CS276374B6 - TSH-116 mouse lymphocyte hybridoma producing IgG2a class anti-human thyreostimulatory hormone monoclonal antibody - Google Patents

Abstract

The solution relates to the mouse lymphocyte hybridoma TSH-116, producing an antibody of the TgG2a class against human thyroid-stimulating hormone. The monoclonal antibody of the hybridoma TSH-116 itself is suitable for sensitive and specific detection of thyroid-stimulating hormone levels by immunoradiometric analysis and related methods.

Description

The invention relates to a novel hybridoma, i.e. a hybrid single-celled organism, constructed by a cell fusion of a mouse myeloma line P3-X63-Ag8.653 and a mouse spleen lymphoid cell producing an antibody against human thyroid stimulating hormone (TSH).

To date, antibodies to thyrestimulatory hormone have been produced by repeatedly injecting thyroid stimulating hormone (TSH) (usually with an adjuvant) into experimental animals, most often rabbits. The serum of the animals thus immunized, collected after a certain period of antigen action, serves as a source of antibodies, used in particular for the determination of the level of thyroid stimulating hormone in the serum by radioimmunoassay (RIA). 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. Production batches of conventional antisera are therefore very difficult to standardize and are based on production in a wide range of quality. Individual antiserum antibodies are directed against various antigenic determinants on the surface of an antigen molecule, making them difficult to use in an immunoradiometric analysis (IRMA) system that is more sensitive than a radioimmunoassay (RIA) system.

For the production of each batch, a highly pure immunization antigen must be prepared, which is isolated by sophisticated techniques from human pituitary glands.

Lymphocyte hybridomas producing antibodies against thyroid stimulating hormone were prepared in the Czechoslovak Socialist Republic. However, these antibodies have too low an association constant and are therefore not useful for immunoradiometric determination of thyroid stimulating hormone levels in body fluids with sufficient sensitivity. The world literature describes hybridomas producing antibodies against thyroid stimulating hormone with sufficient affinity.

However, the expansion of their range allows for further more advantageous combinations suitable for the determination of thyrotropic hormone, for example by immunoradiometric analysis.

The range of monoclonal antibodies against human thyroid stimulating hormone is expanded by a hybridoma stored in the collection of hybridomas of the Scientific Center for Hormonal Proteins and Peptides of the Faculty of Medicine, Charles University in Hradec Králové, Šimkova 870, under the designation TSH-116.

Said hybridoma was obtained in a manner known from the literature (Campbell, AM: Monoclonal antibody technology, Elsevier, 1984, Coding, JW: Monoclonal antibodies: Principles and practice, Second ed., Academic Press, 1986) by cloning a set of fusion cell hybrids. mouse cells of the myeloma line PJěXĎJTAgS.653 and cells obtained from the spleen of BALB / c mice immunized with human thyroid stimulating hormone isolated from the pituitary gland.

The advantage of the TSH-116 lymphocyte hybridoma is that it produces a completely homogeneous antibody, the so-called monoclonal antibody, which specifically reacts with a single antigenic determinant on the human thyroid stimulating hormone molecule and has a high affinity. The TSH-116 hybrid house can be cultured in vitro in media suitable for growing animal cells or in vivo in the peritoneal cavity of BALB / c mice. The TSHslie hybridoma is suitable for long-term storage in cans stored in liquid nitrogen. Monoclonal antibody production can be initiated after thawing the can without the need for additional antigen. Antibody produced by the TSH hybridoma. 116 is able to bind to the antigen molecule simultaneously with both TSH-51 and TSH-61 antibodies, which is a necessary condition

CS 276374 Ββ design of immunoradiometric analysis system (IRMA). The association constant of the TSHβ-106 monoclonal antibody was determined using a radioimmunoassay system with the second antibody by detecting the binding of the decreasing concentration of the international standard thyr reostimulating hormone IRP 80/558 with the addition of labeled hormone (tracer). First, an amount of tracer was added to 100 mIU / L IRP 80/550 to achieve an activity of 100,000 cpm. This material and a series of 8 two-fold dilutions were used in the assay. The tracer concentration determined by the internal standard method was also included in the calculation of the association constant. Based on these measurements, a high association constant of 1.1 x x 10 ¹¹ 1 / mol was found.

The antibody produced by the TSH-116 hybridoma allows the construction of an immunoradiometric analysis (IRMA) system for the determination of thyroid stimulating hormone levels in body fluids with high sensitivity. This is made possible by its unusually high association constant and by reacting with different antigenic determinants than the antibodies produced by the TSH-51 and T5H-61 hybridomas. The low detection limit of the assay will also allow the determination of reduced levels of thyroid stimulating hormone in human serum, which is important for the diagnosis of functional thyroid disorders.

Example

To propagate the hybridoma cells in vivo, 1 x 10 6 cells were applied to the peritoneal cavity of BAtB / c mice. To better adhere the injected cells, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) one week before the transfer of the hybridoma cells. After 17 days of hybridoma growth, ascitic fluid was collected and repeated three times every other day. A total of 13 ml of ascitic fluid containing 7 mg / ml immunoglobulin was obtained. Pure monoclonal antibody was isolated from this ascitic fluid by protein-A-Sepharose CL 4B complex chromatography, dialyzed to 0.01 M sodium phosphate buffer, pH 7.4, and quenched by ultrafiltration to 1 mg / ml. 10 μl of this solution was labeled with radioactive iodine as follows: 10 μl of antibody 125 solution was mixed with 10 μl of iodine I isotope solution having an activity of 0.25 mCi, and 5 μl of chloramine T at a concentration of 1 mg / ml was added. After incubation for 30 s, the reaction was terminated by the addition of 10 μl of a saturated solution of tyrosine in water. The reaction mixture was then transferred to a PD-10 Sephadex G-25 Medium column with 1 ml of 0.1% w / w bovine serum albumin buffer and purified by chromatography. The iodinated monoclonal antibody produced by the TSH-116 hybridoma was used in an immunoradiometric analysis system (IRMA). 200 μl of standards with a known concentration of thyroid stimulating hormone or test sample were pipetted into tubes with anchored antibody produced by TSH-51 hybridoma on solid phase, after 1 hour shaking at room temperature the material was aspirated, the tube was rinsed once with phosphate buffered saline (PBS). , pH 7.5 and in the next step 200 μl of a solution of iodinated monoclonal antibody produced by the TSH-116 hybridoma in PBS buffer, pH 7.5 was added with the addition of albumin 5 wt. The concentration of labeled antibody produced by the TSH-116 hybridoma was prepared so that the number of pulses was 60,000 per minute in a volume of 200 .mu.l. . The results obtained with the standards at a known concentration are summarized by the calibration curve in Fig. 1. The dashed line indicates the absolute number of pulses of labeled antibody bound to the solid phase, the solid line the number of pulses after subtraction of non-specific zero sample binding. The average blank values when measuring 20 tubes and the band of 2 standard deviations above this average are shown by the hatched band. The radioactivity corresponding to the calibration point of 0.05 mIU / l is at the upper limit of this band and thus represents the detection limit of the assay.

CS 276374 Bo

Furthermore, it was determined what values will be detected in this system of immunoradiometric analysis (IRMA) using solutions of other hormones of known concentration, and this addition of other hormones will affect the result of the determination of the level of thyroid stimulating hormone. The results are summarized in Table 1. The immunoradiometric analysis system was not affected by the concentrations of human chorionic gonadotropin and prolactin used, only the international standard LH IRP 68/40 at a concentration of 160 IU / 1 showed a readable value of thyroid stimulating hormone (0.09 mmol / l). However, according to the data, the standard contains 0.1 ml of thyroid stimulating hormone per 77 IU of luteinizing hormone, i.e. 0.129% of the total amount of luteinizing hormone, and the determined value thus corresponds to the presence of thyroid stimulating hormone in the standard.

TSH-116 hybridoma cells have the morphological character of typical myeloma cells. In vitro, they grow in the form of semi-suspension cultures. The basic culture medium is Dulbecco's modification of minimal essential medium (DMEM) supplemented with L-glutamine (2 nM), penicillin, streptomycin and inactivated fetal calf serum (10%). The hybridoma is cultured at 37 ° C in an atmosphere of 5% by volume of carbon dioxide and has a mean generation time of 20 hours. The modal number of chromosomes at 6 months after establishment was 88. The antibody produced is a mouse monoclonal immunoglobulin of the IgG2a class, which binds human thyroid stimulating hormone with high affinity (the association constant in the radioimmunoassay system is 1.1 x 10 .mu.l / mol).

The monoclonal antibody produced by the TSH-116 hybridoma is useful for highly sensitive and strictly specific determination of thyroid stimulating hormone levels in human serum by radioimmunoassay and related immunochemical techniques. It is used in the production of kits for the purposes of functional diagnosis of thyroid disease in medicine.

TABLE No.1

Standard Determined value of TSH mIU / 1 HCG 10,000 IU / l - + 2 mIU / 1 TSH - + 16 mIU / 1 TSH LH 160 IU / l less than 0.05, 2.0 16.0 0.09 t- + 2 mIU / 1 .TSH 2.1 - + 16 mIU / 1 TSH PRL 200 ng / ml + 30 mIU / l TSH 16.0 less than 0.05 30.0

Abbreviations used: HCG - human chorionic gonadotropin LH - luteinizing hormone PRL - prolactin

Claims (1)

9 PATENT CLAIMS Mouse lymphocyte hybridoma TSH-116 producing a class ϊ IgG2a monoclonal antibody against human thyroid stimulating hormone.