CS255643B1 - Mouse lymphocytar hybridome forming monoclonal antibody against tubuline's alpha and beta subunits - Google Patents

Abstract

The present invention relates to a murine lymphocyte hydride producing a monoclonal antibody of the tubulin-α / β-α subunit of a variety of species deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under IMG-CZAS TU-10. The TU-10 hybridoma monoclonal antibody is suitable for use in enzymatic and radioimmunoassay analysis of the tubule, or for visualization of microtubules in cells at the level of optical and electron microscopy.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line Sp2 / 0 and a mouse spleen lymphoid cell producing an antibody against the alpha- and beta-subunit of tubulin (i.e., an oytoskeletal 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 primarily to identify tubulin-containing cell structures. This procedure, called conventional immunization, has several drawbacks. Since tubulin is one of the highly conserved proteins, recurrence 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 range of quality. 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 on both the alpha and beta-subunits of tubulin is likely to be found.

Progress in detailed analysis of microtubular structures, manifested in the simultaneous identification of both tubulin subunits, will be achieved if a hybridoma producing a monoclonal antibody against the alpha- and 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. 1083, under the designation IMG CZAS TU-10.

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

S.S. Scheidegger, O .: 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 Host-Compatibility Antigens Produced by Hybrid Cell Lines. Nature 266: 550-552, 1977) by cloning a set of hybrid cells resulting from the fusion of mouse myeloma line Sp2 / 0 cells and cells obtained from the spleens of BALB / c mice immunized with microtubular proteins of pig brain. Microtubular proteins were prepared by two consecutive cycles of polymerization-depolymerization from brain homogenate (Shelanski, ML, Gaskin, F., Cantor, CR: Microtubule assembly in the absence of added mucleotides. Proc. Natl. Acad. Sci. USA 70: 765-768 , 1973).

The advantage of the hybridoma is that it produces a homogeneous monoclonal antibody that is capable of specifically reacting with the alpha- and beta-subunits of tubulins of different species. The TU-10 hybridoma can be cultured in vitro in suitable animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. Antibody-free antibody production can be initiated from cans stored in liquid nitrogen. The antibody produced by the TU-10 hybridoma reacts specifically with tubulin subunits and there is no need to remove ballast antibodies.

Example g

To expand 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 11 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 2 ml of ascites fluid containing approximately 1 mg / ml immunoglobulin was obtained.

The monoclonal antibody, in the form of an ascites fluid, reacted with a specific antigen (purified porcine tubulin,) in an indirect enzyme immunoassay using a horseradish peroxidase labeled porcine immunoglobulin antibody (Serum and Vaccine Ostav, Prague), to a 1:10 dilution. Binding did not occur when bovine serum albumin, human transferrin 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 the differentiation antigen of mouse teratocarcinoma cells (Dráber, 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-10 specifically reacts with tubulin in poggy.

a) In immunoblotting, proteins were separated by polyacrylamide gel electrophoresis under reducing conditions and electrophoretically transferred from the gel to a nitrocellulose membrane.

After incubation of the replica with the monoclonal antibody (1:10 dilution), the binding of the bound antibody was detected enzymatically immunologically using the peroxidase (mouse monoclonal antibody) complex against horseradish peroxidase (Viklicky, V., Dráber, Pa., Macků, B., Dráberová, E. , Rozprimová,

L .: Monoclonal antibodies against horseradish peroxidase. Fol. Biol. / Prague / 30: 378-382,

1984). Tu-10 antibody bound to alpha- and beta-subunits of purified tubulins of various species (porcine, bovine, mouse, rat, rabbit), in microtubular proteins of pig brain, and in whole lysates of pig brain tissue only bound to proteins in alpha and beta position -subunits of tubulin. In no way did it bind to microtubule-associated proteins (MAP-1, MAP-2, tau proteins). In whole cell lysates of cell lines LEP (human lung fibroblast), T-24 (human bladder cancer), MOLT-4 (human T-lymphoblast),

C-1300 (murine nuetoblastoma), the TU-10 antibody bound only to proteins with electrophoretic mobility corresponding to the position of the alpha- and beta- subunit of tubulin. In control experiments with monoclonal antibody TEC-01, binding did not occur.

b / For indirect immunofluorescence, adherent cells of the LEP line were incubated for 2 min. with 0.15% Triton X-100 in stabilizing buffer for cytoskeleton (Smal, JV, Cell, JE: Filaments arrangements in negatively stained cultured cells: the organization of actin. Cytobiology 16, 308-325, 1978) supplemented with 4% polyethylene glycol 4,000 and fixed for 30 min.

3% paraformaldehyde. Antibody TU-10 (1: 200 dilution) was applied to the prepared cytoskeleton and its binding to cytoskeletal structures was monitored by immunofluorescence microscopy using porcine antibody against murine immunoglobulin with significant fluorescein isothiocyanate (Serum and Vaccine Ostav, Prague). The TU-10 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-10 and mouse monoclonal antibody TU-04 directly labeled with rhodamine, which is directed against the alpha-subunit of tubulin in a number of species (Dráber,

Pa., Dráberová, E., Zicconi, D., Sellitto, C., Viklický, V., Cappuccinelli, P .: Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin. Eur. J. Cell Biol.

41: 82-88 (1986), both antibodies bind to identical microtubule fibers. In control experiments with monoclonal antibody TEC-01, cytoskeletal structures were not shown.

TU-10 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 minimum essential 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, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine germ ( Bioveta, Ivanovice na Hané, 10%).

The hybridoma is cultured at 37 ° C; the mean generation time is 16.4 h and 6 months after construction, the modal number of chromosomes was 80. The antibody produced is an immunoglobulin of the IgM class with kappa light chains.

The monoclonal antibody produced by the TU-10 hybridoma reacts, analogously to conventional antibodies, with tubulins of various species and is therefore useful for detecting cellular microtubular structures. The TU-10 hybridoma can be used industrially as a source of minoclonal antibodies against the alpha- and beta-subunit of tubulin in analytical or preparative methods.

Claims (1)

The murine lymphooytic hybridoma IGM CZAS TU-10, producing a monoclonal antibody of the IgM class against alpha and beta tubulin of a number of species.