CS264947B1 - Mouse lymphocytaric hybridom gf-01 producing antibody against gliophic acid protein central filament - Google Patents

Abstract

Solution that relates to lymphocyte hybridoma, producing an antibody against human the glial acidic protein of the middle filament deposited in the hybridoma collection Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague under the designation IMG CZAS GF-01. Alone the monoclonal antibody hybridomas GF-01 is suitable for use in enzyme immunoassays and radioimmunoassay, for testing tissues, cells and body fluids as well i for visualizing the glial acidic protein in cells at the optical and electron microscope and immunohistochemical detection of glial acid protein on tissue sections.

Description

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

To date, antibodies have generally been produced by immunizing animals such that the antigen is repeatedly administered as a rule together with an adjuvant to test animals, most commonly rabbits, guinea pigs, pigs or goats. The serum of the immunized animals is collected after a period of antigen treatment and serves as a source of antibodies that are used to diagnose and treat a variety of diseases, but also to study the incidence and function of these antigens in cellular pathophysiology. 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. In addition to the antibodies to the desired antigen, the organism generally produces antibodies against the impurities of the antigen preparation, which must be removed from sera in a complicated manner called saturation. In the case of antisera against medium filament proteins, the situation is complicated by their considerable similarity to one another, so preparing conventional antisera against a single medium filament protein by saturation is laborious. Consequently, production batches of conventional antisera are very difficult to standardize and are based on production in a wide quality range. For the production of each batch, a substantially pure immunization antigen and other antigens for saturation of ballast antibodies against impurities or similar molecules such as vimentin, desmin or keratin have to be prepared.

These drawbacks are overcome by the use of a hybridoma producing a completely homogeneous monoclonal antibody against the acidic glial medium filament (GFAP) protein of the invention.

This murine lymphocyte hybridoma GF-01 was obtained by a method known in the literature (Kohler G., Milstein C .: Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity, Nature 256: 495, 1975: Galfre G., Howe SC, Milstein C , Butcher GW, Howard JC: 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 pig brain derived cytoskeletal preparation.

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

Table 1

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

Methanol / acetone metacarn formaldehyde fixation used

Preparation of frozen paraffinized tissue

Resulting + ++ ++ reaction

The results show that this antibody can be used in all pathology departments for routine differential diagnosis to differentiate glial cell tumors.

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

GF-01 antibody was found to react with astrocytes and their modifications in the brain and spinal cord by means of an immunoperoxidase reaction on tissue sections of human tissues, which were also confirmed by the results of the indirect immunofluorescent method. A positive reaction has also been shown in astrocytoma and ependymoma cells. In indirect immunofluorescence using GF-01 antibodies and subsequent incubation with porcine antisera against mouse fluorescein-iso-thiocyanate-labeled immunoglobulins, the intermediate cell filaments of the vimentin, neutral and keratin types were not imaged in the tested cell types of several species. There was also no evidence of reactivity with rat glioma cells. Neither microtubules nor microfilaments distinguished by their characteristic location and sensitivity to colcemid and cytochalasin B reacted with GF-01 antibody. Also, the other structures of the test cells, with the exception of the non-latin structures of 3T3 cells, 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 class of intermediate filaments, since these are completely different structures.

Mouse lymphocyte hybridoma GF-01 is deposited in the collection of hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the name IMG CZAS GF-01.

Example

To expand GF-01 hybridoma cells in vivo, 1x10 6 cells were injected into the peritoneal cavity of mice. To improve attachment of the cells applied, the mouse was treated with paraffin oil (0.5 ml peritoneally) 14 days prior to transfer of the hybridoma cells. After 12 days of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and the produced ascites fluid was collected. A total of 2.5 ml of ascites fluid containing 2.5 mg / ml of immunoglobulin was obtained. The specificity of the obtained antibody was tested on frozen sections of the human brain, both in indirect immunofluorescence using a porcine anti-mouse immunoglobulin labeled with fluorescein isothiocyanate and immunoperoxidase reaction with a porcine anti-mouse Ig antibody labeled with horseradish peroxidase (USSOL, Prague). following electrophoretic transfer of a tissue lysate prepared from the human brain to a nitrocellulose membrane followed by detection by an indirect immunoperoxidase assay (see above). The results are summarized in Table 2.

Table 2

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

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

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Table 2 continued

Tissue Type stř. filament Reaction Brain, astrocytes G + v + Cerebellum, astrocytes. Ber. glie G + v + Brain, neurons N - Cerebellum - ganglia cells N - Spinal cord, astrocytes G + v + Cells, binders IN -

Peripheral nerve cells

knitting N in - Urinary epithelium bladder ^K í 18 ' 19 Dec - Language IN + D + K - Liver TO + IN - Skin TO + IN - thyroid in + TO + D - Blood vessels in + D -

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

Table 3

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

- negativity)

Cytoskeletal protein Reaction v. Immunoblotting alpha-tubulin beta-tubulin vimentin proteins neurofilament

GFAP +

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

Table 4

Reactivity of GF-01 antibody to cells of various lines (V-vimentin, K-keratins, N-proteins, neurofilament, IF-immunofluorescence, IB-immunoblotting)

Species Tissue Origin Name Type interm. Reaction of filament IF IB

LEP V - ^X human pulmonary fibroblast round cell carcinoma

U 1280

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Table 4 continued

Species Tissue origin Name Interm type 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 non-latin structures

GF-01 hybridoma cells have a morphological pattern of typical myeloma cells. They grow in the form of semi-suspension cultures under in vitro conditions. The basic culture medium is RPMI supplemented with L-glutamine (3mM), penicillin, streptomycin, gentamycin and inactivated horse serum. The hybridoma is cultured at 37 ° C, the mean generation time is 19 hours, and the modal number of chromosomes 7 months after hybridoma construction was 76. The antibody produced is a murine IgM class monoclonal immunoglobulin.

The monoclonal antibody produced by the GF-01 hybridoma reacts specifically with both human glial acid fibrillar protein and paraffin-embedded formaldehyde-fixed histological sections and is therefore useful in all histopathological laboratories using various immunohistochemical, enzymoimmunological, immunofluorescence, immunoblotting assays. In addition to medical practice, it is applicable in the field of theoretical research of 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.