CS263450B1 - Mouse lymphocyte hybridoma producing antibody against secretory component - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against a human secretory component, stored in the hybridoma collection of the Research Institute of Clinical Experimental Oncology in Brno under the designation RICEO-SC-05. The monoclonal antibody of the hybridoma RICE0-SC-05 itself is suitable for use in enzyme-immunological and radioimmunological analysis of body fluids, as well as for immunoblotting and for histochemical detection of the secretory component on tissue sections and cells at the optical and electron microscope level.

Description

(54) Mouse lymphocyte hybridoma producing antibody against secretory component

The solution concerns a mouse lymphocyte hybridoma producing an antibody against a human secretory component, stored in the hybridoma collection of the Research Institute of Clinical Experimental Oncology in Brno under the designation RICEO-SC-05. The monoclonal antibody of the hybridoma RICE0-SC-05 itself is suitable for use in enzyme-immunological and radioimmunological analysis of body fluids, as well as for immunoblotting and for histochemical detection of the secretory component on tissue sections and cells at the optical and electron microscope level.

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The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of the mouse myeloma line P3-X63-Ag8.653 and a mouse splenic lymphoid cell, producing an antibody against a human secretory component.

Until now, antibodies against the secretory component have been produced by repeatedly injecting the secretory component as an antigen (usually together with an adjuvant) into experimental animals, most often rabbits or guinea pigs. The serum of animals immunized in this way, collected after a certain period of exposure to the antigen, serves as a source of antibodies, used mainly for the detection of the secretory component in tissues and body fluids. 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. As a rule, the organism produces, in addition to antibodies against the desired antigen, antibodies against impurities of the antigen preparation, which must be removed from the serum in a complex manner called saturation.

In the case of antisera against the secretory component (vis Brandtsaeg Role of <J chain and secretory component in receptor-mediated glandular and hepatic transport of immunoglobulins in man. Scand. J. Immunol. 22, 111 aí 146, 1935) the situation is further complicated by the covalent binding of the secretory component with the immunoglobulin chains of secretory IgA and IgM, which are (possibly with other proteins) a frequent contaminant of purified preparations of the secretory component, so that preparing a conventional antiserum specific exclusively for the secretory component saturation^. is very laborious and rarely leads to a satisfactory result. Production batches of conventional antisera can therefore only be standardized with great difficulty and come out of production with a wide range of quality.

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For the production of each Serge, it is necessary to prepare a highly pure immunization antigen and other antigens to saturate ballast antibodies against impurities or similar molecules, such as the immunoglobulins mentioned above.

The above-mentioned shortcomings of the previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against the secretory component is available, stored in the hybridoma collection of the Research Institute of Clinical and Experimental Oncology in Brno, Yellow Hill 7, under the designation RICE0-SC-05.

The hybridoma was obtained in a manner known from the literature (Pazekas de St. Groth S., Scheidigger Ώ,: Production of monoclonal antibodies with Strategies and tactics, J. Tmtnvinol. Meth. 35« 1 « 21, 1980, Galfré G., Howe S.C., Mílstein C., Butcher G.W., Howard J.O.: Antibodies to major histocompatibility antigens produced by hybrid cell lines, Nature 266: 550 - , 1977) by cloning a set of hybrid cells, resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of BALB/c mice, immunized with a secretory component isolated from human colostrum.

The advantage of the SC-05 lymphocyte hybridoma is that it produces a completely homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with a human secretory component. The SC-05 hybridoma can be cultivated in vitro in media suitable for growing animal cells or under in vivo conditions in the peritoneal cavity of BALB/c mice. The SC-05 hybridoma is suitable for long-term storage in cans stored in liquid nitrogen. The production of the monoclonal antibody can be started after thawing the cell can, without the need for additional antigen. The monoclonal antibody produced by hybridoma SC-05 is specific exclusively for a glycoprotein with a relative molecular mass of approximately 80,000, designated as the secretory component ₍ and does not react with any other secreted protein or other proteins (e.g. serum), as determined by immunoblotting, enzyme immunoassay, indirect immunoperoxidase and indirect fluorescence methods. The evidence was obtained after electrophoretic transfer of a wide range of cell and tissue lysates and human colostrum preparations, after separation of the protein components in a polyacrylamide gel in sodium dodecyl sulfate medium under reducing conditions. After transfer of the separated proteins to a nitrocellulose membrane, the secretory component was specifically visualized using the SC-05 antibody and subsequently rabbit antiserum against mouse immunoglobulins conjugated to horseradish peroxidase. The result was also confirmed enzyme immunoassay, where the binding of the SC-05 antibody to the secretory component was demonstrated, but not to immunoglobulin A, immunoglobulin M or other serum proteins. Using the immunoperoxidase reaction on tissue sections (also confirmed by the results of the indirect immunofluorescence method - see below) of human tissues, the SC-05 antibody was found to be reactive with epithelial tissues of the secretory type producing the secretory component, but not with other tissues.

In indirect immunofluorescence using SC-05 antibodies and subsequent incubation with rabbit or pig antiserum against mouse immunoglobulins labeled with fluorescein isothiocyanate, binding occurred in the tested tissues only to the apical or basolateral membrane of secretory epithelia and to the secreted material (secretion) of exocrine glands, in cells with a high degree of secretion (e.g., the mammary gland in lactation) the reaction was also evident in the cytoplasm of secretory cells. Any other cell types and tissue components were negative.

In the Czechoslovak Socialist Republic, a similar monoclonal antibody has not yet been available. Lymphocyte hybridomas producing antibodies against the secretory component have already been prepared abroad and are also commercially available and are beginning to be used in clinical practice. The antibodies offered in zah.fm J..čí věak lack the unique combination of advantageous properties characteristic of the SC-05 antibody, i.e. they do not combine narrow specificity for the secretory component with recognition of both bound and free forms of this glycoprotein, as well as usability in various application methods including immunoblotting, TCLTSA and RTA and immunohistochemistry on metacarne-fixed paraffin tissue sections (i.e. even in methods in which antibodies offered abroad cannot be used at all)

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Example 1’

In order to propagate hybridoma cells in vivo, 1 x 10^ cells were applied to the peritoneal cavity of RAIiB/c mice. For better attachment of the indicated cells, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 20 days before the transfer of hybridoma cells. After 1& days of hybridoma growth in the peritoneal cavity, the mouse was killed and the formed ascitic fluid was collected. A total of 3.5 ml of ascitic fluid was obtained, which contained 9 mg/ml of immunoglobulin. The antibody reacted with the specific antigen (secretory component) in an etizymoimmunological test up to a dilution of 1 in 10^. The specificity of the obtained antibody was tested for a number of different protein antigens, both in immunoblotting and in an enzyme-linked immunosorbent assay using porcine or rabbit antibodies against mouse immunoglobulin labeled with horseradish peroxidase. The results are summarized in Table 1.

Table 1. Reactivity of the SC-05 antibody with various protein antigens and cell homogenates (+' positivity, - - negativity)

Tested material Presence of a secretory component Reaction Highly purified secretory component , YES + Partially Cleaned — “ — . HOW + Human colostrum A-NO . + Human secretory immunoglobulins (slgA, slgM) YES in 4- Serum immunoglobulins · W. - Serum albumin NO - Human keratins NO - Fibroblast Line Homogenate (LEP) NO w» MOLT 3 leukemia line homogenate RE «» HeLa carcinoma line homogenate KB - U 118 MG glioma line homogenate NO - Melanoma Line Homogenate LT NO , Rhabdonyo sarcoma line homogenate L1168 NO ....... .

From the results presented in Table 1, it is clear that the 3(3-05 antibody reacts with both free and bound secretory component, while being negative for other serum or cellular proteins.

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Example 2

For the in vitro propagation of RICE0-SC-05 hybridoma cells, 1 x 10^ hybridoma cells were inoculated into a Legroux culture flask containing 20 ml of DMEM culture medium with 10% fetal calf serum and antibiotics (see below). After 5 days of growth of the hybridoma cell culture, the culture medium enriched with monoclonal antibody 30-0¾ released into the medium by the hybridoma cells was obtained from the flask. The tissue specificity of the antibody reaction was tested by indirect immunofluorescence and also confirmed by indirect immunoperoxidase reaction, in both cases on tissue sections. The tissue samples listed in Table 2 were taken from surgical or autopsy material, tissue blocks measuring 0.5 X 0.5 x 1 cm were frozen in liquid nitrogen and stored at -70°C. For the actual testing, the frozen blocks were adapted to a temperature of -25 to -30°C and sections 5-7 micrometers thick were prepared from each of them on a cryostat. The tissue sections were then transferred to appropriate histological slides, after air drying the sections were fixed for 10 minutes with a mixture of methanol and acetone (1:1) previously heated to ~15°C and after washing out the remaining fixative for 2 x 10 minutes in phosphate-buffered saline (PBS) pH 7.4, the monoclonal antibody 30-05 was applied to the sections. After an incubation period of 1 hour, the unbound antibody was removed from the section by repeated washing (3 x 5 min) in PBS and rabbit or pig antibodies with fluorescein isothiocyanate were applied to the section. The incubation (40 - 60 min) was terminated by washing three times with PBS for 5 min. The sections were left at room temperature until dry and the preparations were mounted, for example, in a medium with glycerin. The results of the evaluation of the reaction of the SC-05 antibody with selected tissues are shown in Table 2.

Table 2 Reactivity of the SC-05 antibody with normal human tissues (+ positive and - negative reactions)

Tissue Type

Secretory production Reaction components

Secretory epithelium of the mammary gland Mucus of the intestine

YES. +

ABC +

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Table No. 2 - continued.

Type of tissue Production of 3 secretory components Reaction Mucosa of the gallbladder YES + Salivary gland epithelium YES + .Liver - bile ducts YES + Lung epithelium YES + Epidermis NO - Sebaceous gland NO - Structural epithelium j language NO M ““ n j I am NOT ·*» arrow NO •m Ovary NO - Testes NO M Adrenal glands NO 1 thyroid gland NO Striated muscle NO - Smooth muscle NO - Myocardium LE - Cartilage M2 - Trees of various organs THE - Spleen NO - Lymph nodes ?,r*m - Peripheral nerve Z'E Brain NO Cerebellum alcohol - From the spectrum of tissues i.:(vi|s Table No. 2) responding 3 for bodysuit

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SC-C5 shows that all tissues producing the secretory component are positive, whereas all tissues lacking this glycoprotein are negative, which is further evidence of the specificity of antibody 30-05 for the secretory component.

The hybridoma cells ^XCEO-SO-C5 have a morphological appearance of typical myeloma bulbs^.. Under in vitro conditions they grow in the form of semi-suspension cultures. The basic culture medium is Bulbec's modification of minimal essential medium (DUEM) supplemented with

L-glutamine (3mM), penicillin, streptomycin and inactivated

2S3 450 fetal calf serum is cultured at 3 19.2 hours. The produced immunoglobulins are of the IgG1 class,

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I) Sokolnice near Brno. Hybrid medium generation time is mouse >nonclonal immuno binding epitope human secretory components.

The monoclonal antibody produced by the RICSOS0-05 hybridoma reacts with both free and bound secretory components and is useful in various tests including histochemistry on tissue sections, immunoblotting, enzyme-immunological and radioimmunological assays. The SC- ⁰⁵ antibody is therefore particularly suitable for the detection and quantification of the secretory component in various body fluids, not only in the field of immunology), but also in the diagnosis of diseases of the respiratory tract, as well as production ch ad.e n.ocarcinomas • from other types of tumors such as sarcomas, melanomas, lymphomas or carcinomas not producing a secretory component (e.g. carcinomas from multilayered epithelium). In addition to medical practice, its properties can also be used in a number of applications in research, biology of normal and transformed epithelial cells and the study of secretion mechanisms, potentially in the health care, education and Czech Academy of Sciences and Slovak Academy of Sciences.

Claims (1)

SUBJECT Mouse lymphocyte hybridoma RICE0-SC-C9, characterized in that it produces a monoclonal antibody of the IgG1 class against the human secretory component.