CS255843B1 - Mouse lymphocytaric hybridome producing monoclonal antibody class igm against beta-subunit tubuline - Google Patents

Abstract

The present invention relates to mouse lymphocytic monoclonal-producing hybridoma tubulin beta-subunit antibody many species stored in the Collection of Hybridomas Molecular Genetics of CSAV IMG-CZAS TU-06. Monoclonal hybridoma antibody TU-06 is suitable for used in enzymimmunological and radioimmunological tubulin analysis or visualizing microtubules in cells at optical and electron microscopy.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma lineage FO cell and a mouse spleen lymphoid cell, producing an antibody against the beta-subunit of tubulin, i.e. a cytoskeletal protein that is a microtubule building component.

Antibodies to tubulin are prepared in a classical manner by repeatedly injecting tubulin into test animals, most commonly rabbits. The serum of animals thus immunized, taken after a period of antigen treatment, serves as a source of antibodies used in particular to identify tubulin-containing cell structures. This procedure, called conventional immunization, has several drawbacks. Since tubulin is one of the highly conserved proteins, the recruitment of well immunized animals is difficult. There is a heterogeneous mixture of antibodies in the serum, whose spectrum is different and unrepeatable in each individual organism. As a rule, in addition to antibodies against the desired antigen, antibodies against impurities of the antigen preparation are present in the serum; these antibodies must be difficult to remove. Prepared batches of conventional sera are therefore difficult to standardize and have a wide quality range. To produce each batch, a substantially pure immunization antigen should be prepared.

In principle, hybridoma technology removes the disadvantages of conventional sera against tubulin. The hybridoma product - a monoclonal antibody - is directed against a single antigenic determinant. If a sufficiently large set of hybridomas is constructed, it is likely that a hybridoma synthesizing an antibody that recognizes the antigenic determinant present only on the beta-subunit of tubulin is likely to be found.

Progress in detailed analysis of microtubular structures, manifested in the identification of only the beta-subunit of tubulin, will be achieved if a hybridoma producing a monoclonal antibody against the beta-subunit of tubulin is available from the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská 1 083, under the designation IMG CZAS TU-06.

Said hybridoma was obtained by a method of literature (Fazekas de, St., Groth,.

S. S., Scheidegger, 0 .: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth. 35: 1-21, 1980; Galfré, G., Howe, S.C., Milstein, C., Butcher, G.W., Howard,

J. C .: Antibodies to major histocompatibility antigens produced by hybrid cell line. Nature 266: 550-552, 1977) by cloning a set of hybrid cells resulting from the fusion of murine myeloma cell line FO cells and cells obtained from the spleens of mouse strains (BALB / cx B 10.A) immunized with beta-subunits of tubulin isolated from porcine microtubular proteins brain.

Microtubular proteins were prepared by two consecutive cycles of polymerase-depolymerization from brain homogenate (Shelanski, ML, Gaskin, F., Cantor, CR: Microtubule assembly in the absence of added nuoleotides. Proc. Natl. Acad. Sci. USA 70: 765-768 , 1973). After preparative electrophoresis of microtubular proteins in polyacrylamide gel, in the presence of sodium dodecyl sulfate under reducing conditions, polyacrylamide gel strips containing the beta-subunits of tubulin were excised and proteins were isolated from the gel by electroelution (Stralfors, from. PAG slices, Anal. Biochem. 128: 7-10 (1983).

The advantage of the hybridoma is that it produces a homogeneous, monoclonal antibody that is capable of specifically reacting with the beta-subunits of tubulins of different species. The TU-06 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of F4 mice (BALB / c x B 10.A). From cans stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the TU-06 hybridoma reacts specifically with the beta-subunits of tubulin specifically and does not need to remove ballast antibodies.

Example

To expand the hybridoma cells in vivo, 1 x 10 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 intraperitoneally) 14 days prior to transfer of the hybridoma cells. After days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid collected. A total of 3 ml of ascites fluid containing approximately 2 mg / ml immunoglobulin was obtained.

The monoclonal antibody, in the form of an ascites fluid, was reacted with a specific antigen (purified porcine tubulin) in an indirect enzyme immunoassay using a horseradish peroxidase labeled porcine anti-mouse immunoglobulin antibody (serum and vaccine condition, Prague), to a dilution of 1: 10 ^. Binding did not occur when bovine serum albumin or mouse vimentin was used instead of tubulin. Also in control experiments using murine monoclonal antibody TEC-01 (IgM, kappa light chain), which is directed against differentiation antigen of mouse teratocarcinoma cells (Drábeř, Pe., Pokorná, Z .: Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal Cell Differentiation 15: 109-113, 1984) did not bind.

Two approaches were used to demonstrate that the monoclonal antibody TU-06 specifically reacts only with tubulin.

a) In immunoblotting, proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions and electrophoretically transferred from the gel to a nitrocellulose membrane. After incubation of the replica with the monoclonal antibody (1: 10 ³ dilution), the binding of the bound antibody was detected enzymatically immunologically using a mouse peroxidase monoclonal antibody against horseradish peroxidase (Viklický, V., Dráber,

Pa., Macků, B., Dráberová, E., Rozprimová, L .: Monoclonal Antibodies Against Horseradish Preoxidase. Fol. Biol (Prague) 30: 378-382, 1984).

The TU-06 antibody bound to the beta-subunit of purified tubulins of various species (porcine, bovine, mouse, rat), porcine brain microtubular proteins and in whole porcine brain tissue lysates only bound to proteins at the beta-subunit position of tubulin.

In no way did it bind to microtubule-associated proteins (MAP-1, MAP-2, tau proteins).

In whole cell lysates of LEP (human lung fibroblast), HL-60 (human promyelocyte), Cl 300 (mouse neuroblastoma), 3T3 (mouse embryonic fibroblast) lines, in primary Leydig cell culture and in whole cell lysates, Trichomonas vaginalis and Herpetomonas muscarum, the TU-06 antibody only bound to proteins with electrophoretic mobility corresponding to the position of the beta-subunit of tubulin. In control experiments with monoclonal antibody TEC-01, binding did not occur.

b) For indirect immunofluorescence, adherent cells of the 3T3 line were incubated for 2 min.

with 0.15% Triton X-100 in stabilizing buffer for cytoskeleton (Smal, JV, Celis, JE: Filaments arrangements in negatively stained cultured cells: the organization of actin ^ Cytobiology 16, 308-325, 1978), supplemented with 4% polethylene glycol 4000 and fixed for 30 min.

3% paraformaldehyde. Antibody TU-06 (1: 100 dilution) was applied to the prepared cytoskeleton and its binding to cytoskeletal structures was monitored by immunofluorescence microscopy using a porcine anti-mouse immunoglobulin labeled with fluorescein isothiocyanate (Serum and Vaccines, Prague). The TU-06 antibody bound only to microtubular structures of interphase and mitotic cells and no other cytoskeletal structures - microfilaments and intermediate filaments were shown. Typical paracrystals were formed after vinblastine treatment. Indirect / direct immunofluorescence labeling of cytoskeletal structures with monoclonal antibody TU-06 and mouse monoclonal antibody TU-04 directly labeled with rhodamine, which is directed against a tubulin alpha-subunit of a number of species (Dráber, Pa., Dráberová, E., Zicconi, D., Sellitto, C., Viklicky, V., Cappuccinelli, P .: Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin.

J. Cell Biol. 41: 82-88 (1985), both antibodies bind to identical microtubule fibers. In control experiments with monoclonal antibody TEC-01, cytoskeletal structures were not shown.

TU-06 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. They grow in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimal medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (designated as H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomyoin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum (TU-10 hybridoma). Bioveta, Ivanovice na Hané, 10%). The hybridoma is cultured at 37 ° C; the mean generation time is 17.2 h and 8 months after construction, the modal number of chromosomes was 84. The antibody produced is an immunoglobulin of the IgM class with kappa light chains.

The monoclonal antibody produced by the TU-06 hybridoma reacts, analogously to conventional antibodies, with tubulins of various species and is therefore useful for detecting cellular microtubular structures. The TU-06 hybridoma can be used industrially as a source of monoclonal antibodies against the beta-subunit of tubulin in analytical or preparative methods.

Claims (1)

OBJECT OF THE INVENTION The IMG CZAS TU-06 murine lymphocyte hybridoma, producing a monoclonal antibody of the IgM class against the beta-subunit of tubulin in a number of species. Severography, n Price 2,40 Kčs