HYBRIDOMA

From Big Medical Encyclopedia

HYBRIDOMA (Latin hibrida, hybrida a hybrid + - oma) — the malignant cell received by merge (hybridization) of a tumor cell to its normal analog, which inherited ability to unlimited growth from a malignant cell, and from a normal cell ability to synthesis of specific protein, most often antibodies.

For the first time in 1975 G. Kohler and Milstayn (S. of Milstein) showed that individual clones (see. Clone ) antitelosekretiruyushchy cells can be turned in immortal at merge to myeloma cells. The line of the malignant cells capable to infinite growth in culture or in an organism and producing results antibodies (see) the set specificity.

Spontaneous cell fusion happens exclusively seldom. However practically any cells can be merged, using the parainfluenza Sendai virus, and also lysolecithin or polyethyleneglycol. Slrg-yanrsh of cells with formation of the general cellular cover leads to emergence of so-called heterokaryon — cells with two or more kernels, to-rye merge, combining the chromosomal complements and forming a hybrid cell (see. Genetics of somatic cells ). Merge of kernels, even under the influence of polyethyleneglycol happens not often. By means of special culture medium not merged cells eliminirutsya, and culture of cells (see. Cultures of cells and fabrics ) through a nek-swarm time later hybridizations (see) consists practically only of hybrid cells. Therefore for G.'s receiving it is important to create the selection mediums providing growth only of hybrid cells. A theoretical basis of a method of selection is the following. The main way of biosynthesis of purines and pyrimidines can be blocked by the antagonist folic to - you aminopterine. In this case the cell can synthesize DNA in the collateral way depending on enzymes of a thymidinekinase (KF 2.7.1.21) and gi-poksantin-fosforiboziltransferazy (KF 2.4.2.8) capable to provide proliferation of a cell in the presence of thymidine and hypoxanthine. In the absence of one of these enzymes synthesis of DNA stops. The cell can avoid it by merge to other cell capable to supply it with missing enzyme. Thus, if to achieve merge of a normal cell of a spleen, edges has activity of a thymidinekinase and hypoxanthine-phosphoribosyl-transferase with the myeloma cell deprived of activity of these enzymes and therefore perishing at cultivation on the Wednesday with hypoxanthine, aminopterine and thymidine (so-called GHAT Wednesday), in culture of cells only hybrid cells will breed.

A number of lines of the mutant myeloma cells deprived of activity of hypoxanthine-phosphoribosyl-transferase is received. Selection of mutants is quite simple since cytostatics like thioguanine or azaguanine join in molecule DNA by means of this enzyme; therefore in the presence of azaguanine in culture only mutant cells survive. Synthesis hypoxanthine-fosforiboziltrans-ferazy is coded by the gene located on X-chromosome. In cells of mammals only one X-chromosome therefore the only mutation is enough for complete cessation of synthesis of enzyme is active. It is significantly more difficult to select the cells mutants deprived of a thymidinekinase since existence of two rare mutations at the same time for this purpose is required.

In order that the hybrid cell could prove as functionally differentiated cell, both parent cells shall be at approximately identical stages of a differentiation, otherwise the hybrid cell quickly loses this ability. Therefore for G.'s receiving capable to cosecrete antibodies, normal antibodies ©секретирующие cells (V-lymphocytes) merge only with myeloma cells (for simplification of selection of the necessary clones use the mutant myeloma cells not only deprived hypoxanthine-fosfori-boziltransferazy, but also which are not cosecreting immunoglobulins). For receiving G. keeping function of T lymphocytes (see. Immunocompetent cells ), the last merge with mutant cells of a thymoma. Of cells of a liver is received merge of normal cells of an embryonal or adult liver to mutant cells of a gepatoma etc. However most widely use G. cosecreting antibodies. Most often as a source of parent cells take cells of a mouse or a rat. Interspecies hybrids are not stable since the chromosomes bearing genes of immunoglobulins eliminirutsya quickly.

Usual antiserums contain the antibodies produced by cells of hundreds and even thousands of clones. At the same time many antibodies are directed against the antigens which are accidentally present at the immunizing material and are undesirable to the experimenter. Drugs of many highly active biol. substances, napr, hormones, it is difficult to clear of ballast proteins, contents to-rykh in these drugs is one thousand times higher, than the content of hormones. All this limits possibilities of a usual serology. The Gibridomny technology solved these difficulties — with its help any quantities of almost homogeneous monoclones can be received even if the immunizing material is not purified.

In the late seventies are received 20 century the first G. of the person, the mutant line of the myeloma cells deprived hypoxanthine-fosforibo-ziltransferazy is allocated. However it is difficult to receive normal antitelosekretiruyushchy cells of the person. If antigen is harmless, immunize patients, the Crimea the splenectomy is shown. Use also cells regional in relation to a tumor limf, nodes, believing that among them there can be cells, immune against tumoral antigen. And still the main type G. a mouse — a mouse or a rat — a mouse (the first donor animals of normal cells are specified).

Efficiency of hybridization is higher if use proliferating antitelosekretiruyushchy cells. Therefore carry out not really hyperimmunization: cells of a spleen receive in 4 days after the first or second administration of antigen when percentage among them actively sharing immunoblasts (blasts) highly.

The procedure of selection of the clones cosecreting the set antibodies is most difficult. Cells right after merge are very vulnerable and badly grow out of an organism. The sufficient density of cells in culture is required that is reached by means of various nutritious underlayers of cells of type of thymocytes, macrophages, to-rye in culture do not breed (so-called feeders). Even in these conditions it is necessary to grow up a certain mass of hybrid cells, edges, however, it should not be too big since usually not cosecreting cells grow quicker than cosecreting and force out the last. Therefore any G.'s receiving needs bystry testing of the clones producing antibodies and repeated cloning with selection of the necessary clones. It is necessary to consider that perished-ridomnye cells are unstable and about a half of the selected clones perishes that demands a constant reklonirovaniye already received.

Stable lines G. conduct or in culture of cells, or by passages on mice. Cells of such lines produce huge quantities of antibodies: cultural liquid contains to 50 mg of monoclonal (purest) antibodies in 1000 ml; in blood serum of mice, at to-rykh G. grow, the maintenance of antibodies makes 300 — 1500 mg / 100 ml. Often grow in a type of an astsitny tumor if one week prior to a passage to enter to the recipient intraperitoneally pristan or full adjuvant. It is possible to receive from each mouse up to 10 ml of astsitny liquid, the maintenance of antibodies in a cut same, as well as in blood serum. Thus, the gibridomny technology allows to receive almost unlimited number of the high-homogeneous antibodies which are not needing cleaning.

The monoclones produced by G. apply in many fields of biology and medicine. So, they are used for studying of fine structure of proteins, radioimmunol, definitions of hormones, testings of fabrics for histocompatability, definitions of antigens on a surface of various cells, for definition of localization and for classification of tumors and hemoblastoses, for an immunotherapy of malignant diseases (sometimes connected to toxins), for hromatografichesky cleaning of molecules of the corresponding antigens etc.

By means of gibridomny technology it is possible to make immortal practically any cell cosecreting the set protein. Already now G. are received, to-rye cosecrete the hormones regulating a hemopoiesis («colonies - the stimulating factor»), many proteins of a liver etc. Works on receiving G. producing blood-coagulation factors, insulin, interferon, etc. are conducted.



Bibliography: Brodsky F. M and. lake of Monoclonal antibodies for analysis of the HLA system, Immun. Rev., v. 47, p. 3, 1-979; Fazekas S., Groth St. Schei-d e g g e r D. Production of monoclonal antibodies, J. Immunol. Methods, v. 35, p. 1, 1980; Go ding J. W. Antibody production by hybridomas, ibid., v. 39, p. 285, 1980; Kohler G. a. M i 1 stein C. Continuous culture of fused cells secreting antibody of predefined specificity, Nature (Lond.), v. 256, p. 495, 1975; Lymphocyte hybridomas, ed. by F. Melchers and. lake, V. a. o., 1978; R a s with h-k e W. Page of Plasmacytomas, lymphomas and hybridomas, Biochem, biophys. Acta (Amst.), v. 605, p. 113, 1980; T r u with with about M. M. and. lake of Murine monoclonal antibodies against HLA structures, Immunol. Rev., v. 47, p. 219, 1979; Ziegler A. Monoclonal antibodies as tools in hematology, Blut, v. 41, p. 1, 1980.


I. Chertkov.

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