RU2355762C2 - STRAIN OF HYBRIDE CULTURED ANIMAL CELLS Mus musculus L-PRODUCER OF MONOCLONAL ANTIBODIES AGAINST DIFFERENT ISOFORMS OF ALPHA 2-FERTILITY MICROGLOBULIN/GLYCODELIN - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: present invention relates to biotechnology. An A-5D1 strain is obtained from fusing mouse myeloma cell line SP2/0.Ag14 with mouse lymphocyte line Balb/c, immunised by introduction into pouches of a purified preparation of alpha 2-fertility microglobulin, extracted from amniotic fluid, and deposited in the collection of passaged culture of mammal cells of the state research institute of human morphology (Russian Academy of Medical Sciences) under the number 144/2002. The strain of hybredome A-5D1 synthesises the IgG1 class monoclonal antibody, which specifically interacts in enzyme-linked immunosorbent assay and immunohistochemical assay with isoforms of alpha 2-fertility microglobulin of endometrial or special origin. Monoclonal antibodies do not have cross reaction with placental α 1-microglobulin, trophoblastic β1-globulin, human chorionic gonadotropin, α-fetoprotein, bovine serum albumin, and C-reactive protein. Monoclonal antibodies detect alpha 2-fertility microglobulin/glycodelin in cells and tissue of female and male reproductive organs.

EFFECT: use of monoclonal antibody A-5D1 as an immunodiagnostic system allows for quantitative analysis of isoforms of alpha 2-fertility microglobulin/glycodelin in body fluids with high sensitivity (1 ng/ml) and specificity.

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Description

The invention relates to the field of biotechnology, reproductive medicine, immunohistochemistry.

The inventive strain was obtained by fusion of murine myeloma cells SP2 / 0.Ag14 with lymph node cells of Balb / c mice immunized with the introduction of a purified preparation of AMGF isolated from amniotic fluid into the paw pads. Hybrid breeding was performed on HAT medium. The strain synthesizes monoclonal antibodies (MCAs) that specifically interact in enzyme-linked immunosorbent assay and immunohistochemical analysis with AMHF both endometrial and sperm origin. The antibody titer in the culture fluid is 1: 500-1: 1000, in ascites fluid up to 1: 1 × 10 ⁷ . Monoclonal antibodies can be used to construct immunobiotechnological test systems for the quantitative determination of AMHF in biological fluids and for immunohistochemical study of tissues.

AMGF / glycodelin protein is a glycoprotein, which is a homodimeric complex with a molecular weight of 50-60 kDa and a molecular weight of 28 kDa subunits. In the years 1976-1985. By several independent research groups, the protein was isolated from the early human placenta, decidual membrane, amniotic fluid, seminal plasma and was characterized under different names depending on the source and physico-chemical properties: placental α2-globulin (Petrunin D.D., Gryaznova I.M. ., Petrunina Yu.P., Tatarinov Yu.S. Immunochemical identification of organ-specific human placental α2-globulin and its content in amniotic fluid. BEBiM, 1976, No. 7, p. 803-804); α2-microglobulin of fertility (Petrunin D.D., Pshenichnikova T.Ya., Shevchenko O.P., Piganova N.L. Immunochemical study of α2-microglobulin fertility in the endometrium. Akush. and gin., 1983, No. 9, p. 27-28); placental protein 14, PP14 (Bohn H., Kraus W., Winckler W, New soluble placental tissue proteins: their isolation, characterization, localization and quantification. In: "Immunology of Human Placental Proteins", ed. Klopper. Placental Suppl (4 ) Praeger, NY, 1982, p. 67-82); endometrial α2-globulin, α2-PEG (Bell SC, Hales MW, Patel S., Kiwan PH Protein synthesis and secretion by the human endometrium and decidua during early pregnancy. Br. J. Obstet. Gynaecol., 1985a, v. 92, p.793-803); pregnancy-associated endometrial protein 15, EP15 (Bell SC, Patel S., Hales MW Immunochemical detection and characterization of pregnancy-associated endometrial alpha-globulins secreted by the human endometrium. J. Reprod. Fertil., 1985b, v. 74, p .261); progestogen-dependent endometrial protein, PEP (Joshi S.G., Ebert K.M., Swartz D.R. Detection and synthesis of a progestogen-dependent protein in human endometrium. J. Reprod. Fertil., 1980, v. 59, p. 273); α-uterine protein, AUP (Sutcliff R.G., Joshi S.C., Patterson W.F. Serological identity between human alpha-uterine protein and human progestogen-dependent endometrial protein. J. Reprod. Fertil., 1982, v. 65, p.207.). In immunological tests, the antigenic identity of these proteins was established (Bohn H., Kraus W., Winckler W. New soluble placental tissue proteins: their isolation, characterization, localization and quantification. In: "Immunology of Human Placental Proteins", ed. Klopper. Placental Suppl; (4) Praeger, NY, 1982, p. 67-82; Bell SC, Bohn H. Immunochemical and biochemical relationship between human pregnancy-associated secreted endometrial alphal- and alpha2-globulins (alphal- and alpha2-PEG) and the soluble placental proteins 12 and 14 (PP12 and PP14). Placenta, 1986, v. 7, N4, p. 283-294). For unification of nomenclature Dell et al. (Dell A., Morris HR, Easton RL Structural analysis of the oligosaccharides derived from glycoprotein with potent immunosuppressive and contraceptive activities. J. Biol. Chem., 1995, v.270, N41, p.24116-24125) proposed the modern designation " glycodelin ", reflecting the unique property of the protein - gender-dependent glycosylation and the presence of its various glycoforms depending on the place of synthesis: amniotic - glycodelin-A, sperm - glycodelin-S (Seppala M., Taylor RN, Koistinen H., Koistinen R., Milgrom E. Glycodelin: A major lipocalin protein of the reproductive axis with diverse action in cell recognition and differentiation. Endocrin. Reviews, 2002, 23 (4): 401-430), follicular - glycodelin-F (Chiu PC, Koistinen R. , Koistinen H. et al. Zona binding inhibitory factor-1 from human follicular fluid is an isoform of glycodelin. Biol. Reprod., 2003, 69: 365-372), cumulus - glycodelin-C (Chiu R.S., Chung M.K., Koistinen R. , et al. Cumulus oophorus-associated glycodelin-C displaces sperm bound glycodelin-A and -F and stimulates spermatozoa-zona pellucida binding. J. Biol. Chem., 2007, 282 (8): 5378-88). The level of AMHF / glycodeline in biological fluids reflects the functional state of the organs of the human reproductive system, its quantitative determination can be used for differential diagnosis and monitoring the effectiveness of treatment of reproductive health disorders.

The invention relates to hybridoma biotechnology and can be used to create immunodiagnostic test systems for the quantitative determination of AMHF / glycodeline in biological fluids and immunohistochemical study of tissues.

The purpose of the invention is to obtain a strain of hybridoma cells synthesizing MCA against AMHF / glycodeline with high proliferative activity and productivity. Foreign literature describes the preparation of MCA for PP14 proteins (Riitinen L., Narvanen O., Virtanen et al. Monoclonal antibodies against endometrial protein PP 14 and their use for purification and radioimmunoassay of PP 14. J. Immunol. Methods, 1991, 136: 85-90), α2-PEG (Bell SC, Jackson J., Dore-Green F. et al. Development and validation of a radioimmunoassay for human α2-PEG and detection in serum during pregnancy Hum. Reprod., 1987, 25 ( 5): 389-97), glycodeline (Eschke U., Kuhn S., Mylonas I. et al. Development and characterization of monoclonal antibodies for the immunohistochemical detection of glycodelin A in decidual, endometrial and gynecological tumor cells. Histopathology, 2006, 48 (4): 394-406). However, when obtaining strains of hybrid cells, another source of antigen for immunization was used (purified endometrial cytosolic extract - Bell (Bell SC, Jackson J., Dore-Green F. et al. Development and validation of a radioimmunoassay for human α2-PEG and detection in serum during pregnancy Hum. Reprod., 1987, 25 (5): 389-97); another method of immunization (subcutaneous administration) and hybridization partners are splenocytes and X63-Ag8.653 myeloma, Riitinen et al. (Riitinen L. , Narvanen O., Virtanen et al. Monoclonal antibodies against endometrial protein PP 14 and their use for purification and radioimmunoassay of PP 14. J. Immunol. Methods, 1991, 136: 85-90); the resulting MCAs were directed to carbohydrate structures glycodelin and reacted only with protein isolated from amniotic fluid, but not with sperm glycodeline (Eschke U., Kuhn C., Mylonas I. et al. Development and characterization of monoclonal antibodies for the immunohistochemical detection of glycodelin A in decidual, endometrial and gynecological tumor tissues. Histopathology, 2006, 48 (4): 394-406), therefore, there is no identity with analogues.

Domestic strains of hybridomas producing MCA against various isoforms of AMGF / glycodelin were obtained by us for the first time.

The strain of hybridoma cells 4f8 was adopted as the prototype (Boltovskaya M.N., Starosvetskaya N.A., Marshitskaya M.I., Nazimova S.V., Stepanov A.A., Shevchenko V.V. Production and practical use of monoclonal antibodies against alpha-2-microglobulin of fertility Bull. esperim biol. and honey. - 1997. - T.124, No. 9. - C.319-322), producing IgG1 class MCA, directed against one of the epitopes of the AMGF molecule, revealing this protein in enzyme-linked immunosorbent assay with a sensitivity of up to 5 ng / ml. Unlike the prototype obtained by hybridization of the spleen lymphocytes of Balb / c mice immunized with 4-fold intraperitoneal injection of 10 μg AMHF isolated from amniotic fluid with final intravenous immunization, the hybridoma strain A5D1 was obtained using another immunization method, the sensitivity of detection of AMHF in enzyme-linked immunosorbent assay (ELISA) using MCA A5D1 is 1 ng / ml

Strain A5D1 obtained in the following way.

Balb / c mice are immunized with 4-fold administration of 10-40 μg AMHF (sequentially in complete, incomplete Freund's adjuvant and in physiological saline) in the paw pads. 3 days after the final immunization, the popliteal lymph node lymphocytes hybridize with Sp2 / 0.Ag14 line syngenic myeloma cells in a ratio of 1:10 in 50% solution of polyethylene glycol-4000 (Merck) with 10% dimethyl sulfoxide in RPMI 1640 medium for 1 min. After hybridization, the cells are resuspended in a selective HAT medium - hypoxanthine, aminopterin, thymidine (Flow) based on RPMI 1640 with the addition of 20% fetal calf serum (Flow) and 4 mM glutamine and plated into 96-well flat-bottom microplate culture plates (Costar, Linbro). Three weeks after hybridization (completion of the metabolic selection stage), primary hybridomas and / or clones are transferred to NT medium that does not contain aminopterin, and NT is excluded after a week of cultivation. 7-14 days after hybridization in the wells, colony growth of hybrid cells is observed.

For selection of hybridomas producing MABs of a given specificity, a culture supernatant was selected from wells with growing colonies and tested using the solid-phase ELISA method in an embodiment that precludes the possibility of selecting MABs for a conformationally altered antigen. For this, ELISA microplates are sensitized with affinity-purified polyclonal rabbit antibodies against mouse IgG at a concentration of 2 μg / ml in carbonate-bicarbonate buffer (0.05 M, pH 9.5), non-specific binding is blocked with 1% solution of bovine serum albumin in pH-buffered saline 7.4. Then, 100 μl of test samples of the culture supernatant were added to the wells and incubated for 1 h at room temperature on a shaker. A conjugate of highly purified AMHF with horseradish peroxidase at a dilution of 1: 1000 obtained in the laboratory of cellular immunopathology and biotechnology of the Scientific Research Institute of MCH RAMS according to the Nakane method (incubation for 1 h at room temperature on a shaker) is used as a detector. After each incubation, microplates are washed 5 times from unbound components. The reaction was manifested with 4 mM tetramethylbenzidine di hydrochloride with 0.01% hydrogen peroxide in 100 mM phosphate citrate buffer pH 4.5 (20 min in the dark at room temperature) and stopped with monoform sulfuric acid. The optical density is recorded using a Multiskan Titertek (Flow, UK) scanning multichannel spectrophotometer at a wavelength of 450 nm.

Hybridomas in the supernatant of which anti-AMGP-MCA were detected are cloned by limiting dilutions in the liquid phase on the feeder layer of thymocytes 2-4 times until the frequency of positive clones reaches 100%. AMHF-positive clones are grown in 24-well culture plates, selecting the supernatant to assess the specificity of the produced MCAs. The specificity of MCA is determined by indirect ELISA using purified preparations of endometrial and placental proteins (placental α1-microglobulin, trophoblastic β1-globulin, chorionic gonadotropin), α-fetoprotein, bovine serum albumin, C-reactive protein adsorbed on a solid phase from solutions with 1 mcg / ml. Clones that react with AMHF and do not cross-react with control proteins are propagated in vitro and in vivo.

Strain A-5D1 is characterized by the following properties.

Cultural properties of the strain: hybrid cells grow in suspension in vitro or as an ascites tumor in the peritoneal cavity after administration of Balb / c syngeneic mice.

In vitro strain cultivation conditions: RPMI 1640 medium, 10% fetal calf serum, 2 mM glutamine, temperature 37 ° C, gas phase 5-7% CO ₂ , glass or plastic bottles. The cell concentration during sowing 2 × 10 ⁵ / ml, the frequency of passage - 2-3 times a week, the maximum density of -10 ⁶ / ml. The strain is inoculated on an antibiotic-free medium. Contamination by bacteria and fungi is not detected.

Cultivation in the animal organism: Balb / c mice pretreated with paraffin oil (0.5 ml intraperitoneally 10-30 days before the introduction of hybrid cells), the dose of the introduced cells 2-5 × 10 ⁶ / mouse, the formation of ascites 7-14 days, the volume of ascites fluid is 3-7 ml.

Monoclonal antibody titers as determined by enzyme-linked immunosorbent assay 1: 500-1: 1000 in culture supernatants, 1:10 ⁶ -1: 10 ⁷ in ascites fluid.

Recommended conditions for freezing. Cells are suspended in RPMI 1640 medium containing 20% fetal calf serum and 10% dimethyl sulfoxide, diluted to a concentration of 1-2 × 10 ⁶ cells / ml and transferred to plastic cryovials. Vials in a thick-walled foam container are placed overnight in a low-temperature refrigerator (-70 ° C), after which they are transferred to liquid nitrogen.

The product produced by strain A-5D1 is monoclonal antibody. A marker feature is the production of MCA against AMHF / human glycodeline.

The activity (productivity) of the strain: hybridoma culture supernatant contains 3-5 μg / ml of MCA, ascites fluid - 2-5 mg / ml of MCA. A method for determining the activity of a strain is spectrophotometric determination of the concentration of immunoglobulins in the culture medium and ascites fluid, determination of the titer of MKA in ELISA with antigen adsorbed on the solid phase.

MCAs produced by strain A-5D1 belong to the class of IgG1 immunoglobulins according to ELISA with typing monospecific antisera against mouse immunoglobulin classes.

The karyotype of the hybrid cells of the murine strain, the modal number of chromosomes was not determined.

The scope of strain A-5D1: biotechnology, reproductive medicine, immunohistochemistry.

Examples of the use of a strain of hybrid cells (hybridoma)

Example 1. The use of MCAs produced by strain A-5D1 for immunohistochemical (IHC) studies of the organs of the female reproductive system

Material for the IHC study of the endometrium was obtained by artificial termination of a physiological pregnancy (4-10 weeks). Scrapes from the uterine cavity were fixed in 10% neutral formalin in phosphate-buffered saline and processed according to the standard procedure for the preparation of histopathological preparations containing paraffin. The visualization of AMGF in tissues was performed by indirect immunoperoxidase staining using purified A-5D1 MCAs at an initial concentration of 4.0 mg / ml. The dewaxed sections were rehydrated and treated for 30 minutes with a 0.03% solution of H ₂ O ₂ in absolute methanol to block the activity of endogenous peroxidase. To reduce nonspecific antibody binding, sections were incubated with a 5% BSA solution in phosphate-buffered saline for 30 minutes at room temperature. MCA A-5D1 was layered on sections at 1: 500-1: 1000 dilutions and the glass was exposed in a humid chamber at 4 ° C for 18-20 hours. After washing three times with buffer, rabbit anti-mouse antibodies conjugated to horseradish peroxidase diluted 1: 1000 (30 minutes at room temperature) were applied to the sections. To manifest the reaction, 3.3-diaminobenzidine (Sigma, USA) was used as a chromogen. After the reaction, the sections were dehydrated by a standard method and enclosed in Canadian balsam. To exclude non-specific reactions, a negative control of reagents was performed: primary MCAs were excluded, replacing them with phosphate-saline buffer; as primary antibodies used MCAs of the same subclass (IgG1) to antigens that are obviously absent in the test tissue (C-reactive protein, alpha-fetoprotein); instead of primary MCAs, non-immune murine immunoglobulins were applied to sections.

On sections of endometrial tissue, MCA A-5D1 revealed AMHF in the cells and lumen of the uterine glands. The number of glands producing AMHF, and the intensity of their immunostaining increased in the gestational interval of 4-9 weeks. Thus, the obtained MCAs react with endometrial AMHF after tissue fixation with formalin, paraffin embedding and routine histological processing. The use of MCA A-5D1 does not require special methods of tissue fixation and processing, which makes them widely applicable for immunohistochemical studies of clinical material.

Example 2. The use of MCAs produced by strain A-5D1 for IHC studies of the organs of the male reproductive system

The tissue of the seminal vesicles for immunohistochemical studies was obtained by autopsy performed no later than 3 hours after the sudden death of a man at the age of 21 years. Processing of the material and immunohistochemical reaction was carried out as described in Example 1. AMHF was detected in the glands of the seminal vesicles in the form of intense fine-grained staining of epithelial cell cytoplasm on sections of seminal vesicles of MCA A-5D1. Thus, A-5D1 MCAs obtained by immunization with AMHF isolated from amniotic fluid (AMHF-A) also react with AMHF produced in the male reproductive organ, i.e. with sperm AMGF (AMGF-S). This indicates that A-5D1 MCAs react with the protein and not the carbohydrate part of the AMGF molecule and can be used for immunodetection of AMGF-C in the cells and tissues of the organs of the male reproductive system, in contrast to the MCA for glycodeline obtained by Eschke et al . (2006).

Example 3. The use of MCA produced by strain A-5D1, to create an enzyme-linked immunosorbent assay system for the quantitative determination of various glycoforms AMGF / glycodeline in biological fluids.

For large-scale production of MCA, the A-5D1 hybrid cell strain was cultured in Balb / c syngeneic mice and ascites fluid was obtained. MCA A-5D1 was isolated from ascitic fluid by salting out with 50% ammonium sulfate, followed by purification by ion exchange chromatography on columns with DEAE cellulose. MCA A-5D1 (trap) was adsorbed on the bottom of a flat-bottomed 96-well polystyrene microplate for enzyme-linked immunosorbent assay (ELISA) from a solution of 10 μg / ml in carbonate-bicarbonate buffer (pH 9.5), non-specific binding was blocked with a 1% solution of bovine serum albumin in phosphate buffered saline (pH 7.4). To select an MAB directed against another epitope of the AMGF molecule, a panel of AMGF-specific MAB was conjugated to horseradish root peroxidase and used as detecting MAB when introduced into the test system with various dilutions of the antigen standard in (1-100 ng / ml). The use of MCA A-5D1 for immobilization on a solid phase in the sandwich ELISA instead of the prototype MCA 4f8 increased the sensitivity of the test system to 1 ng / ml. When samples of blood serum, amniotic and follicular fluid of women, seminal fluid of men MCA A-5D1 were introduced into the test system, various glycoforms of AMHP produced in the organs of the male and female reproductive systems were connected, providing the possibility of their quantification in biological fluids with high sensitivity and specificity .

Claims (1)

The strain of hybrid cultured animal cells Mus musculus L., deposited in the collection of transplantable mammalian cell cultures of the Research Institute of Human Morphology RAMS under the number 144/2002, is a producer of monoclonal antibodies against different isoforms of alpha2-microglobulin fertility (AMGF) / glycodelin.