strain ALMA-7 on soaps is not intertwined.

Product Feature

The class of produced immunoglobulins is igG2a. The antibodies produced are characterized in a point immunoassay method (TIFM).

Isozymes.

In 250-500 ml culture mattresses, ALMA-7 hybrid cells are sown at a concentration of 150000– 200000 cells / ml in Dulbecco’s sodium pyruvate medium, 15% fetal caloric, 4 g / l glucose, 2 mM glutamine and 50 µg / ml gentamicin. Cultures were incubated in a steady state for 3-4–4 days. JQ Cells are separated by centrifugation. The supernatant is stored at minus 20 ° C and used as QL samples. The cell pellet is resuspended in 1 ml of medium without serum and injected into the cavity of the syngeneic membrane. After 12–15 days, an ascites tumor is formed, which is used as a sample.

Example 2. Determination of the specific activity of monoclonal antibodies TIFM.

On nitrocellulose filters, nano with 20 μl of purified protein of 27- 32 kD AL, dried and washed with 1 r of solution containing 20% isopropyl alcohol and 7% acetic acid, 3 times with distilled water. At the same time, a buffer — 0.05 M Tris-HC1, pH 7.4 (buffer O, containing 0.5% gelatin) is applied to the filters. After contact, the test samples are applied to the filters and placed in a thermostat at 37 ° C. for 1 hour. 5 times in buffer 1 containing 0.05% Triton X-100 (buffer 2) and 1 time with buffer 1. Apply anti-immunoglobulin labeled

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The mobility of glucose-6-phosphate dehyde-55 by peroxidase for 1.5 h and placed in

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Contamination

No bacteria were detected, fungi and yeast were not detected, mycoplasmas were not detected, viruses — particles of type A and C were found, similar to particles of the parental strain of X63-Ag 8/653 cells.

Cell preservation.

ALMA-7 cells are frozen at if 24 and 26 passages. The total number of ampoules 40 to 5-7 million cells in the ampoule. The protection medium is a growth medium with 50-60% fetal calf shank and 10% glycerol. Survival rate when defrosting 74%.

PRI and MER 1. Cultivation of hybrid cells and the accumulation of monoclonal antibodies in culture (QOL) and ascitic fluids (AF).

In 250-500 ml culture mattresses, ALMA-7 hybrid cells are sown at a concentration of 150000– 200000 cells / ml in Dulbecco’s sodium pyruvate medium, 15% fetal caloric, 4 g / l glucose, 2 mM glutamine and 50 µg / ml gentamicin. Cultures are incubated at steady state for 3–4 days. JQ Cells are separated by centrifugation. The supernatant is stored at minus 20 ° C and used as QL samples. The cell pellet is resuspended in 1 ml of serum-free medium and injected into the cavity of the syngeneic membrane. After 12-15 days, an ascites tumor is formed, which is used as a sample.

Example 2. Determination of the specific activity of monoclonal antibodies TIFM.

20 µl of purified protein of 27- 32 kD AL are applied to nitrocellulose filters, dried and washed once with a solution containing 20% isopropyl alcohol and 7% acetic acid, and 3 times with distilled water. Then, a buffer — 0.05 M Tris-HC1, pH 7.4 (buffer 0, containing 0.5% gelatin) is applied to the samples. After contact, the samples are applied to the filters and placed in a thermostat at 37 ° C. for 1 hour. 5 times in buffer 1 containing 0.05% Triton X-100 (buffer 2) and 1 time with buffer 1. Apply anti-immunoglobulin labeled

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-55 peroxidase for 1.5 hours and placed in

rogenase and lactate dehydrogenase in the cells of clone APMA-7 is characteristic of murine cells.

thermostat at 37 C. Wash 6 times with buffer 2, 1 time with buffer 1 and 1 time with distilled water. Immersed in

a solution of 3 4-diaminobenzidine from 0.005% for 5 min and washed off with water 5-6 times.

The reaction is considered positive if a brown spot is formed on the filter.

The results of the studies are shown in the table.

The results obtained indicate that monocloidal antibodies only specifically react with the pgp 27-32 kD ALSP protein and do not react with the protein with the same molecular weight obtained from normal CNS tissue.

The obtained strain of hybrid cells ALMA-7 synthesizes highly specific monoclonal antibodies to the protein pgp - the causative agent of amyotrophic leukospongiosis of the class IgG, subclass 2a, with a titer in TIFM 1: 10000 (AF).

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Thus, monoclonal antibodies can be used as raw materials for diagnostic preparations.

Monoclonal antibodies are indispensable for the method of affinity chromatography of highly purified proteins to study the relationships between different members of the group of prions. They may be useful for studying the pathogenesis of the disease and the development of new methods for in vivo diagnosis.

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Claims (1)

Invention Formula The strain of hybrid cultured animal cells Mus musculus VSKK (P} 20 No. 223D - producing monoclonal antibodies to the pathogen of amyotrophic leukospongiosis.