CS264946B1 - Mouse lymphocyte hybridoma NF-01 producing antibody against the 210 kDa protein neurofilament - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against the human 210 kDa neurofilament protein, stored in the hybridoma collection of the Molecular Genetics Collection of the Czech Academy of Sciences in Prague under the designation IMG CZAS NF-01. The monoclonal antibody of the hybridoma NF-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis, testing of tissues, cells and body fluids, as well as for visualization of the 210 kDa neurofilament protein in cells at the optical and electron microscope level and also for immunohistochemical detection of 210 kD and neurofilament protein on tissue sections.

Description

(57) The solution concerns a mouse lymphocyte hybridoma producing an antibody against the human 210 kDa neurofilament protein, stored in the hybridoma collection of the Molecular Genetics Collection of the Czech Academy of Sciences in Prague under the designation IMG CZAS NF-01. The monoclonal antibody of the hybridoma NF-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis, testing of tissues, cells and body fluids, as well as for visualization of the 210 kDa neurofilament protein in cells at the optical and electron microscope level and also for immunohistochemical detection of 210 kD and neurofilament protein on tissue sections.

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The invention relates to the murine lymphocyte hybridoma NF-01 producing a monoclonal antibody against the 210 kDa neurofilament protein.

Until now, antibodies have generally been produced by immunizing animals by repeatedly administering 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 neurofilament proteins, the situation is complicated by their considerable mutual similarity, so preparing a conventional antiserum against a single neurofilament protein by saturation is laborious. Production batches of conventional antisera are therefore very difficult to standardize and come from 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 the ballast antibodies against impurities and similar molecules such as vimentin, desmin or keratins.

These shortcomings are overcome by using a hybridoma producing a homogeneous monoclonal antibody against the 210 kDa neurofilament protein according to the invention.

This mouse lymphocyte hybridoma NF-01 was obtained in a manner known from the professional 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. Immunol. Meth. 35:1 to 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 NF-01 is that it binds to the target neurofilament structure, which is stable even during 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. the antibody against neurofilament proteins from Labsystems, Helsinki, Finland, react on similarly prepared sections only after enzymatic digestion or require a more gentle special fixation. None of the antibodies against the human 210 kDa neurofilament protein produced by the hybridomas described so far have the ability to react on paraffin, undigested sections. The NF-01 antibody also gives very good results on frozen tissue sections. Table 1 shows the strength of the reaction of the NF-01 antibody when using different methods of tissue processing:

Table 1

Intensity of the NF-01 antibody reaction on tissue sections fixed in three different ways (++ 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 neurons.

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Hybridoma NF-01 can be cultured in vitro in media suitable for growing animal cells or in vivo in the peritoneal cavity of BALB/c mice. Hybridoma NF-01 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 hybridoma NF-01 reacts with the human 210 kDa neurofilament protein and does not react with any other intermediate filament protein or other human cellular proteins, as determined by immunoblotting, enzyme immunoassay, indirect immunoperoxidase and indirect immunofluorescence methods.

This specificity was demonstrated by electrophoretic transfer of a wide range of cell and tissue lysates and isolated cytoskeleton preparations from various human tissues and cell lines, separated by sodium dodecyl sulfate polyacrylamide gel separation under reducing conditions. After transfer of the separated proteins to a nitrocellulose membrane, the 210 kDa neurofilament protein was specifically visualized (detected) using NF-01 followed by a horseradish peroxidase-conjugated porcine antiserum against mouse immunoglobulins (OSOL, Prague). A monoclonal antibody against neurofilament proteins 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, reactivity of the NF-01 antibody was found on the processes of ganglion cells of the spinal cord and the cerebellar cortex.

In indirect immunofluorescence using NF-01 antibodies and subsequent incubation with fluorescein-isothiocyanate-labeled porcine antiserum against mouse immunoglobulins, intermediate filaments of vimentin, glial and keratin types were not visualized in the tested cell types of several species. Neither microtubules nor microfilaments, distinguishable by their characteristic localization and sensitivity to colcemid and cytochalasin B, reacted with the NF-01 antibody.

The mouse lymphocytic hybridoma NF-01 is stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague under the designation IMG CZAS NF-01.

Example

In order to propagate NF-01 hybridoma cells in vivo, 1x10 ⁶ 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 14 days of hybridoma growth in the peritoneal cavity, the mice were sacrificed and the produced ascitic fluid was collected. A total of 2.0 ml of ascitic fluid was obtained, which contained 3.0 mg/ml of immunoglobulin. The specificity of the obtained antibody was tested on frozen sections of human brain, both in indirect immunofluorescence using a pig antibody against mouse immunoglobulin with fluorescein isothiocyanate and immunoperoxidase reaction with a pig 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 indirect immunoperoxidase test (see above). The results are summarized in Tables 2 and 3.

Table 2

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

V-vimentin, D-desrain, G-GFAP, N-pro^eins neurofilaments, K-keratins).

Tissue Type of filament Reaction

Cerebellum, neurons Cerebellum, astrocytes

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Table 2 continued

Tissue Type middle filament Reaction Spinal cord, neurons N + Spinal cord, astrocytes G - Ependymal cells In - The connective tissue and blood vessels of the soft diaper V + D - Tongue - epithelium, glands To - Tongue - muscles, blood vessels D,V - Language - connective tissue In - Tongue - peripheral nerves N Rectum - epithelium To - Rectum - muscles, blood vessels V,D - Rectum - connective tissue In - Rectum - peripheral nerves N + ¹ Pancreas - endocrimal parenchyma To - Pancreas - exocrine gland To - Pancreas - vessels, connective tissue V + D - Pancreas - Peripheral nerves N Trachea - epithelium, glands To - Trachea - blood vessels, ligaments V + D Trachea - cartilage In - note 1 - frozen sections int- ί , i external reaction ) u 1 k a 3 NF-01 antibody reactivity with tumors human central nervous system : ta, - negativity, K-keratin, . V-vimentin, G-GFAP, N- -neurophil proteins. Tumor Type middle filament Reaction Papillary ependymoma K,V - Medulloblastoma V,N - Astrocytoma G, V - Ganglioglioma G,N + ¹ Meingeom In -

- in the ganglion part

From the results presented in Tables 2 and 3 it is clear that the NF-01 antibody reacts exclusively with human neurofilament proteins. The narrow specificity of the antibody was confirmed in immunoblotting, where a strong reaction was found with the 210 kDa protein, while the other cytoskeletal proteins tested were negative (see Table 4).

Table 4

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

- negativity)

Cytoskeletal protein

Immunoblotting reaction alpha-tubulin beta-tubulin vimentin

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Table 4 continued

Cytoskeletal protein Reaction in immunoblotting

neurofilament proteins - 68 kDa - - 160 kDa - - 210 kDa + GFAP -

The specificity of the obtained antibody was further tested on a panel of cells 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 an indirect immunoperoxidase assay. The results are summarized in Table 5.

Table 5

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

Type Tissue origin Name Type interm. filament Reaction IF IB person lung fibroblast LED In - - squamous cell carcinoma U1280 K + N + + epidermoid carcinoma A 431 To mouse fibroblast 3T3 Swiss In - - neuroectoderm C 1300 N - - rat glioma C6 G + V - -

The NF-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, gentamycin and inactivated horse serum. The hybridoma is cultured at 37 °C, the mean generation time is 18.5 hours and the modal chromosome number 7 months after hybridoma construction was 79. The produced antibody is a mouse monoclonal immunoglobulin of the IgM class.

The monoclonal antibody produced by the NF-01 hybridoma reacts specifically with the human 210 kDa neurofilament protein 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 can be used in the field of theoretical research on human intermediate filaments.

Claims (1)

SUBJECT OF THE INVENTION IMG C-ZAD NF-01 murine lymphocyte hybridoma producing a monoclonal antibody of the IgM class against human 210 kDa protein neurofilament.