CS239761B1 - Mouse lymphocyte hybridoma IMG CZAS T3-01 producing anti-triiodothyronine antibody - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against triiodothyronine, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation IMG CZAS T3-01. The monoclonal antibody of the hybridoma T3-01 is suitable for use in radioimmunological analysis and in immunohistochemical examination of microscopic preparations of the thyroid gland.

Description

(54) Mouse lymphocyte hybridoma IMG CZAS T3-01 producing antibody against triiodothyronine

The invention relates to a murine lymphocyte hybridoma producing an anti-triiodothyronine antibody deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the name IMG CZAS T3-01. The T3-01 monoclonal antibody is suitable for use in radioimmunoassay and immunohistochemistry for microscopic thyroid preparations.

IN

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line FO cell and a mouse spleen lymphoid cell producing an anti-triiodothyronine antibody.

To date, anti-triiodothyronine antibodies have been produced such that the triiodothyronine-protein conjugate, such as serum albumin or immunoglobulin, is repeatedly administered as an antigen to test animals, most commonly to rabbits.

The serum of the immunized animals collected after a certain period of exposure to the antigen serves as a source of antibodies used in particular for the quantitative determination of plasma triiodothyronine by radioimmunological analysis.

This procedure, called conventional immunization, has several drawbacks. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum is different and unrepeatable in each individual organism.

As a rule, the body produces antibodies which, in addition to triiodothyronine, react poorly with thyroxine, reverse triiodothyronine, diiodothyronine, monoiodothyronine and other tyronine derivatives.

Manufacturing batches of mercantile sera are difficult to standardize and are based on production in a wide quality range. For each batch, the triiodothyronine-protein conjugate should be prepared in the highest purity possible.

The above mentioned drawbacks of the above-mentioned and hitherto used procedure will be eliminated if hybrids producing monoclonal antibody against triiodothyronine, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vienna No. 1083, available IMG CZAS T3-01.

Said hybridoma was obtained by a method known in the literature / Fazekas de St. Groth S., Scheidigger D: Production of monoclonal antibodies: Strategies and taotics. J. Immunol. Meth. 35, 1-21 (1980), Galfré G., Howe S. C., Milstein C., Butcher G. W., Howard J C., Antibodies to Major Histo-Compatibility Antigens Produced by Hybrid Cell Lines. Nature 266, 550, 1977].

Immunization of Balb / c mice was performed by repeated administration of the triiodothyronine-bovine immunoglobulin conjugate prepared by the Gharib / Gharib H. method, Ryen RJ, Mayberry WE, Hookert T .: Radioimmunoassay | for triiodothyronine (T ₃ ).

I. Affinity and specificity of the antibody for T ^. J. Clin. Endocr. Metab. 33, 509-516 (1971)]. In this conjugate, 17 residues of triiodothyronine were bound to the protein molecule. Hybrid cells were produced by fusion of mouse myeloma lineage FO cells and spleen cells of mice immunized with a triiodothyronine conjugate with a bovine immunoglobulin.

Hybridoma selection was achieved by radioimmunoassay testing of cell culture supernatants of individual hybrid populations / Nedvidkova J., Felt V .: Radioimmunological Assays of Serum Triiodothyronine. Time. Medicine. čes. 120, 1565 (1981)].

The advantage of hybridomas is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with triiodothyronine. The T3-01 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of Balb / c mice.

From cans stored in liquid nitrogen, antibody production can be started without additional antigen and immunization of animals. The antibody produced by the T3-01 hybridoma only reacts with triiodothyronine, does not react with thyroxine, reverse triiodothyronine, or even with diiodothyronine.

Example

2.5 χ 10 ⁶ cells were injected into the peritoneal cavity of mice to expand hybridoma cells in vivo. Mice were administered intraperitoneally 10 ml prior to hybridoma administration for 0.5 ml of paraffin oil to improve attachment of the cells.

After 12 days of hybridoma growth in the peritoneal cavity, mice were killed and ascites fluid was collected. An average of 4.2 ml of ascites fluid was obtained per mouse. The ascites fluid contained 3 to 4 mg / ml immunoglobulin.

Immunoglobulin was isolated from ascites fluid by the method of Hořejší and Smetana / Hořejší J., Smetana R .: 0 the effect of rivanol on plasma proteins. Chem. Leaves 48, 756-764 (1954) and, for further use, made into a solution at a concentration of 5.3 mg / ml in saline and preserved by the addition of sodium azide.

The specificity of the T3-O1 monoclonal antibody was verified by radioimmunoassay method / Nedvidikova J., Felt V .: Radioimmunological assay of serum triiodothyronine. Time. Medicine. čs. 120,

1565, 1981].

The antigen-antibody complex was precipitated with high molecular weight polyethylene glycol mh 6000. ^17β -triiodothyronine was used in Maarai provenance (Institute for Isotopes of Maarai Academy of Sciences). 0.1 ml of T3-01 antibody diluted in veronal buffer to a concentration of 80 µmol / l 125 binds 50% with 66 µg, 1-triiodothyronine.

The monoclonal antibody T3-01 at a concentration of 14 nmol / L is capable of detecting 10 nmoles of triiodothyronine in a sample. The ascitation constant of triiodothyronine binding was calculated according to Jacobs / Jacobs S., chang K.J., Cuatrecasas P .: Biochem. Biophys. Res. Commun. 66,687,

1975 (a) is 3.9 x 10 ⁷ 1 / mol.

Monoclonal antibody T3-01 showed a specific reaction with triiodothyronine in a method of indirect immunohistochemical enzyme immuno-detection with microscopic preparations prepared from human thyroid. The antibody still gave a positive reaction at a concentration of 15 µg / ml.

T3-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells. Cytoplasm contains a large number of polyribosomes, mitochondria and endoplasmic reticulum.

They grow in vitro as semi-suspension cultures.

The basic culture medium is RPMI 1640, which is supplemented with L-glutamine (2 mmol / l), sodium pyruvate (1 mM), penicillin, streptomycin, gentamycin, HEPES buffer (11 mM) and inactivated bovine serum for T3-01 hybridoma culture. (Bioveta, Ivanovice na Hané, 10%).

The hybridoma is cultured at 37 ° C in an incubator with an atmosphere containing 5% carbon dioxide. The mean generation time is 13.4 h. 4 months after construction, the modal number of chromosomes was 89. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass, with type X light chains.

The monoclonal antibody produced by the T3-01 hybridoma reacts positively with triiodothyronine in the radioimmunoassay, it does not have a measurable cross reaction with reverse triiodothyronine, diiodothyronine. Cross-reactivity with thyroxine is less than 0.5%.

The T3-01 hybridoma can be used industrially as a source of a monoclonal antibody against triiodothyronine. The monoclonal antibody against triiodothyronine can be used for the analytical determination of triiodothyronine in the manufacture of pharmaceutical preparations or in endocrinological research.

Another possible use of a monoclonal antibody against triiodothyronine is in thyroid immunohistochemistry. The image after visualization of the antibody in a microscopic preparation with an anti-mouse antibody peroxidase conjugate shows the content and relative distribution of triiodothyronine depending on the current thyroid condition.

Claims (1)

SUBJECT OF THE INVENTION IMG CZAST3-O1 mouse lymphocyte hybridoma, producing a monoclonal antibody of subclass IgG1 against triiodothyronine.