CS264947B1 - Mouse lymphocyte hybridoma GF-01 producing antibody to glial acid protein of middle filaments - Google Patents

Abstract

The solution relates to a lymphocyte hybridoma producing an antibody against human glial acidic protein of intermediate filaments, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague under the designation IMG CZAS GF-01. The monoclonal antibody of the hybridoma GF-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis, for testing tissues, cells and body fluids, as well as for visualization of glial acidic protein in cells at the optical and electron microscope level and also immunohistochemical detection of glial acidic protein on tissue sections.

Description

The invention relates to the murine lymphocyte hybridoma GF-01 producing a monoclonal antibody of the IgM class against human glial intermediate filament acidic protein (GFAP).

Until now, antibodies have generally been produced by immunizing animals by repeatedly applying the antigen, usually together with an adjuvant, to experimental animals, most often rabbits, guinea pigs, pigs or goats. The serum of animals immunized in this way is collected after a certain period of exposure to the antigen and serves as a source of antibodies that are used for the diagnosis and treatment of a number of diseases, but also for studying the occurrence and function of these antigens in cellular pathophysiology. This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable in each individual organism. In addition to antibodies to the desired antigen, the organism usually produces antibodies against impurities of the antigen preparation, which must be removed from the serum in a complex process called saturation. In the case of antisera against intermediate filament proteins, the situation is complicated by their considerable mutual similarity, so preparing a conventional antiserum against a single intermediate filament protein by saturation is laborious. Therefore, production batches of conventional antisera are very difficult to standardize and come out of production with a wide range of quality. For the production of each batch, it is necessary to prepare a highly pure immunizing antigen and other antigens to saturate ballast antibodies against impurities or similar molecules such as vimentin, desmin or keratins.

These shortcomings are overcome by using a hybridoma producing a completely homogeneous monoclonal antibody against glial intermediate filament acidic protein (GFAP) according to the invention.

This mouse lymphocytic hybridoma GF-01 was obtained in a manner known from the literature (Kohler G., Milstein C.: Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495, 1975; Galfré G., Howe S. C., Milstein C., Butcher G. W., Howard J. C.: Antibodies to major histocompatibility antigen produced by hybrid cell lines, Nature 266:550, 1977; Fazekas de St. Groth S., Scheidigger D.: Production of monoclonal antibodies: Strategies and tactics, J. Immuol. Meth. 35: 1-21, 1980) by cloning a set of hybrid cells, resulting from the fusion of cells of the mouse myeloma line Sp 2/0 and cells obtained from the spleen of BALB/c mice, immunized with an isolated cytoskeletal preparation obtained from pig brain.

The advantage of the mouse lymphocyte hybridoma GF-01 is that it binds to the target structure GFAP, which is stable even with relatively drastic fixation of human material with formaldehyde and subsequent embedding in paraffin, which is a routinely used fixation of histological material in all histopathological laboratories. Other antibodies known abroad, e.g. anti GFAP antibody from Labsystems, Helsinki, Finland, react on similarly prepared sections only after enzyme digestion or require more gentle special fixation. None of the antibodies against human glial intermediate filament acidic protein produced by the hybridomas described so far have the ability to react on paraffin, undigested sections. The GF-01 antibody also gives very good results on frozen tissue sections. Table 1 shows the strength of the reaction of the GF-01 antibody when using various methods of tissue processing:

Table 1

Intensity of the GF-01 antibody reaction on tissue sections fixed in three different ways (+ moderate reaction, ++ strong reaction, - non-reactivity)

Fixation used methanol/acetone metacarb formaldehyde

Tissue preparation frozen paraffin embedded paraffin embedded

Resulting reaction + ++ ++

The results show that this antibody can be used in all pathology departments for routine differential diagnosis to distinguish tumors originating from glial cells.

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The GF-01 hybridoma can be cultured in vitro in media suitable for growing animal cells or in vivo in the peritoneal cavity of BALB/c mice. The GF-01 hybridoma is suitable for long-term storage in cans stored in liquid nitrogen. Monoclonal antibody production can be initiated after thawing the cell can without the need for additional antigen. The monoclonal antibody produced by the GF-01 hybridoma reacts with human GFAP and does not react with any intermediate filament protein or other cellular proteins, as determined by immunoblotting, enzyme-linked immunosorbent assay, indirect immunoperoxidase, and indirect immunofluorescence. This specificity was demonstrated by electrophoretic transfer of a wide range of cell and tissue lysates and preparations of isolated cytoskeleton from various human tissues and cell lines, after separation of protein components in a polyacrylamide gel in sodium dodecyl sulfate under reducing conditions. After transfer of the separated proteins to a nitrocellulose membrane, GFAP was specifically visualized (detected) using GF-01 and subsequently a porcine antiserum against mouse immunoglobulins conjugated with horseradish peroxidase (ÚSOL, Prague). A monoclonal antibody against GFAP from Labsystems, Helsinki, Finland, which gave similar results, served as a positive control.

Using the immunoperoxidase reaction on tissue sections of human tissues, which was also confirmed by the results achieved with the indirect immunofluorescence method, the GF-01 antibody was found to be reactive with astrocytes and their modifications in the brain and spinal cord. A positive reaction was also demonstrated in astrocytoma and ependymoma cells. In indirect immunofluorescence using GF-01 antibodies and subsequent incubation with swine antiserum against mouse immunoglobulins labeled with fluorescein-iso-thio-cyanate, intermediate filaments of the vimentin, neutral and keratin types were not visualized in the tested cell types of several species. Reactivity with rat glioma cells was also not demonstrated. Microtubules or microfilaments distinguishable by their characteristic localization and sensitivity to colcemid and cytochalasin B did not react with the GF-01 antibody. Other structures of the tested cells also did not react, with the exception of nematin structures in 3T3 cells, which may be identical or similar to GFAP (Willingham M.

C., Richert W. D., Rutterford A. V.: Experimental Cell Research 171: 285-295, 1987). However, this positive finding can in no way lead to confusion in the identification of the intermediate filament class, as these are completely different structures.

The mouse lymphocyte hybridoma GF-01 is stored in the hybridoma collection of the Molecular Genetics Collection of the Czechoslovak Academy of Sciences in Prague under the designation IMG CZAS GF-01.

Example

In order to propagate GF-01 hybridoma cells in vivo, 1x10^ cells were injected into the peritoneal cavity of mice. In order to improve the adhesion of the injected cells, the mice were treated with paraffin oil (0.5 ml peritoneally) 14 days before the transfer of hybridoma cells. After 12 days of hybridoma growth in the peritoneal cavity, the mice were sacrificed and the produced ascitic fluid was collected. A total of 2.5 ml of ascitic fluid was obtained, which contained 2.5 mg/ml of immunoglobulin. The specificity of the obtained antibody was tested on frozen sections of human brain, both in indirect immunofluorescence using a swine antibody against mouse immunoglobulin labeled with fluorescein isothiocyanate and immunoperoxidase by reaction with a swine antibody against mouse Ig labeled with horseradish peroxidase (ÚSOL, Prague), and after electrophoretic transfer of tissue lysate prepared from human brain to a nitrocellulose membrane with subsequent detection by an indirect immunoperoxidase test (see above). The results are summarized in Table 2.

Table 2

Reactivity of the GF-01 antibody with normal human tissues (+ positivity, - negativity,

V-vimentin, D-desmin, G-GFAP, neurofilament N-proteins, K-keratins).

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Table 2 continued

Tissue Type middle filament Reaction Brain, astrocytes G + in + Cerebellum, astrocytes. Ber. glia G + in + Brain, neurons N - Cerebellum - ganglia cells N - Spinal cord, astrocytes G + in + Cells, connective tissue In -

Peripheral nerve cells

knitting N in - Urinary epithelium bladder ^K í 18' 19 - Language In + D + K - Liver To + In - Skin To + In - thyroid gland in + To + D - Vessels in + D -

The results presented in Table 2 show that the GF-01 antibody reacts exclusively with human glial fibrillary acidic protein. The narrow specificity of the antibody was confirmed by immunoblotting, where a strong reaction with GFAP was found, while other cytoskeletal proteins tested were negative (see Table 3).

Table 3

Reactivity of the GF-01 antibody with cytoskeletal proteins from human brain (+ reactivity,

- negativity)

Cytoskeletal protein Reaction in immunoblotting alpha-tubulin beta-tubulin vimentin proteins neurofilament

GFAP +

The specificity of the obtained antibody was further tested on a panel of cells from tissue cultures, both in indirect immunofluorescence from tissue cultures, both in indirect immunofluorescence using a swine antibody against mouse immunoglobulin labeled with fluorescein isothiocyanate, and after electrophoretic transfer of cell lysates to a nitrocellulose membrane with subsequent detection by indirect immunoperoxidase assay. The results are summarized in Table 4.

Table 4

Reactivity of the GF-01 antibody with cells of different lineages (V-vimentin, K-keratins, N-proteins, neurofilament, IF-immunofluorescence, IB-immunoblotting)

Species Tissue origin Name Type interm. Reaction filament IF IB

LEP V - ^X human lung fibroblast round cell carcinoma

At 1280

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Table 4 continued

Type Tissue origin Name Type interm. filament IF IB reaction epidermoid carcinoma A 431 To - mouse fibroblast 3T3 Swiss In x neuroectoderm C 1300 N - rat glioma C6 G + V

x positivity of nematin structures

The GF-01 hybridoma cells have a morphological appearance typical of myeloma cells. They grow in vitro as semi-suspension cultures. The basic culture medium is RPMI supplemented with L-glutamine (3mM), penicillin, streptomycin, gentamicin and inactivated horse serum. The hybridoma is cultured at 37 °C, the median generation time is 19 hours and the modal chromosome number 7 months after the hybridoma was constructed was 76. The antibody produced is a mouse monoclonal immunoglobulin of the IgM class.

The monoclonal antibody produced by the GF-01 hybridoma reacts specifically with human glial acidic fibrillary protein even on formaldehyde-fixed paraffin-embedded histological sections and is therefore usable in all histopathological laboratories using various immunohistochemical, enzyme-immunological, immunofluorescence, and immunoblotting tests. In addition to medical practice, it is useful in the field of theoretical research on human intermediate filaments.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS GF-01, producing a monoclonal antibody of the IgM class against the human medium filament glial acid protein.