CS267035B1 - Interspecies lymphocyte hybridoma producing antibody against stage specific embryonal antigen tec-5 - Google Patents

Abstract

The IMG CZAS TEC-05 Hybridoma Constructed fusion of mouse myeloma lineage P3-X63-Ag8.653 and rat spleen cells membrane-producing antibody antigen TEC-5. The hybrid can be used as a monoclonal source antibodies for cell identification embryonic TEC-5 antigen and for biochemical and target antigenic biological analysis structure.

Description

The present invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag8.653 and a rat spleen cell producing an antibody against the membrane antigen TEC-5 present on the surface of mouse embryonic and embryonic carcinoma cells.

The study of many aspects of developmental biology, cell differentiation and oncology requires the use of highly specific antibodies recognizing stage-specific embryonic antigens. Antibodies to cell surface antigens that do not exhibit intraspecific polymorphism cannot be prepared by direct immunization of an animal of one species with cells or their membrane fraction of another species, i.e., conventional immunization. Conventional immunization has the inherent disadvantage that sera contain antibodies against a number of membrane antigens, some of which are present on a number of cell types. Therefore, conventional sera cannot normally be used to fine-tune cell types. The preparation of pure antigens, suitable for conventional immunization, is laborious and usually unsuccessful.

The disadvantages of conventional sera against membrane antigens are in principle eliminated by the use of hybridoma technology. The hybridoma product - a monoclonal antibody - is always directed against a single antigenic determinant. If a sufficiently large set of hybridomas is prepared, it is likely that it will be possible to find a hybridoma synthesizing an antibody that recognizes an antigen with the properties of stage-specific embryonic antigens, ie that it occurs only on cells of a certain stage of embryogenesis and cell lines. which have some properties of embryonic cells.

A number of stage-specific embryonic antigens are found on the surface of embryonic and embryonic carcinoma cells. According to published results (collectively: Feizi, T .: Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are oncodevelopmental antigens. Nature 314, 53-57, 1985; Muramatsu, T .: Cell surface glycoproteins as markers in monitoring in vitro differentiation Cell Differ. 15, 101-108, 1984) it can be judged that monoclonal antibodies can be prepared that specifically react only with a certain stage of embryogenesis.

Substantial progress in the analysis of mouse embryogenesis, cell differentiation and oncology problems, reflected in the broadening of the range of signaling traits used, will be achieved if a hybridoma producing a monoclonal antibody against a newly identified antigenic determinant of embryonic and embryonic carcinoma cells is available. Collection of hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská 1083, under the designation IMG CZAS TEC-05 ·

Said hybridoma was obtained according to a method known from the literature (Kohler, G., Milstein, C .: Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256, 495-497, 1975; Kennett, RH, Me Kearn, TJ, Bechtol , B.. B. (eds.) Monoclonal antibodies, hybridomas: A new dimension in biological analysis, Plenum Press, New York, London, 1980).

Cells of the mouse myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of a Lewis rat strain immunized with cells of the embryonic carcinoma line P19S18C1A1 were used for the fusion. The TEC-05 hybridoma can be cultured in vitro in sieve cell media and is adapted for growth in vivo in the peritoneal cavity of thymic nu / nu mice. From cans stored in liquid nitrogen, antibody production can be initiated without the need for immunizations and complex antibody specificity testing.

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The advantage of said hybridoma is that it produces a homogeneous (monoclonal) antibody that specifically reacts with the later developmental stages of mouse embryos, starting with cicells and other stages of preimplantation development (cell nuclei, morulas, early and late blastocysts) and does not react with any young embryos. developmental stages or eggs.

The floor

In order to propagate hybridoma cells in vivo, 5x 10 ⁶ hybridoma cells were applied to the peritoneal cavity of athymic nu / nu mice. To increase the production of ascitic fluid, the mouse was treated with 2,6,10,14-tetramethylpentadecane oil (0.5 ml intraperitoneally) for 14 days before applying the hybridoma cells. After 14 days of hybridoma growth in the peritoneal cavity, 4 ml of ascitic fluid containing approximately 8 mg / ml immunoglobulin was collected from the mice.

In an indirect immunofluorescence assay, the monoclonal antibody reacted with eight-cell mouse embryos and all other stages of preimplantation development.

Absorption analysis showed that TEC-5 antigen is present only in a limited number of adult mouse tissues (brain, kidney, lung, stomach) and in some mouse emryonic carcinoma cell lines (Table 1). ’

During the differentiation of these cells (induced by retinoic acid), the TEC-5 antigen disappears from their surface. TEC-05 monoclonal antibody was found to bind to embryoglycan; the antigenic determinant defined by this antibody differs from all embryoglycan-specific monoclonal antibodies described so far.

The binding of TEC-05 antibody to embryonic carcinoma (EC) cells can be inhibited by 3 mM lactose and 47 mM galactose, but cannot be inhibited by a number of other sugars, including fructose, N-acetylglucosamine and N-acetylgalactosamine.

The TEC-05 antibody is particularly useful for studying the structure and function of developmentally regulated cell surface structures because it reveals subtle differences in the sugar components of embryonic carcinoma cells.

Table 1

Reactivity of TEC-05 antibody with cell lines ^ ·

1 ................... • Cell line Characteristics (origin) 2 * % cell-bound radioactivity Mice, derivation, from teratocarcinoma cells P19X1 EC (from P19) 6.9 (5.3-8.6) P19XT.1.1 EC (from P19X1) <0.2 P19S1801A1 EC (from PÍ9) 4.9 (4.1-6.4) F9 EC (129 / Sv) 8.8 (8.1-10.0) 0TF9-63 EC (from F9) 18.3 (13.3-24.3) PYS-2 Pariental endoderm (129 / Sv) 0.2 Mice transformed C13OC / E7 Neuroblastoma <0.2 BW5147 Thymom (AKR) <0.2 X63-Ag8.653 Myeloma (BALB / c) <0.2

CS 267 035 B1 ^ Origin and culture conditions and expression of other stage-specific embryonic antigens! are listed in Dráber P., Pokorná Z .: Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies. Cell. Differ. 15, 109-113,

1984.

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A direct radioimmunoassay was used in which an I-labeled monoclonal antibody (ICO 000 cpm) was incubated with 0.25 x 10 6 cells and the% cell-bound radioactivity was determined as described previously (Dráber P., Stanley P .: Cytotoxicity of plent lectins for mouse embryonal carcinoma cells (Somat. Cell Genetics 10, 435-443, 1984).

The monoclonal antibody recognizes an antigenic determinant that has been designated TEC-5 and differs from all antigenic determinants of stallion-specific embryonic antigens described so far in the world literature. The following approaches were used to define the uniqueness of the antigenic determinant TEC-5:

1) The monoclonal antibody reacted in a direct radioimmunological and indirect immunofluorescence assay with a panel of embryonic and carcinoma cell lines and did not react with cell lines derived from differentiated cells. The antibody reacted with eight 16-cell embryos, morulas, early and late blastocysts, and did not react with eggs or two- and four-cell embryos.

2) In absorption tests with tissues from adult animals, the monoclonal antibody reacted only with homogenate from brain, kidney, lung and stomach and very weakly with homogenate from testes and epididymis; did not react with homogenates from muscle, heart, liver, spleen and thymus.

3) When inducing the differentiation of embryonic carcinoma cells with 10 ^- retinoic acid doSlc to reduce the binding of the antibody to the cells with morphological manifestations of differentiation.

4) The monoclonal antibody precipitated a number of cell surface glycoproteins with a molecular weight> · 100,000. The antigenic determinant is located on a high molecular weight glycan called embryoglycan. This antibody does not react with cells that are defective in embryoglycan expression.

TEC-05 hybridoma cells have the morphological features typical of myeloma cells. They are grown in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimal essential medium with Hanks' salt mixture, supplemented with non-essential amino acids, 1-glutamine (3 mM) and sodium pyruvate (1 mM). This medium (designated H-MEMD, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin and temperature (56 °, 30 min.) Inactivated bovine serum (Bioveta, Ivanovice na Hané), 10Ϊ for culturing the TEC-05 hybridoma. The hybridoma is cultured at 37 ° C. The median generation time is 17 hours. Four months after construction, the modal number of chromosomes was 75. The monoclonal antibody produced is a rat IgM class immunoglobulin.

The monoclonal antibody produced by the TEC-05 hybridoma reacts specifically with the late preimplantation stages of mouse embryos and with embryonic carcinoma cells.

The hybridoma can be used industrially as a source of monoclonal antibody to identify cells carrying the stage-specific embryonic antigen TEC-5 and for biochemical and biological analysis of the target antigenic structure.

Claims (1)

An interspecies lymphocyte hybrid IMG CZAS TEC-05 constructed by fusing a mouse myeloma cell line P3-X63-Ag8.653 and a rat spleen cell producing an antibody against the TEC-5 membrane antigen.