RU2049816C1 - Strain of hybrid cultured murine cells mus musculus l used for preparing monoclonal antibodies to thyroxine - Google Patents

Abstract

FIELD: medicinal biotechnology. SUBSTANCE: method involves preparing the strain of hybrid cultured murine cells which produces monoclonal antibodies to thyroxine. Strain is prepared by hybridization of murine splenocytes of line BALB/c with myeloma X63.Ag 865.3 cells. Monoclonal antibodies show affinity constant 3·10⁹M^-1, their crossed reactivity with triiodothyronine is 1% EFFECT: improved method of hybrid cells preparing. 2 dwg

Description

 The invention relates to biotechnology and can be used to obtain monoclonal antibodies (mAbs) to thyroxine.

 For the correct and timely diagnosis of diseases associated with impaired thyroid function, as well as to monitor the effectiveness of treatment, it is necessary to draw up a fairly accurate idea of the concentration of thyroid stimulating hormone, thyroxine (T4) and triiodothyronine.

 Most immunoassay methods for the determination of thyroxine have been developed on polyclonal sera. As a rule, there are no special problems with their specificity. However, many serums have a relatively low titer and binding constant, which imposes some limitations on their suitability for use in immunoassays, especially when attempts are made to measure free hormone.

Monoclonal antibodies to thyroxine were obtained in several laboratories, the binding constants for the most affinity antibodies were 1.5 ˙ 10 ⁹ to 2.6 ˙ 10 ⁹ M ^-1 (Murray R. Burkhart F. 1990, Clin. Chem. 36, 1077; GR Siebert, NCRaleyh, J. Armstrong US Patent, 1989, 4888296).

It is known to obtain a hybridoma producing monoclonal antibodies to thyroxin by fusing splenocytes of BALB / c mice immunized with chicken thyroxin-gamma globulin conjugated antigen to X63 mouse myeloma cell lines. Ag 865.3. Antibody affinity constant 2.6 ˙ 10 ⁹ M ^-1 , cross-reactions with thyroxine 0.12%. To use these antibodies in thyroxine immunoassay, however, it turned out to be insufficient and the authors of the patent to use them in a mixture with other monoclonal antibodies also claimed in this patent.

 The aim of the invention is to obtain hybrid cultured mouse cells of the BALB / c line producing mAB having high affinity, good specificity, relative indifference to various blocking agents, especially 8-anilino-1-naphthalenesulfonic acid, which is commonly used in thyroxine immunoassay. MKAT should be suitable for the development of immunological test systems for determining the concentration of T4 in biological fluids.

 To obtain hybridoma cells producing mAb, cells of the murine X63 myeloma line were fused. Ag 8653 and splenocytes of a BALB / c mouse immunized with conjugated T4 antigen with hemocyanin from mollusk keyhole limpet.

 Mice were immunized for 3-4 months, periodically, animal serum was analyzed for the presence of antibodies to T4. The analysis was carried out by enzyme-linked immunosorbent assay, conjugated T4 antigen with bovine serum albumin (T4-BSA) was used as antigen. In animals with a high titer of antibodies to T4, spleens were taken and cells were isolated from them for fusion.

 Myeloma line X63. Ag 865.3. a stable non-secreting immunoglobulin myeloma line was selected as the second fusion partner. Cell fusion was performed using polyethylene glycol. The ratio of the number of spleen cells to the number of myeloma cells varied from 5: 1 to 1: 1. After the fusion, the cells were diluted and cultured in a selective medium containing aminopterin, hypoxanthine, and thymidine until growing clones appeared. Supernatants were analyzed by enzyme-linked immunosorbent assay, T-BSA conjugate was used as antigen, antibodies were detected using rabbit anti-mouse IgG labeled with horseradish peroxidase, so that only clones producing class G antibodies were detected.

 An additional analysis of the synthesized antibodies was performed by inhibition of T4 and its analogs of antibody binding to the sorbed T4-BSA conjugate. Positive clones were re-cloned.

The most important parameter in the selection is affinity. Of the positive clones, clone 1B7 was selected, producing antibodies with Kaff 3 ˙ 10 ⁹ M ^-1 , which exceeds the Kaff analog. The cross-reactivity of these antibodies with respect to triiodothyronine is 1%, which is higher than the cross-reactions of the analogue, but it is enough to develop any immunoassay. The range of serum thyroxine concentrations is normally 50-120 ng / ml, and triiodothyronine 0.8-2 ng / ml, so the error that is possible in immunoassay using these antibodies is negligible and there is no need to obtain antibodies with very low values crosshairs. Technicon, for example, uses anti-thyroxine antibodies with a cross of 1.7%, Baxter with a cross of 3%
Antibodies 1B7 belong to the IgG1 subclass.

 Technological is that hybridoma that is stable and grows well in ascites tumors. Hybridoma 1B7 satisfies both requirements. To obtain large quantities of antibodies, it was administered intraperitoneally to mice previously sensitized by the pier. Antibodies from ascites liquids were isolated by DEAE cellulose ion exchange chromatography.

 The obtained μAt were used in the enzyme immunoassay for the determination of T4. The method is based on the competition between thyroxin from a sample and solid-state thyroxin for binding to peroxidase-labeled thyroxine antibodies. Using antibodies 1B7, you can measure the concentration of thyroxine in the entire concentration range in which it is necessary to determine this hormone (from 10 to 400 ng / ml), the calibration curve is linear in this entire area.

 The effect of 8-anilino-1-naphthalenesulfonic acid (ANS) on the binding of 1B7 antibodies to thyroxine was studied and it was shown that ANS to a concentration of 0.05%, which is commonly used in thyroxine immunoassay, does not affect the results of the analysis.

 The strain is obtained as follows.

 1. Immunization.

 BALB / c mice, females, were immunized intraperitoneally with 100 μg of conjugated T4 hemocyanin antigen suspended in 100 μl of saline and 200 μl of complete Freund's adjuvant. The injection was repeated after 6 weeks and then after 2-4 weeks with incomplete adjuvant. 7 days after the last immunization, blood was taken from mice and the sera were analyzed for the presence of antibodies by enzyme-linked immunosorbent assay. For final immunization, mice were selected whose serum showed the highest titer of thyroxine antibodies. 3 days before hybridization, 100 μg of antigen without adjuvant was administered to such mice.

 2. Obtaining conjugated antigens.

 2.1. Obtaining a conjugate of thyroxine with hemocyanin from the mollusk fissurella (KLH).

1 mg of thyroxine was dissolved in 0.05 M NaOH and mixed with 1 mg of hemocyanin suspended in 1 ml of distilled water. Was added under vigorous stirring, 26 .mu.l of 25% glutaraldehyde and incubated for 12 hours at ⁴ ° C was added 0.1 mg of NaBH 50 .mu.l, were incubated for 1 hour at room temperature with stirring, and then dialyzed against distilled water for a week . According to spectrophotometric analysis, the conjugate contained 130 moles of thyroxine per protein molecule.

 2.2. Obtaining conjugate thyroxine-bovine serum albumin (BSA).

1 mg of thyroxine dissolved in a mixture of 0.5 ml of dimethyl sulfoxide and 0.5 ml of water was mixed with 1 mg of bovine serum albumin in 0.5 ml of water and 3 mg of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide was added to the mixture with stirring hydrochloride. The reaction mixture was incubated for 12 hours at 4 ^{° C} and dialyzed against water. The conjugate contained 17 molecules of thyroxine per protein molecule according to spectrophotometry.

 3. Enzyme-linked immunosorbent assay for the detection of antibodies to thyroxine.

The conjugated antigen thyroxine-BSA dissolved in 0.05 M carbonate-bicarbonate buffer, pH 9.6 at a concentration of 1 ug / ml and adsorbed on polystyrene 96-well plates at 100 l per well, at ⁴ ° C overnight, then the solid the phase was washed with a 0.05% solution of triton X-100 and water. 90 μl of 0.01 M sodium phosphate buffer containing 0.15 M NaCl, 0.05% tween-20 and 0.2% BSA (buffer A) were added to the wells. 10 μl of serum was added to the first well and a series of ten-fold dilutions was made until the end of the row. After incubation for 1 h at room temperature, the plate was washed and 90 μl of rabbit antibodies against mouse immunoglobulins labeled with horseradish peroxidase dissolved in buffer A were added to each well. The plate was incubated for 45 min, washed and a substrate solution was added to measure enzyme activity (4 μl of 30% H202 and 4 mg of orthophenylenediamine per 100 ml of 0.1 M sodium citrate buffer (pH5), 90 μl each, after 10 minutes, 45 μl of a 10% sulfuric acid solution was added to stop the enzymatic reaction and the absorbance was measured at 492 nm.

 4. Hybridization.

 The spleen was taken from an immunized mouse under sterile conditions and a suspension of splenocytes was prepared. Cells of the myeloma line RZ-X63. Ag 8.653. washed with serum-free medium and poured with splenocytes in a 1: 1 ratio using 30% polyethylene glycol 6000, 1 ml. The fused cells were washed with serum-free medium and suspended in 50 ml of RPM1 1640 medium containing 10% fetal calf serum, 2 mM glutamine, 5-10 5 M 2-mercaptoethanol, 1 mM sodium pyruvate, 100 units of penicillin and streptomycin, 4-10-7 M aminopterin, 10-4 M hypoxanthine, 1.6 ˙ 10-5 thymidine and poured into 96-well plates containing a feeder layer of murine peritoneal macrophages at 50 μl per well. After a week, the plates were scanned daily, supernatants from actively growing clones were selected and analyzed for the presence of antibodies to thyroxine. Active clones were transferred to 24-well plates, some of the cells were frozen.

 5. The selection of positive clones.

 For selection of positive clones, the enzyme-linked immunosorbent assay was used. The conjugated thyroxin-BSA antigen was sorbed in 96-well plates as described in claim 3. Serum of mice containing antibodies to thyroxin (1: 200 dilution) was used as positive controls, and serum of unimmunized mice and supernatants from myeloma lines were used as negative controls.

 Primary screening: 50 μl of assay buffer was added to the wells of the T4 protein antigen plate and 50 μl of supernatants were added. As a positive control, 50 μl of immunine mouse serum at a dilution of 1: 200 was placed in one of the wells, and culture medium was used as a negative control. The tablets with these solutions were incubated for 1 h at room temperature, washed, treated for 30–40 min with a solution of rabbit anti-mouse IgG antibodies associated with horseradish peroxidase, and washed with water. After this, the enzymatic activity was determined as described in section 3. Those clones that gave a signal 10 times higher than the background were selected. 29 positive clones were identified.

 Next, the ability of antibodies produced by these clones to bind free thyroxine was studied. The anti-thyroxine antibody was determined by inhibiting the binding of antibodies to the immobilized antigen in the presence of free thyroxine.

 Secondary Screening: 500 μl of supernatants that showed high binding to the sorbed antigen were placed in the wells of the antigen plate. 50 μl of assay buffer was added to half of the wells, and 50 μl of a thyroxine solution at a concentration of 500 ng / ml in the same buffer was added to the other half. Further analysis was carried out as described above. 6 clones were selected that actively secreted antibodies to thyroxine. These clones were analyzed in a similar manner for the ability to bind the thyroxine analog triiodothyronine. For further work, clone 1B7 was selected, which has the highest efficiency and the least cross-reaction with respect to triiodothyronine.

 6. Cloning by the method of limiting dilutions.

 Cells of the selected clone were cloned by limiting dilution in a 96-well plate containing the macrophage feeder layer at a rate of 0.3 cells per well. After clones appeared, supernatants were tested for the presence of monoclonal antibodies. All grown clones continued to produce anti-thyroxine antibodies.

 7. Production of monoclonal antibodies in the form of ascites.

To obtain large amounts of antithyroxin antibodies, hybridoma cells were injected intraperitoneally to BALB / c mice, 10 days before receiving injections of 0.5 ml pristan, 3x10 cells per animal. After the appearance of ascites tumors in mice (5-14 days) were collected ascites fluid, the cells were removed by centrifugation, ascites fluid was frozen and stored at -20 ° ^C. From each mouse received 5-10 ml ascites containing from 5 to 8 mg / ml of antibodies. The obtained strain of hybrid cultured cells 1B7 is stored in the Specialized collection of transplantable somatic vertebrate cells of the All-Union collection of cell cultures under the number VSCK (P) 622D. The strain is characterized by the following features:
Cultural properties of clone 1B7.

 Cells are cultured in a monolayer at a concentration of 5 ˙ 10 cells / ml, the subculture time was about 48 hours.

Cryopreservation of hybridoma is carried out in a mixture of equal volumes of RPMI 1640 medium and calf embryonic serum containing 15% dimethyl sulfoxide with 10 ⁶ cells per ampoule. The first three days ampoules stored at ^-70 ° C, then transferred to liquid nitrogen. After defrosting, the cells did not lose their growth properties.

Characterization of monoclonal antibodies:
The affinity constant of antibodies 1B7 3 ˙ 10 ⁹ M ^-1 .

Cross reactions with triiodothyronine 1%
Antibodies from ascites were isolated by salt deposition with ammonium sulfate and subsequent chromatography on DEAE cellulose in 0.01 M phosphate buffer, pH 8.2 using a NaCl gradient from 0 to 0.5 M NaCl. The homogeneity of the isolated antibodies was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Antibodies synthesized by clone 1B7 belong to the IgG1 subclass, as determined by enzyme-linked immunosorbent assay using goat antibodies to mouse IgG subclasses. The use of the strain is illustrated by the following example.

 PRI me R 1. The use of the obtained monoclonal antibodies for the determination of thyroxine in serum by enzyme immunoassay.

 In this solid-phase method, the conjugated thyroxin-protein antigen is adsorbed on the solid phase, and the measurement is based on the competition of thyroxin from the sample and immobilized thyroxin for binding to antithyroxin antibodies labeled with horseradish peroxidase enzyme.

 Thyroxin-BSA conjugated antigen was dissolved in 0.05 M sodium carbonate-bicarbonate buffer (pH 9.6) at a concentration of 1 μg / ml and sorbed on NUNK polystyrene 96-well plates, 120 μl / well, 3 h at room temperature and at 4 ° C. overnight, then the solid phase was washed three times with a 0.05% X-100 triton solution, the tablet was then dried.

 Monoclonal antibodies purified by ion exchange chromatography on DEAE cellulose were oxidized with sodium periodate and labeled with horseradish peroxidase. For this, 1 mg of peroxidase was dissolved in 0.1 ml of water, and 1 mg of sodium periodate in 25 μl of water was added. The mixture was incubated for 2 h in the dark at room temperature, passed through a 0.5 cm layer of Sephadex G-25 and mixed with 4 mg of antibodies in 0.1 M carbonate-bicarbonate buffer, pH 9.5. After 3 hours, 0.1 mg of sodium borohydride was added, incubated for 1 hour and the conjugate was dialyzed against water. Then the conjugate was precipitated with a 75% solution of ammonium sulfate, centrifuged and dissolved in water. After dialysis against saline, the conjugate was stored in PBS at a concentration of 10 mg / ml with the addition of 0.01% merthiolate.

Serum thyroxine standard solutions were prepared as follows:
Donor pool serum or horse serum 100 ml was centrifuged at 15 m / min, inactivated at ⁵⁶ ° C, then passed through a column of 1.5 cm with activated charcoal. The serum thus prepared was passed through an affinity column with monoclonal antibodies 1B7 to thyroxine, after which the depleted serum was filtered through a membrane with a pore diameter of 3.0 nm. Thyroxine was dissolved in DMSO at a concentration of 1 mg / ml and standard solutions in serum were prepared from it at a concentration of 0; 12.5; 25; fifty; 100; 200; 400 ng / ml. Standard serum was poured into test tubes and freeze-dried, before use, the standards were diluted with distilled water.

 Analysis.

 Aliquots of 10 μl from the patient sera and standard solutions of thyroxine in donor serum containing a known amount of the hormone were added to the wells of the tablet and 50 μl of a solution of 0.06% 8-anilino-1-naphtholsulfonic acid (ANS) in 0.05 M were added. veronal-medial buffer pH 8.6 with 0.05% tween-20 and after 2 min 50 μl of a solution of peroxidase-antibody conjugate in the same buffer, dilution from a concentration of 10 mg / ml 1: 10000. The mixture was thoroughly shaken, incubated for 30 min at room temperature, the solid phase was washed and 100 μl of a substrate solution were added to each well (4 μl of 30% H202 and 4 mg of orthophenylenediamine per 10 ml of 0.1 M sodium citrate buffer, pH 5.0 ), incubated for 10 min in the dark, detection at 492 nm. Based on the absorption values obtained for standard solutions, a calibration curve was constructed (Fig. 1). All measurements were performed in duplicates.

 The concentration of thyroxin in the serum of patients was determined by a calibration curve. For example, an absorption of 1.0 corresponds to a serum thyroxine concentration of 90 ng / ml. The coefficient of variability for parallel measurements did not exceed 8%. The minimum detectable thyroxine concentration was 10 ng / ml. As can be seen from the calibration curve, the developed method allows you to determine thyroxin in the range from 0 to 400 ng / ml.

 Triiodothyronine at a concentration of 200 ng / ml did not affect the results of the analysis.

The developed method was used to measure the concentration of thyroxine in 64 sera of patients and donors. At the same time, the concentration of the hormone was measured by the radioimmune method, a kit produced by the Minsk Institute of Bioorganic Chemistry. The results of the analysis found a good match, the correlation coefficient between measurements made by different methods was 0.96 (Fig. 2),
The data presented confirm that the obtained 1B7 monoclonal antibodies can be successfully used in thyroxine immunoassay.

Claims (1)

 The strain of hybrid cultured mouse cells of Mus musculus L. BCCK / P / N 622 D used to obtain monoclonal antibodies to thyroxine.

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