CS261305B1 - Intergeneric (sewer-rat x mouse) lymphocyte hybridome producing antibody against embryonal tec-4 antigen in specific stage - Google Patents

Abstract

The present invention relates to an interspecific (rat x mouse) lymphocyte hybridoma producing an antibody against the stage specific embryonic antigen, deposited in the hybridoma collection of the Molecular Molecule genetics of CSAV under the designation CZG TAS-04. Monoclonal antibody hybridomas is suitable for identification cells bearing the site specific embryonic TEC-4 antigen (early preimplantation biochemical and biological target antigenic structure analysis.

Description

The invention relates to a novel hybridoma, i. 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-4 present on the surface of mouse embryonic and embryonic carcinoma cells.

Studying many aspects of developmental biology, cell differentiation and oncology requires the use of highly specific antibodies recognizing stage-specific embryonic antigens. Antibodies against cell surface antigens that do not exhibit intraspecific polymorphism cannot be prepared by directly immunizing an animal of one species with cells or their membrane fraction of another species, i. by conventional immunization. Conventional immunization has the inevitable 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 be routinely used to fine-tune cell types. The preparation of a pure antigen suitable for conventional immunization is laborious and mostly unsuccessful.

In principle, the use of hybridoma technology eliminates the disadvantages of conventional sera against membrane antigens. The hybridoma product - a monoclonal antibody - is directed against a single antigenic determinant. If a sufficiently large set of hybridomas is prepared, it is likely to be able to identify a hybridoma synthesizing an antibody that recognizes an antigen having the characteristics of a stage-specific embryonic antigen, i.e., only occurs on cells of a particular 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 in vitro differentiation monitoring of embryonic carcinoma cells. Cell Differ. 15: 101-108 (1984) suggests that monoclonal antibodies can be prepared that specifically react only with a particular stage of embryogenesis. All so far prepared and characterized monoclonal antibodies against stage-specific differentiation antigens recognize sugar determinants that are found either on glycolipids or on glycans with a molecular weight of more than 10,000, called an embryoglycan. ·

Significant progress in the analysis of mouse embryogenesis, cell differentiation and oncology problems, manifested in the broadening of the spectrum of signaling features used, is achieved when a hybridoma producing a monoclonal antibody against the newly identified antigenic determinant of embryonic and embryonic carcinoma cells is available. of hybrids of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská 1 083, under the name IMG CZAS TEC-04.

Said hybridoma was obtained by a method known in the literature (Köhler, G., Milstein,

C .: Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity, Nature 256: 495-497, 1975; Kennett, R.H., McKearn, T.J., Bechtol, Κ. B. (eds.): Monoclonal antibodies, hybridomas: A new dimension in biological analysis, Plenum Press, New York and London, 1980). P3-X63-Ag8.653 mouse myeloma cell line and cells obtained from the spleen of a Lewis rat immunized with embryonic carcinoma cell line P19XT.1.1 defective in embryoglycan expression were used for fusion.

The advantage of said hybridomas is that it produces a homogeneous (monoclonal) antibody that specifically reacts only with eggs and early pre-implantation stages of mouse embryos and embryonic carcinoma cells. The TEC-04 hybridoma can be cultured in vitro in a mammalian cell culture medium and is adapted for growth in vivo in the peritoneal cavity of athymic nu / nu mice. From preserves stored in liquid nitrogen, antibody production can be initiated without the need for immunization and extensive testing for antibody specificity.

Example

To expand the hybridoma cells in vivo, 5 x 10 ⁶ hybridoma cells were injected into the peritoneal cavity of athymic nu / nu mice. To increase production of ascites fluid, mice were treated with 2,6,10,14-tetramethylpentadecan oil (0.5 ml intraperitoneally) 14 days prior to hybridoma cell administration. After 12 days of hybridoma growth in the peritoneal cavity, 3 ml of ascitic fluid containing 6 mg / ml immunoglobulin was collected from the mouse. The monoclonal antibody reacted in an indirect immunofluorescence assay with non-fertilized eggs, fertilized eggs and two-cell stages of mouse embryos and did not react with 16-cell stages, morulae and early and late blastocysts of Balb / c mice. Direct radioimmunoassay and indirect fluorescence assay results showed that the antibody reacts only with embryonic carcioma cells and not with differentiated cells. Retinoic acid-induced differentiation of embryonic carcinoma cells resulted in complete disappearance of antigen in all cells with morphological manifestations of differentiation within three days of culture (see table)

Table

Reactivity of antibody produced by TEC-04 hybridoma with cell lines.

Cell line ¹ Test used to detect reactivity radioimmunological ³ immunofluorescence ⁴ (bound activity) cpm (even positive cells)

Embryonic carcinoma cells P19S1801A1

PCC4

Differentiated cells P19S1801A1-RA-3 days ² PYS-2

LEC 12

000 6 800

800 200 300 untested and the culture conditions and expression of other stage-specific embryonic entigens are reported in Dráber, P., Pokorná, Z .: Differentiation antigens of the teratocarcinoma fly stem cells defined by monoclonal antibodies. Cell Differ. 15, 109-113, 1984.

² Cells of line P19S1801A1 in which differentiation was induced by 10 ⁶ M retinoic acid, as described previously (Dráber, P., Stanley, P .: Isolation and characterization of lectin-resistant F9 cells. Somat. Cell Genetics 10, 445-454 , 1984).

12 5

A direct radioimmunoassay was used in which the I-labeled monoclonal antibody β

(100,000 cpm) was incubated with 0.25x10 5 cells, and cell bound radioactivity was measured as previously described (Dráber, P., Stanley, P .: Cytotoxicity of plant lectins for fly embryonic carcinoma cells. Somat. Cell Genetics 10, 435-443 (1984).

⁴ was used indirect immunofluorescence assay in which cells were incubated sequentially with the monoclonal antibody, rabbit antibody against the monoclonal antibody and FITC-labeled anti-rabbit antibody.

The monoclonal antibody recognizes an antigenic determinant, which has been designated TEC-4, which differs from all previously described antigenic determinants of stage-specific embryonic antigens described in the world literature. To define the uniqueness of antigens. Five approaches were used to determine the uniqueness of the TEC-4 antigenic determinant.

1) The monoclonal antibody reacted in a direct radio-immunological and indirect immunofluorescence assay with a panel of embryonic carcinoma cell lines and did not react with cell lines derived from differentiated cells. The antibody also reacted with unfertilized and fertilized eggs and two-cell stages of mouse embryos and did not react with 16-cell stages, morulae and early and late blastocysts.

2) In tissue uptake assays from adult animals, the monoclonal antibody only reacted with a homogenate of kidney and did not react with a homogenate of brain, liver, lung, thymus, spleen, testes, epididymis and muscle.

3) Retinic acid differentiation of embryonic carcinoma cells of line P19S1801A1 10 resulted in a decrease in antibody binding to cells with morphological manifestations of differentiation (see table).

4) The monoclonal antibody precipitated a cell surface glycoprotein having a molecular weight of approximately 230,000, demonstrated by electrophoretic analysis in a polyacrylamide gel in the presence of sodium dodecyl sulfate.

5) The monoclonal antibody reacted with embryonic carcinoma cell mutant cells that are defective in embryoglycan expression. That is, the TEC-4 epitope, unlike the epitopes of other stage-specific embryonic antigens found on high molecular weight carriers, is not bound to the embryoglycan.

TEC-04 hybridoma cells have morphological features typical of myeloma cells. They grow in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimum essential medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3mM) and sodium pyruvate (ImM). This medium (called H-MEMd, the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin and tempered (56 ° C, min) inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%) for TEC-04 hybridoma culture. The hybridoma is cultured at 37 ° C. The mean generation time is 17 h. 4 months after construction, the modal number of chromosomes was 78. The monoclonal antibody produced is a rat IgG immunoglobulin.

The monoclonal antibody produced by the TEC-04 hybridoma reacts specifically with eggs and early pre-implantation stages of mouse embryos and embryonic carcinoma cells.

The TEC-04 hybridoma can be used industrially as a monoclonal antibody source to identify cells carrying the stage-specific embryonic TEC-4 antigen and for biochemical and biological analysis of the target antigenic structure.

Claims (1)

Interspecific (rat x mouse) lymphocyte hybridoma IMG CZAS TEC-04, producing an antibody against the stage-specific embryonic antigen TEC-4.