SU1671687A1 - Strain of hybridous cultured mammalian cells mus musculus l - a producer of monoclonal antibody for c-terminal site a- chain of human fibrinogen - Google Patents

Abstract

The invention relates to hybridoma technology and can be used to determine the content of fibrinogen in human plasma. The purpose of the invention is to obtain a monat-producing hybridoma strain that interacts with fibrinogen but does not interact with the products of its degradation under the action of plasmin. The strain is obtained by hybridization of myeloma X63, AG8.653 cells with spleen cells and BALB / C mice immunized with partially purified fibrinogen fragments. The strain is cultured in RPMI 1640 medium with 10% bovine fetal serum, 4 mM L - glutamine, 0.05 mM mercaptoethanol and antibiotics, in an atmosphere with 5% CO ₂ . The strain is inoculated into the abdominal cavity of syngeneic mice. MonAT is a subclass of IGGI. The specificity of binding to the α α target of fibrinogen is determined by the methods of enzyme-linked immunosorbent assay and immunoblotting. The strain deposited under the number VSKK (P) N 323 D.

Description

one

(21) 4663268/13

(22) 03/20/89

(46) 08.23.91. Bksh. No. 31

(71) All-Union Cardiology Research Center

(72) G.P.Samokhin, O.V.Iitkevich, Z.A.Streltsova, T.N. Vlasik, G.F. Kalantarov and I.N. Trakht

(53) 578.085.23 (088.8)

(56) Thurlow P.I., Connellan I.M., Kenneally D.A. - Thrombosis Res., 1987, 47, p. 427-439.

(54) 11US MUSCULUS L. HYBRID CULTIVATED ANIMAL CELL STRAIN - PRODUCER OF MONOCLONAL ANTIBODY

TO THE C-END PLOT AO (-CHAIN FIBRINOGEN PERSON

(57) The invention relates to hybridoma technology and can be used to determine the content of fibrinogen in human plasma.

The purpose of the invention is to obtain a PONAT producing hybridoma strain interacting with Fibrinogen but not interacting with the products of its degradation under the action of plasmin. The strain is obtained by hybridization of X63 Ag8.653 myeloma cells with spleen cells and VAB3 / c mice immunized with partially purified Librinogen cells. The strain is cultured in RPMI 1640 medium with 10% bovine fetal serum, 4 mM L-glutamine, 0.05 mM mercaptoethanol and antibiotics, in atmosphere with 5% CO. The strain is inoculated into the abdominal cavity of syngeneic mice. MonAT refers to a subclass of IgGl. The specificity of binding to the Abrin-chain of Librinogon ™ was not determined by methods of enzyme-linked immunosorbent assay and immunoblotting. The strain deposited under the number VSKK (II) No. 323D.

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The invention relates to hybridoma technology and can be used to determine the content of fibrinogen in human sprinkled plasma.

The purpose of the invention is to obtain a monat-producing hybridoma strain that interacts with fibrinogen but does not interact with the products of its degradation under the action of plasmin.

Strain hybridoma 5A2 VSKK (II) ff 323D was prepared as follows.

BALB / c mice are immunized with intraperitoneal administration of 100 μg of the preparation of partially purified human cyanogen bromide fragments.

(fraction from 20 to 70 kD) in 0.5 ml of physiological saline solution, labeled with Lospat (SFR) (5 mM Na - (brothel buoyer; pH 7.2 - 7.4; 150 mMaC), in full adjuvant Freund. Immunization was repeated after 23 days, intraperitoneal injection of 100 µg of the same preparation into PBS without adjuvant. Three days after that, 1x10 immune cells of the spleen mice were hybridized with 6 x XY myeloma cells of X63-Ag 8.653 mice with 0.4 ml 45 % solution of polyethylene glycol (PEG) mol. mass 2000, containing 10% dimethyl sulfoxide, for 1 minute. After hybriO1

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dzatstsi and otmynki kchggok from 1G) G cells BMcoii.V vr in FH -)) The panel of 2x10 cells per well. For the cultivation and hybridization of hybrids, the PFM1-1 medium is used: 640 g with the delivery of 10% horse and 10 calves

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serum, 10 M hypoxanthine, 4 x UM ampnoterbin and 1.6x10 II thymidine. GM-breeding producers clone 2 times by the method of limiting dilutions, seeding 1 cell per well, containing 4x10 spleen cells. After two cloning almost 100%

To put the total amount of MonAt in the amount required for immunochemical studies, ascites fluid is released from the cells by centrifugation and sodium sulfate is added to a final concentration of 18%. The precipitate is separated by centrifugation, dissolved in 0.02 M

subclones produce antibodies to Fib-15 (pH 8.0) and dialyze against the same

35

rhinogenu. Antibody production is maintained for at least 40 passages in culture.

Ylam is characterized by the following properties.

The culture medium is RP11I-1640 medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 µg / ml penicillin, 100 µg / ml streptomycin, and 0.05 mmM 25 mercaptoethanol at 37 ° C in an atmosphere of 5% carbon dioxide.

Seed dose - 10 cells in 1 ml. After 3-4 days, the concentration of the antibody in the culture liquid reaches 30–20– 30 µg / ml. Contdination by bacteria, fungi, and mycoplasma was not detected. For long-term storage, hybridoma cells are frozen in fetal bovine serum supplemented with 10% dimethyl sulfoxide. The freezing mode is 4 ° C in 1 min to + 4 ° C, and then - 1 ° C in 1 minute to -40 ° C, after which the cells are transferred to liquid nitrogen. The cells are thawed, transferring ampoule 40 from liquid nitrogen to a water bath at + 37 ° C.

In order to grow the hybridoma strain in ascites form, the cells are injected intramuscularly to BALB / c mice, and we will process 2 to 5 x 10 cells per wash. Ascites ripens in 12 to 14 days. MonAt secretion by the hybridoma strain is maintained for i passages for 3 passages in ascithnon form 50m. MonAT, produced by cells of strain 5A2, belongs to the 1p, O1-subclass and binds to the epitope located in the C-Cone region of A & b Fibrinogen human strain.

The specificity of the MonAT interaction is determined by the method in which human fibrinogen or its fragments are used as antigens,

buber. After dialysis, the solution is applied to a 16x200 mm column with a DEAE-Toyopearl 650 I, equilibrated with the same buffer. Antibodies were eluted with a linear gradient of NaH2P04 (pH 8.0 - 1 10 - 160 mM). Immunoglobulins of the IgGl class elute in the range of 60–30 mM, the purity of the preparation according to polyacrylamide gel electrophoresis is at least 95%.

Testing the specificity of the interaction of MonAT with antigens by the method of enzyme-linked immunosorbent assay.

For a polyvinyl chloride 96-cell panel (Flow), microtiter sorbent antigen - 2 µg per well in 50 µl of PBS at 4 ° C overnight. Thereafter, 200 µl casein solution (2 mg / ml) containing 0.1% Tween-20 in PBS is incubated in the panel cells for 1 hour to reduce non-specific binding of the antibodies to the plastic. Then, 50 µl of a 5A2 antibody solution in 0.05 M Tris buffer (pH 7.4) containing 0.15 M MaCl and 2 mg / ml casein is added to the wells and incubated for 1 hour at 20 ° C.

After that, the panel is washed 3 times with a stream of water and 50 µl of a solution of an alkaline phosphatase conjugate with rabbit antibodies against mouse immunoglobulins diluted 1000 times with the same buffer is added and incubated for 1 hour. After 3 times cell washing 100 µl p-nitrophenyl phosphate solution (1 mg / ml) in a 10% solution of diethanolamine (pH 9.8) containing 1 mM magnesium chloride and incubated until a yellow color of the solution is developed in the cells. Optical density of solution at wavelength

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immobilized on ii i, ir-i ika ((, - Phase immuno-enzyme analysis) or separated by polyacrylamide gel electrophoresis and transferred onto a sheet of nitrocellulose (immunoblotting).

To put the total amount of the MonAt preparation necessary for immunochemical studies, ascites fluid is released from the cells by centrifugation and sodium sulfate is added to a final concentration of 18%. The precipitate is separated by centrifugation, dissolved in 0.02 M

15 (pH 8.0) and dialyzed against the same

five

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5 0 g

buber. After dialysis, the solution is applied to a 16x200 mm column with a DEAE-Toyopearl 650 I, equilibrated with the same buffer. Antibodies were eluted with a linear gradient of NaH2P04 (pH 8.0 - 1 10 - 160 mM). Immunoglobulins of the IgGl class elute in the range of 60–30 mM, the purity of the preparation according to polyacrylamide gel electrophoresis is at least 95%.

Testing the specificity of the interaction of MonAT with antigens by the method of enzyme-linked immunosorbent assay.

For a polyvinyl chloride 96-cell panel (Flow), microtiter sorbent antigen - 2 µg per well in 50 µl of PBS at 4 ° C overnight. Thereafter, 200 µl casein solution (2 mg / ml) containing 0.1% Tween-20 in PBS is incubated in the panel cells for 1 hour to reduce non-specific binding of the antibodies to the plastic. Then, 50 µl of a 5A2 antibody solution in 0.05 M Tris buffer (pH 7.4) containing 0.15 M MaCl and 2 mg / ml casein is added to the wells and incubated for 1 hour at 20 ° C.

After that, the panel is washed 3 times with a stream of water and 50 µl of a solution of an alkaline phosphatase conjugate with rabbit antibodies against mouse immunoglobulins diluted 1000 times with the same buffer is added and incubated for 1 hour. After 3-fold cell washing 100 µl of a solution of p-nitrophenyl phosphate (1 mg / ml) in a 10% solution of diethanolamine (pH 9.8) containing 1 mM magnesium chloride, and incubated until a yellow color of the solution develops in the cells. The optical density of the solution at wavelength

40S im opr mrplyu i in the first place rti i po- spectrum gomer.

Fibronectin is used as a control antigen.

The results show that the antibodies bind efficiently to human Fibriogen and hardly interact with Fibropectin.

Determination of the specificity of antibodies 5A2 in relation to the chains of Fibrinogen.

The human fibrinogen preparation was separated by polyacrylamide gel electrophoresis with an exponential gradient of polyacrylamide concentration from 7 to 18% under reducing conditions, and the separated Fibrinogen chains were electrophoretically transferred onto a nitrocellulose membrane. The membrane is stained by immunoblotting using a 5A2 antibody concentration of 5 μg / ml. When this is observed staining of the strip with mol. weighing 70 kd, which corresponds to the Aftt-chain of fibrinogen. Thus, the resulting antibody interacts with the determinant located on the Ab-chain of Fibrinogen.

The study of the specificity of antibodies 5A2 in relation to proteolytic fragments of Fibrinogen.

To a solution of Fibrinogen (3 mg / ml) in 0.05 M tris-chloride, 0.15 M sodium chloride and 5 mM calcium chloride (pH 7.4), plasmin is added to 0.2 units / ml and incubated at room temperature . After 1, 2, 10, 30 and 60 min, the cleavage is stopped by adding aprotinin to 100 kallikrein units per ml. The resulting preparations of fibrinogen of various degrees of cleavage are separated by gel electrophoresis, transferred to nitrocellulose and stained by immunoblotting using 5A2 antibodies. In this case, the staining of the bands with mol. Masses 340,45,22, 20,17 and 15 k is observed. The first of them corresponds to intact, undigested fibrinogen, the others correspond to the products of its degradation. At the same time, after 30 minutes only a strip with a molar mass of 15 kDa remains, and after 60 minutes even it disappears. In accordance with known data, that the C-terminal region of the Aob chain is most sensitive to the action of plasmin, it is concluded that the antigenic determinant recognized by antibody 5A2 is located in this region. Characteristic feature of the determinants in

TIP, THO 0 | 1L .. H gnostyo is split pch.mshmm.

Example 1. VCHPNRNR Lngite m 5L2 for cross-linking 06-cps n Fiornma Nykprom XIII.

When a blood clot forms in the plate, a specific transgch g-taminase (Factor XIII) is activated, which links the Fibrin chains to form G-D-dimer and fri-chain to form polymers, the mass of which may exceed 1000 kDa. The 6-chain cross-link is located in the C-terminal part

O-chains, therefore, study the effect of antibody 5A2 on this process.

An equal volume of 5A2 antibody solution (2 mg / ml) in 0.05 M Tris-Chloride buffer (pH 7.4) containing 0.15 M sodium chloride is added to 500 µl of donor plasma. Calcium chloride to 20 mM and thrombin to 1 U / ml are then added to the plasma and incubated overnight at 37 ° C. The resulting clot is ground and washed several times.

a buffer. Thereafter, an electro-aoretical analysis of the obtained preparation is carried out under reducing conditions. Samples are used as controls.

in which, instead of 5A2 antibodies, antibodies are added that do not interact with the components of the clot, or they do not add antibodies.

In a preparation without added antibodies or with antibodies that do not interact with the clot, electrophoregram contains two main bands. First with a pier. weighing about 90 KD corresponds to the Y-dimer, the second with the mol.

mass 53 to corresponds to the β-chain of fibrin. od -Fibrin chain is represented by high molecular weight polymers that are not part of the gel. In the presence of 5A2 antibodies, three additional bands appear on the electrophoregram. Stripes with a pier. masses of 50 and 25 kD correspond to the heavy and light chains of mouse immunoglobulin. Band with mol. Mass 67 kD corresponds to unstitched06-chains

Fibrin

 The results indicate that 5A2 antibody, specifically interacting with Fibrin, is included in the clot and is not removed during washing. In addition, this antibody inhibits the binding of the L & chain of Fibrin by HSh factor, without affecting the y-chain linking. Since it is assumed that the stitching of the Rb chains occurs with

involving glutamia 328 and 366, it can be assumed that the determinant for antibody 5A2 is between these residues, so that the antibody bound to fibrinogen (fibrin) blocks the access of factor XCh to both glutamines.

Example 2: Determination of concentration change of intact fibrinogen-0 on human plasma.

Plasmin is added to the human plasma so that its concentration is 1.7 mg / ml and incubated at room temperature. Plasma samples are taken: 15 before the introduction of plasmin through 5,20,60, 1800 and 3600 s. To stop the reaction, aprotinin up to 100 Kaplikrein units per ml is added to the samples. In a polyvinyl chloride 96-well microtiter patch coated pre-coated with 5A2 antibodies (1 µg per well), followed by blocking non-specific binding sites,

into the cell). After 20 min, the reaction is stopped by adding 50% sulfuric acid 50% per well. The optical density of the solutions is determined using an automatic microspectrophotometer. In parallel, in the same samples (but with a dilution of 5,000 times), the concentration of fibrinogen is determined using enzyme immunoassay. based on the use of polyclonal antibodies to human fibrinogen.

The results of measurements of optical density at 492 nm show that already after 5 s of incubation with plasmin, the concentration of fibrinogen, determined using MonAT 5A2, drops by a factor of 2, whereas, according to the data obtained using diagnosticum based on polyclonal antibodies, the concentration fibrinogen for 5 seconds is almost not reduced. With an incubation time of more than 3 minutes, 50 µl of samples are taken in samples each time, Fibrinogen, detected using 5A2 antibodies, is not found, while the polyclonal diagnostic gives a decrease in concentration by no more than 30-40%. Thus, AA2 antibodies can be used to determine intact fibrinogen in plasma.

100 times in Tris-NaCl buffer with casein (pH 7.6), and incubated for 1 hour at room temperature. After thoroughly washing the panel cells, cells of cells in which 30 times the plasma was diluted 100 times were incubated, 50 µl of the 5A2 antibody conjugate with peroxidase was added and incubated for 1 hour at room temperature. The cells were thoroughly washed and filled with a substrate solution (in 2 ml of 0.02 M citrate buffer; pH 4.7), 20 µl of a solution of orthophenylenediamine in methanol with a concentration of 10 mg / ml was dissolved and

divided with the help of antibodies 5A2, then yes, as a polyclonal diagnosticum, the concentration is reduced by no more than 30–40%. Thus, AA2 antibodies can be used to determine intact fibrinogen in plasma.

Claims (1)

Invention Formula The strain of hybrid cultured cells of animals Hus rausculus L. BCKK (II) No. 323D is a producer of monoclonal antibodies to C-terminal learning. 1 μl of 30 hydrogen peroxide (100 μl of 40 CTKU of the human fibrinogen AA chain. into the cell). After 20 min, the reaction is stopped by adding 50% sulfuric acid 50% per well. The optical density of the solutions is determined using an automatic microspectrophotometer. In parallel, in the same samples (but with a dilution of 5,000 times), the concentration of fibrinogen is determined using enzyme immunoassay. based on the use of polyclonal antibodies to human fibrinogen. The results of measurements of optical density at 492 nm show that already after 5 s of incubation with plasmin the concentration of fibrinogen, determined using MonAT 5A2, drops by a factor of 2, whereas, according to data obtained using the diagnosticum based on polyclonal antibodies, the concentration of fibrinogen in 5 seconds it practically does not decrease. With an incubation time of more than 3 minutes, there is practically no Fibrinogen in the samples, which is detected using 5A2 antibodies, while the polyclonal diagnosticum gives a decrease in concentration by no more than 30-40%. Thus, AA2 antibodies can be used to determine intact fibrinogen in plasma. Invention Formula The strain of hybrid cultured cells of animals Hus rausculus L. BCKK (II) No. 323D is a producer of monoclonal antibodies to C-terminal learning.