RU2097426C1 - Strain of the hybrid cultured mammalian cells mus musculus l - a producer of monoclonal antibodies to a-determinant of hepatitis b virus surface antigen - Google Patents

Abstract

FIELD: biotechnology, virology. SUBSTANCE: invention relates to preparing the strain of hybrid cultured murine cells HBSV-4 producing monoclonal antibodies to a-determinant of hepatitis B virus surface antigen. Strain is obtained by hybridization of myeloma cells P3-NS1-Ag 4-1 with murine splenocytes of strain BALB/c immunized with hepatitis B virus surface antigen isolated from serum blood of patients with chronic hepatitis. The obtained strain produces monoclonal antibodies of class IgG1 interacting with conformational epitope on group-specific a-determinant HBSAg and showing high stability. Monoclonal antibodies ensure to detect HBSAg in solutions and biological fluids of organism. Invention can be used for hepatitis B diagnosis. EFFECT: strain indicated above. 1 dwg

Description

 The invention relates to hybridoma technology and can be used in the diagnosis of hepatitis B.

 Hepatitis B is a serious human infectious disease, HBsAg (hepatitis B surface antigen) is the main marker of the disease, confirming the presence of an acute or chronic form of infection or carriage. The creation of test systems for the diagnosis of hepatitis B requires the presence of monoclonal antibodies (MCAs) to HBsAg.

A number of authors [1,5] obtained collections of hybridomas producing MAB (monoclonal antibodies) to HB _s Ag. However, only a few of the strains possess the properties necessary for producer strains suitable for the production of diagnostic test systems with high growth characteristics, a high yield of antibodies to the mouse in ascites fluid, the stability of produced antibodies in solution and adsorbed on the solid phase, and high activity in the reaction antigen-antibody, specificity for a specific site “a” of the antigenic determinant and, most importantly, the ability to detect HBsAg in low concentrations [3,4]
It is known that the group-specific a-determinant of the HB _s Ag molecule is complex and contains several antibody binding sites [6] The presence of MCAs to different sites on the a-determinant of the HB _s Ag molecule could allow constructing diagnostics using the two-site method in which the interaction of immunospecific reagents is carried out in one stage, using the independence of the binding of MAB to different epitopes. When designing a test system using the two-site method, higher sensitivity is possible [3] The closest to the claimed one is the ZhAK-22 hybridoma, which has the following properties: ascitic fluid titer in ELISA 4x10 ⁵ , the content of specific antibodies 9.7 mg / ml [1,2 ] sensitivity in the determination of HB _s Ag in RIA 2 ng / ml, in ELISA 1 ng / ml [3,4] Compared with ZhAK-22, HBSV-4 has a higher ascitic fluid output to the mouse, higher ascitic fluid titers in ELISA, higher sensitivity in the determination of HB _s Ag, another site of a-determinants. The aim of this invention is to obtain a strain of hybrid cultured animal cells of Mus. musculus producing MAB to a specific site of the group-specific a-determinant of HB _s Ag. The goal is achieved by obtaining collections of hybridomas producing MAB for HB _s Ag (by reaction in ELISA) and consisting of 50 different clones. Hybridomas are compared in terms of growth characteristics, stability of antibody production, grafting in syngeneic mice, the volume of ascites fluids obtained, antibody titers of ascites and culture fluids in different ELISA variants, as well as in RPHA and immunodiffusion. Antibodies produced by hybridomas are also compared in the reaction of competitive ELISA, which allows them to be divided into several competition groups. The reaction with adw and avw HB _s Ag genetically engineered variants allows them to be divided into several competition groups. The reaction with adw and ayw genetically engineered variants of HB _s Ag allows the determination of MCAs complementary to the a-determinant of the HB _s Ag molecule.

As a result of the analysis, a strain was selected that was complementary to a specific site of the a-determinant of HB _s Ag and possesses good characteristics of the strain-producer of MCA.

Hybridomas are obtained by fusion of P3-NS1-Ag4-1 myeloma cells with splenocytes from the BALB / c mouse immunized with HB _s Ag.

A BALB / c mouse (female, 8 weeks old) was immunized intraperitoneally with HB _s Ag isolated from serum of chronically sick people in an amount of 20 μg of 0.5 ml phosphate buffered saline solution (PBS) containing 50% of Freund's complete adjuvant and after 8 weeks 200 μg HB _s Ag in 0.5 ml PBS intraperitoneally. 3 days after the second immunization, spleen cells of the immune mouse hybridize with mouse myeloma cells P3-NS1-Ag4-1 (ratio 3 1) in 1 ml of a solution containing 50% polyethylene glycol (mm 4000 “Merk”) and 5% dimethyl sulfoxide . After hybridization and washing from polyethylene glycol, cells are plated in 96-well plates with 3 x 10 ⁵ cells per well. For cultivation and selection of hybrids, DMEM medium (m) with the addition of 10% calf embryonic serum, 10 ^-4 M hypoxanthine, 4 • 10 ^-7 aminopterin and 1.6 • 10 ^-5 M thymidine is used. Producing hybrids are selected by ELISA and enzyme-linked immunosorbent assay twice cloned by limiting dilution.

 The productive strain was designated HBSV-4.

 The strain was deposited in the Specialized collection of transplantable somatic vertebrate cells of the All-Union collection of cell cultures on 11/30/93 under the number VSCK / P / 628 D. The strain was deposited with a scientific description (passport).

 The strain is characterized by the following features.

 The pedigree of the strain.

The hybridoma was obtained by fusion of P3-NS1-Ag4-1 myeloma cells with BALB / c mouse splenocytes immunized with HB _s Ag using polyethylene glycol (mm 4000). Hybrid breeding was performed on NAT medium. Cells were cloned twice, the percentage of positive clones after the 2nd cloning of 100% The number of passages at the time of deposit: 35 passages.

 Standard growing conditions.

DEMEM medium with 10% fetal calf serum, 40 μg / ml gentamicin, 37 ^o C, 5% CO ₂ .

 Cultural properties.

 The hybridoma is cultivated in glass or plastic bottles with a volume of 0.05-1.5 L in a stationary state, the cells are removed with a mixture of trypsin solutions (25 μg / L) and Versen in a ratio of 1: 2, inoculative dose of 120 thousand cells per ml, transfer rate 1 5 or 1 6, subculture time 2 3 days.

 The cultivation of hybridomas in the body of the animal.

The hybridoma can be cultured in the body of BALB / c mice, sensitized intraperitoneally in 5-40 days, 0.5 ml pristan or paraffin oil. The dose of cells when vaccinated is 1 -10 • 10 ⁵ . Ascites is formed in 5-18 days. Hybridoma may interrupt.

 Morphological signs.

 The culture consists of large rounded cells, similar in morphology and size to the original parental myeloma P3-NS1-Ag4-1.

 Species data.

 A hybridoma is a mouse obtained by fusion of mouse myeloma cells and mouse splenocytes; it can be cultured in the mouse organism; the MCAs secreted by it interact with antiserum to mouse immunoglobulins.

 Marker signs and methods for their assessment.

The hybridoma is a monolayer-suspension cell culture and produces antibodies with the following marker characteristics of the antibody belong to the IgG1 class, weakly interact with Staphylococcus aureus Protein A (according to ELISA and affinity chromatography), react with ELISA with HB _s plasma antigen and HB _s - antigen genetically engineered adw and ayw serotypes give a positive response in the diffuse precipitation reaction in the gel, RPHA, and ELISA sandwich, interacting simultaneously with HB _s Ag assembled on the solid phase and with HBs _s Ag conjugated bath with peroxidase and in solution.

 Contamination data.

 Bacteria and fungi were not detected, control for contamination with viruses and mycoplasmas was not carried out.

 Characterization of the biosynthesis of useful products.

MAB secreted by the hybridoma have a titer of about 1 • 10 ^-4 in the culture fluid and 1 • 10 ^-6 1 • 10 ^-7 in the ascites fluid (according to ELISA); antibody production stability is maintained for 35 passages in the cell culture (observation period).

 The method of cryopreservation.

Cells are frozen on growth medium with 40% fetal calf serum and 10% dimethyl sulfoxide at a temperature of -70 ^o C in a foam container with a wall thickness of 1.5 cm for 1 2 min at a temperature of 37 40 ^o C, washed from cryoprotective medium and cultivated on a growth medium in glass or plastic bottles, cell survival during thawing is 70–80% (by trypan blue stain).

 The invention is illustrated in the drawing.

 On the abscissa axis is the number of three-fold breeding of a competitor. The competitors in the reaction are MCA HBSV-4 from ascites (2) and JAC-22 from ascites (1). Labeled antibodies MKA ZhAK-22. The initial concentration of MAB HBSV-4 200 μg / ml, MAB Jacques-22 4 μg / ml.

 Example 1. Determination of the specific activity of antibodies.

HB _s Ag from the plasma of sick people or a genotype engineering ayw serotype is sorbed into the wells of polystyrene plates from a PBS solution containing 10 μg / ml antigen overnight; non-specific binding sites on plastic are blocked with 0.1% casein in PBS; titration of the culture and ascites fluid with a dilution of 1/30 with a multiplicity of 3 in blocking buffer; the reaction time of the antigen-antibody overnight at room temperature; after washing, antibodies against mouse immunoglobulins conjugated to horseradish peroxidase are added to the wells. After incubation (1 h, 37 ^° C), unbound reaction products are washed and the bound conjugate is quantified by cleavage of the substrate (H ₂ O ₂ ) in the presence of the orthophenylenediamine chromogen. A positive reaction is manifested by brown staining and calculated at 492 nm. The titer is considered the dilution of the antibody solution, in which there is an optical absorption of 0.2 pu / ml at a wavelength of 492 nm.

When reacting with plasma HB _s Ag, the titers of the culture fluid are 10 ^-4 , the titers of ascites fluid are 10 ^-6 10 ^-7 . The ratio of reaction titers with HB _s Ag plasma and genetically engineered ayw serotype 1:27.

Example 2. The reaction of MCA with different serotypes of genetically engineered HB _s Ag (adw b ayw), MCA is sorbed from a PBS solution containing antibodies at a concentration of 10 μg / ml in wells of polystyrene plates, blocked with a 0.1% casein solution and adw is added and ayw variants of genetically engineered HB _s Ag in blocking in blocking buffer and at a concentration of 100 1.3 ng / ml with a dilution ratio of 3. Stabilized staphylococcus aureus A affinity chromatography is used for sorption [8] Antigen-antibody reaction is carried out for nights at room temperature. After washing, HBSV-4 MCA was added conjugated to horseradish peroxidase for 1 h at 37 ^° C. The positive reaction was determined as described above. HBSV-4 MCAs react with both adw and ayw serotypes of genetically engineered HB _s Ag with a titer ratio of 1 1.

 Example 3. Carrying out a competitive ELISA.

The method is based on competition between horseradish peroxidase labeled and non-labeled MCA with limited antigen concentration. MAB conjugates are prepared by the periodic method [7] Competitive antibodies are added to the wells of polystyrene plates with sorbed HB _s Ag in a series of 3-fold dilutions and incubated for 0.30 h at 43 ^° C, after which labeled MABs were introduced for 2 h at 43 ^° C Dilution of labeled MCAs is selected so that they, in the absence of a competitor, give an extinction value of 492 nm 1 pfu / ml in ELISA. The amount of bound label is determined as described above.

где А величина связывания метки в отсутствии конкурента;
В связывание метки в присутствии немеченного МКА.The percentage of competition is calculated by the formula:

where A is the label binding value in the absence of a competitor;
In label binding in the presence of unlabeled MCA.

 Figure 1 shows the competition curves of clones HBSV-4 and ZhAK-22. From FIG. 1 it follows that HBSV-4 MCAs do not compete for binding to labeled MCA JAC-22, while labeled and unlabeled MCAs JAC-22 compete.

 Example 4. Determination of the stability of the MCA in HBSV-4 solutions.

The stability of the MCA is determined by the ratio of titers in ELISA of culture and ascites liquids after treatment from the following methods: heating at 37 ^° C for 48 hours, heating at 60 ^° C for 1 hour, triple freezing and defrosting.

 It was shown that with all processing methods, the titers of specific antibodies determined by ELISA did not change in culture and ascites fluids.

Example 5. Determination of HB _s Ag in solutions.

HBSV-4 MCAs, purified by affinity chromatography on Staphylococcus aureus Protein A, are conjugated to horseradish peroxidase. In the wells of polystyrene plates, purified HBSV-4 MCAs are sorbed from a PBS solution containing 10 μg / ml MCA overnight; nonspecific binding sites are blocked with a 0.1% solution of 3FR casein and washed three times with water and HB _s Ag is added in a series of three dilutions at a concentration of 100 0.01 ng in a volume of 100 μl and conjugate HBSV-4 MCA with horseradish peroxidase in a dilution of 1/1000 in volume 50 μl for 3 hours at 43 ^° C. The reaction is performed as described above. A reaction is considered positive if the extinction value at 492 nm exceeds the extinction value for the control well (without HB _s Ag) by 0.04 p.u. The method allows determination of up to 0.3 ng / ml HB _s Ag in solution.

The strain differs from other similar strains in the following set of features: the MABs produced by it react in ELISA with the following HB _s Ag variants: plasma from the serum of ill people, genetically engineered vaccine ayw and adw serotypes; do not compete for the binding site on the HB _s Ag molecule with JAC-22 MAB; does not bind to HB _s Ag denatured with 2-mercaptoethanol; therefore, MCAs are complementary to the confarmation epitope on the group-specific "a" antigenic determinant of the HB _s Ag molecule (this region of the a-determinant of the HB _s Ag molecule is designated B); MCAs refer to IgG1; can be purified by affinity chromatography on protein A of Staphylococcus aureus with high yield, have high stability when stored in solutions and sorption on plastic; the cells have excellent growth characteristics, are stable according to the production of MCA (observation period of about 45 passages), graft well in animals and give a good yield of ascites fluid to the mouse (3-4 ml) with a high content of specific antibodies (5-10 mg / ml) and with high titers of specific antibodies in ELISA (10 ⁶ 10 ⁷ ); MCAs are suitable for detecting HB _s Ag in solutions in human serum and adsorbed on plastic.

 These properties allow the use of strain HBSV-4 for the production of diagnostic test systems.

Sources of information
1. Kushch A. A. Momot A.M. Shumai E.P. et al. Preparation and characterization of hybridomas producing monoclonal antibodies to the hepatitis B virus surface antigen, Issues in Virology, N 4, 1987, p. 448,541.

 2. Zhdanov V.M. Kushch A.A. Momot A.M. et al. Properties of monoclonal antibodies interacting with the determinants of the hepatitis B virus surface antigen, Molecular Genetics, Microbiology, Virology, N 12, 1986, p. 37 40.

 3. Kushch A. A. Momot A. M. et al. Comparison of various test systems for radioimmunological determination of hepatitis B virus surface antigen using monoclonal antibodies, Issues of Virology, N 5, 1987, p. 544-548.

 4. Vorobyov S.M. Simonov V.I. Kushch A.A. The use of monoclonal antibodies for enzyme immunoassay to determine the surface antigen of hepatitis B virus, Biotechnology, N 6, 1987, p. 720 724.

5. Wands JR Zurawski VR High affinity monoclonal antibodies to hepatitis B surface antigen (HB _s Ag) produced by somatic cell hybrids, Gastroenterology, 1981 yv 80, p. 225,232.

 6. Wand J. R. et al. Signature analisis of hepatitus B viral antigenic structure: analisis by monoclonal radioimmunoassays, PNAS USA, 1984 y. v. 81, p. 2237 2241.

 7. Nakane P.K. and Kawaoi A. Histochem. Cytochem. 1974 y. v.22, p. 1084.

 8. Ey P.L. Prowse S.J. and Jenkin C.R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose, Immunochemistry, v. 15, p. 429,436.

Claims (1)

 The strain of hybrid cultured cells of the animal Mus musculus L. VSCC (II) N 6280 producer of monoclonal antibodies to the α-determinant of the hepatitis B surface antigen.