CS263123B1 - Mouse lymphocytaric hybridome producing antibody against human alpha-fetoproteine - Google Patents

Abstract

The present invention relates to mouse lymphocytic antibody-producing hybridomas human alpha-fetqprotein deposited in collection of hybridoma U3tavu molecular genetics C3AV under the designation IMG CZAS AFP-01. Hybridoma monoclonal antibody alone AFP-01 is suitable for use in enzyme immunoassay and radioimmunoassay to determination of alpha-fetoprotein and for purification human alpha-fetoijrotein by affinity chromatography. To increase enzyme sensitivity and radioimmunoassays a sandwich test in combination can be used with other monoclonal antibodies against human alpha-fetoprotein (AFP- -03 or AFP-11) prepared at the Institute molecular genetics.

Description

The invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line Sp2 / O-Ag14 and a mouse spleen lymphoid cell producing an antibody against human alpha-fetoprotein.

Alpha-fetoprotein antibodies can be produced by alpha-fetoprotein being injected repeatedly as an antigen into test animals, most commonly rabbits. The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies used, in particular, for the quantitative determination of plasma alpha-fetoprotein by radioimmunoassay or enzyme-immunoassay analysis. This procedure, called conventional immunization, has several disadvantages. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum varies and does not repeat in each individual organism. Generally, in addition to antibodies to the desired antigen, the organism will produce antibodies against the impurities of the antigen preparation. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range.

The disadvantage of conventional sera against alpha-fetoprotein is in principle eliminated by the use of hybridora technology. The hybridoma product - a monoclonal antibody - is directed against a single antigenic determinant. If a sufficiently large set of hybridomas is constructed, it is likely that a hybridoma synthesizing an antibody that is directed against a particular antigenic determinant of a human alpha-fetoprotein molecule is likely to be found. This means that molecular antibody clones can be constructed against various antigenic determinants of the alpha-fetoprotein molecule chain. The advantage of obtaining monoclonal antibodies against several antigenic determinants is mainly in the possibility of using combinations of antibodies directed against different detectors to increase the sensitivity of analytical assays for the determination of alpha-fetoprotein in body fluids. This approach, ie, the preparation of hybridomas producing monoclonal antibodies specifically recognizing alpha-fetoprotein, has been used by several authors (eg Tsung, Y.-K., Milunsky,

A., Alpert, E .: Derivation and characterization of monoclonal hybridoma antibody specific for human alpha-fetoprotein, J. Immunol. Meth. 39: 363-368, 1980; Stenman, U.H., Sutinen, M.L., Selander, R.K., Tontti, K., Schroder, J .: Characterization of Monoclonal Antibody to Human Alpha-Fetoprotein and Its Use in Affinity Chromatography, J. Immunol. Meth. 46: 337-445, 1981; Uotila, M., Ruoslahti, E., Engvall, E., Two-site Sandwich Enzyme Iramunoassay with Monoclonal Antibodies to Human Alpha-Fetoprotein, J. Immunol. Meth. 42:11 (^ 15, 1981; Hunter, WM, Bennie, JG, Brock, DJH, Van Heyningen, V .: Monoclonal Antibodies for Use in Immunoradiometric Assays for (X-fetoprotein, J. Immunol. Meth. 50: 1) Van Heyningen, V., Barron, L., Brock, DJH, Crichton, D., Lawrie, S .: Monoclonal Antibodies to Human α-β Ethoprotein: Analysis of the Behavior of Three Different Antibodies, J Immunol. Meth.

Two groups also used prepared monoclonal antibodies to prepare an antigen, i.e., alpha-fetoprotein, by immunoaffinity chromatography on an immobilized monoclonal antibody (Stenman et al .; Van Heyningen et al., Supra). Pure antigen is used as a standard for determination in serum samples and other biological materials.

Solutions of high concentration of chaotropic substances (magnesium chloride or urea) are used to release the antigen bound to the immunosrobent, which can damage the native alpha-fetoprotein structure and thus affect the reliability of the obtained standard.

Significant progress will be achieved when monoclonal antibodies produced by the hybridoma deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4 are used to isolate alpha-fetoprotein by immunoaffinity chromatography.

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Vídeňská δ. 1083, under the designation IMG CZAS AFP-01. Using this monoclonal antibody to isolate alpha-fetoprotein, very gentle antigen wash conditions can be used.

Said hybridoma was obtained by a method known in the literature (Fazekas de St. Groth, S., Scheidigger, D .: Production of monoclonal antibodies: Strategies and Tactics,

J. Immunol. Meth., 35: 1, 21, 1980; Galfré, G., Howe, SC, Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977) by cloning a set of hybrid cells resulting from fusion of mouse myeloma cells Sp2 / O-Ag14 line and cells obtained from the spleen of BALB / c mice immunized with human alpha-fetoprotein.

The advantage of hybridomas is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with human alpha-β etoprotein. The AFP-01 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALE / c mouse mice. From preserves stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the AFP-01 hybridoma reacts with human alpha-fetoprotein and does not need to be deprived of ballast antibodies.

Example 1

To expand the hybridoma cells in vivo, 5 x 10 6 cells were injected into the peritoneal cavity of mice. To improve attachment of the cells applied, the mice were treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells. After 10 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 2 ml of ascites fluid containing 6.5 mg / ml immunoglobulin was obtained. The AFP-01 antibody reacted with a specific antigen (human

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- 4 263 123 alpha-fetoprotein) in an enzyme-immunoassay (using a horseradish peroxidase-labeled anti-mouse antibody) up to a 1:10 dilution and in a radioimmunoassay (using a rabbit antiserum against mouse immunoglobulin and polyethylene glycol to precipitate antigen-monoclonal antibody complexes) up to a 1:10 dilution. This hybridoma produces a monoclonal antibody that can also be used to determine the alpha-fetoprotein concentration in a sandwich enzyme immunoassay in combination with other alpha-fetoprotein monoclonal antibodies (AFP-11 or AFP-03), either using immobilized AFP- Peroxidase-conjugated AFP-11 or AFP-03 antibodies, or vice versa, using immobilized AFP-11 antibodies, respectively. AFP-03 and peroxidase conjugate. The monoclonal antibodies AFP-01, AFP-03 and AFP-11 react with different sites (antigenic epitopes) on the human alpha-fetoprotein molecule.

Example 2

A globulin fraction was prepared from ammonium sulfate precipitation from 4 ml of ascites fluid containing AFP-01 and bound to 2 ml of CNBr-activated Sepharose 4B. The column prepared from this immunosorbent was washed with 5 ml of 0.1 M glycine-HCl buffer pH 2.8 and then with saline. Thereafter, 40 ml of neonatal cord blood serum was filtered through this column over 4 hours at 4 ° C, the column was washed with 20 ml of saline containing 0.5 M NaCl and the specifically adsorbed alpha-fetoprotein was washed out of the column at 37 ° C with saline containing 50 ml. mM ethylenediaminetetraacetic acid (pH adjusted to 7.2). The overall yield was 2.9 mg of alpha-fetoprotein, the purity of which by electrophoretic and high performance liquid chromatography was at least 90%. Immunoglobulin, which contaminated some of the preparations obtained, could be removed by gel chromatography.

AFP-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells. In vitro they grow as semi-suspension

26T 12T culture. The basic culture medium is Eagle's minimum essential medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (called KEMI, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES (N- (2-hydroxyethyl) piperazine-N * -2 acid) for the cultivation of AFP-01 hybridomas. -ethanesulfone) (10 mM) and inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 19.8 hours. The antibody produced is a monoclonal immunoglobulin of the IgG1 subclass of light chain type K, its isoelectric point is in the range of pH 6.6 6 6.8.

The monoclonal antibody produced by the AFP-01 hybridoma reacts specifically with human alpha-fetoprotein.

The AFP-01 hybridoma can be used industrially as a source of monoclonal antibody against human alpha-fetoprotein in analytical or preparative methods.

The monoclonal antibody of AFP-01 hybridomas can be used for the analytical determination of alpha-fetoprotein in clinical diagnosis in healthcare facilities, particularly for the diagnosis of cancer (hepatoma, teratoma, etc.) or congenital malformations (cleft spine - fetal spina bifida). Immobilized monoclonal antibodies can be used to purify alpha-fetoprotein by affinity chromatography from neonatal cord blood serum.

Claims (1)

SUBJECT OF THE INVENTION Murine hybridoma cell IMG Czasy AFP-01, wherein _R that produces a monoclonal antibody of subclass IGOL against human alpha-fetoprotein.