RU2271010C2 - Method for immunoenzyme analysis for assay of von willebrand factor, monoclonal antibody to von willebrand factor (variants) and strain of hybrid cultured mammal cells mus musculus l - producer of monoclonal antibodies to von willebrand factor (variants) - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: invention relates to immunoenzyme analysis and can be used for assay of von Willebrand factor. Method involves immunoenzyme analysis wherein monoclonal antibody 5C3 is used as an immobilizing antibody, and a mixture of biotin-labeled monoclonal antibodies 2H2 and 7D12 is used as a detecting antibody. Also, invention relates to monoclonal antibodies produced by the strain of hybridoma cultured cells Mus musculus L. and directed against von Willebrand factor, and to strains of hybrid cultured cells Mus musculus L. producing indicated monoclonal antibodies. Invention provides the development of highly sensitive method for assay of von Willebrand factor.

EFFECT: improved method for analysis.

9 cl, 1 tbl, 2 dwg, 3 ex

Description

The invention relates to the field of biotechnology, and in particular to methods of enzyme-linked immunosorbent assay (ELISA) and monoclonal antibodies (monoAT) for their implementation, in particular to systems for analysis of von Willebrand factor (FI), and technology for their preparation.

Willebrand factor (PV) is a multimeric glycoprotein (GP) of blood plasma (0.5-20 × 10 ⁶ D), which plays a significant role in the processes of stopping bleeding (hemostasis), namely in the reactions of vascular-platelet and plasma-coagulation units of hemostasis. [Barkagan Z.S. Hemorrhagic diseases and syndromes., M., 1988. S. 216-234; Papayan L.P. Modern problems of clinical coagulology., L., 1985., p.21-27; Zimmerman TS, Ruggeri ZM Hum. Path., 1987, 18: 140-152]. This protein is synthesized in the vascular endothelium and bone marrow megakaryocytes. The synthesized protein is contained in the intracellular granules of endothelial cells and platelets (formed from megakaryocytes) and is secreted from them into the blood plasma.

One of the main functions of PV is to ensure the adhesion (attachment) of platelets to damaged areas of the vascular wall. PV plays the role of a molecular glue, interacting simultaneously with the subendothelial structures of the vessel and its main receptor on the surface of platelets - GP Ib. On the surface of activated platelets, PV can also bind to another receptor, GP IIb-IIIa. Due to this interaction, he, along with fibrinogen, is involved in the formation of molecular bonds between activated platelets, i.e. in their aggregation.

The participation of PV in the coagulation unit of hemostasis is ensured by its ability to form a complex in plasma with coagulation factor VIII, in which PV plays the role of a carrier protein and provides smaller factor VIII stability during circulation in the bloodstream, increasing its lifetime in the vascular bed and facilitating its transportation in places of damage to blood vessels.

Hereditary disorders, expressed in a decrease in the number or change in the functional properties of PV, are called von Willebrand disease. This disease is one of the most common pathologies of the hemostatic system. [Cm. Reviews: Barkagan Z.S. Hemorrhagic diseases and syndromes., M., 1988, p.216-234; Shiffman F.D. Pathophysiology of blood, "Publishing House Binom" - "Nevsky dialect", M.-SPb., 2000, p.233-8; Sandler J.E., Davie E.W. Von Willebrand factor and von Willebrand disease. In "The Molecular basis of Blood Disease", eds. Stamatoyannopoulos S., Majerus P.W., Perlmutter R.M., Varmus H. W.B. Saunders Company., Philadelphia, London, New York, St. Louis, Sydney, Toronto., 2001, p. 697-718; Ruggeri Z.M. Thromb. Haemost, 1999, 82: 576-84]. The main manifestations of a decrease in the level and / or functional activity of PV in patients with von Willebrand disease are pronounced violations of indicators characterizing the viability of platelet-vascular hemostasis (increased bleeding time, decreased platelet adhesion and their aggregation in the presence of the ristocytin antibiotic), as well as profuse bleeding from mucous membranes for a defect in the formation of a primary platelet plug. Observations of patients with von Willebrand disease also showed that they may have reduced factor VIII activity due to quantitative and / or qualitative defects in the carrier protein - PV. Clinically, this is manifested by bleeding that resembles those in hemophilia A patients who have a true defect in the synthesis of factor VIII.

It is also known that elevated PV levels can be a risk factor for thrombotic exacerbations of coronary heart disease, such as myocardial infarction and unstable angina [Thompson S.G., et al. N. Engl. J. Med., 1995, 332: 635-41; Voskoboy I.V. and other Cardiology, 2002, 42: 4-11]. An increase in plasma PV levels is one of the markers of activation and / or damage to endothelial cells and platelets, which is regarded as one of the significant factors provoking the development of thrombotic complications in cardiovascular diseases.

Based on the foregoing, determining the level of plasma PV is important for the diagnosis of von Willebrand disease and the differentiation of some forms of this disease with hemophilia A, as well as for the prognosis of thrombosis. In this regard, it seems relevant to create systems for the quantitative determination of PV in plasma.

Currently, a wide range of methods are used to determine the PV. Thus, the plasma PV content can be determined by measuring its so-called ristocytin-cofactor activity, i.e. the ability of this protein to cause platelet aggregation due to interaction with GP Ib in the presence of the antibiotic ristocytin. In various embodiments of this method, native or fixed platelets and various methods of measuring aggregation are used. In addition, various immunochemical methods are used to determine the PV, such as Laurell rocket electrophoresis and variants of radioimmunoassay and enzyme immunoassay using polyclonal and monoclonal antibodies (monoAT) [Schiffman FD Pathophysiology of blood, “Binom Publishing House” - “Nevsky Dialect”, M.-SPb., 2000, p.233-8: Sandier J.E., Davie E.W. Von Willebrand factor and von Willebrand disease in "The Molecular basis of Blood Disease", Eds. Stamatoyannopoulos S., Majerus P.W., Perimutter R.M., Varmus H. W.B. Saunders Company., Philadelphia, London, New York, St. Louis, Sydney, Toronto., 2001, p.697-718].

The determination of ristocytin cofactor activity is a laborious method, requiring the receipt of donor platelets, the use of special equipment to measure their aggregation and not allowing simultaneous testing of a large number of samples. In addition, this method is significantly lower in sensitivity of most immunochemical methods, and the results obtained with its help make it possible to judge more about the activity of PV (the ability to bind to Ib GP), rather than its concentration. Therefore, with variants of von Willebrand disease with impaired function of this protein, this method can only be used in combination with immunochemical methods.

In recent years, a number of methods for studying PV have been developed, based on the interaction of a soluble antigen with specific antibodies and precipitation of the resulting complex. In particular, immunoelectrophoretic and immunodiffusion methods were proposed, the disadvantages of which are the duration of the study (up to several days), a difficult assessment of the results, and most importantly low sensitivity (the detection limit of PV is 5-10% of normal values in blood plasma). Immunoradiometric and enzyme immunoassay methods are devoid of these drawbacks and make it possible to detect an insignificant amount of PV protein - less than 1% of its amount in the plasma of healthy donors [Casonato A., Girolami A. Folia haemat (Lps.), 1986, 113: 670-684; Sandier J.E., Davie E.W. Von Willebrand factor and von Willebrand disease in "The Molecular basis of Blood Disease", Eds. Stamatoyannopoulos S., Majerus P.W., Perimutter R.M., Varmus H. W.B. Saunders Company., Philadelphia, London, New York, St. Louis, Sydney, Toronto., 2001, p.697-718]. However, the most effective and convenient is enzyme-linked immunosorbent assay (ELISA), because it is less labor intensive and, unlike radioimmune methods, does not require the use of radioactively labeled reagents.

Closest to the claimed method according to the technical nature and the achieved effect is a method for determining PV by ELISA using monoAT from a hybridoma strain 380 F2. [Toropova V.G. et al. Lab. business, 1990, 12: 52-55]. The proposed ELISA involves fixation in the wells of a plastic tablet of polyclonal rabbit anti-PV antibodies to PV produced by Dakopetts (Denmark), washing them with phosphate-buffered saline, sequentially introducing into the wells of the studied plasma in various dilutions, mouse monoAT to PV (in the form of culture fluid strain 380 F2) and peroxidase conjugate (anti-mouse IgG labeled with horseradish peroxidase), incubating the plate and determining the activity of peroxidase, which is part of the conjugate, using a chromogenic substrate at a wavelength of 490 nm. Non-specific binding is monitored with normal murine immunoglobulins. The disadvantages of the method is the use of polyclonal antibodies, because the properties of polyclonal antibodies from batch to batch may vary. In addition, when using monoAT, it is possible not only to increase the reproducibility of the method, but also to avoid the time-consuming and expensive affinity purification of specific polyclonal antibodies from serum.

Along with those mentioned in the previous work, other monoAT to PV are known. In particular, the monoATs MA-82D1E1 and MA-82D6A3 are known that are capable of selectively reacting with PV [Tornai I. et al. Haemostasis, 1991, 21: 125-134]. MonoATs were obtained by traditional methods and purified from the ascites fluid of BALB / c mice after culturing producer cells in the abdominal cavity using affinity chromatography on protein A-Sepharose. It was shown that during ELISA they turned out to be more promising than polyclonal antibodies, but their characteristics are not described in detail in the work. Also close antibodies and strains to the claimed are monoAT MAb 53 and MAb D7 [Bradley L. et al., Clin. Chem., 1984, 30: p. 87-92), which were obtained by a method including immunization with FVIIIR: Ag (factor VIII related antigen - previously used name of the PV antigen) of BALB / c mice, hybridization of spleen cells of immune mice, selection and cloning of hybridoma cells producing antibodies MAb D7 and MAb 53. There is no detailed description of producer strains and purified monoAT to PV.

The problem solved in the framework of this patent was the creation of a more effective method for determining the PV by the ELISA method. It was suggested that the sensitivity of the method can be significantly increased when three monoATs are used to determine the PV, one for immobilization and two biotin-labeled for detection of the immobilized antigen. To do this, all three antibodies used must not compete with each other for binding to the defined antigen - PV.

The method proposed in this case consists in the use of monoAT 5C3 as an immobilizing PV antibody, and a mixture of biotin-labeled monoAT 2H2 and 7D12 as a detecting reagent.

The use of a mixture of these monoATs (2H2 and 7D12) for the detection of immobilized PV makes it possible to increase the sensitivity of the method. However, it is fundamentally possible in some cases that do not require high detection sensitivity to use each of these antibodies separately as a detecting reagent.

The 5C3, 2H2, and 7D12 monoAT used in the method were obtained on the basis of mouse hybridoma cell strains 5C3 (No. RCCC (P) 684D), 2H2 (No. RACC (P) 683 D) and 7D12 (No. RCCC (P) 685 D), respectively .

The main properties of monoAT 5СЗ are:

- belongs to the IgGI class;

- binds to PV;

- does not compete for binding to PV with monoAT 2H2 and 7D12.

The main properties of monoAT 2H2 are:

- belongs to the class IgG2b;

- binds to PV,

- does not compete for binding to PV with monoAT 5C3 and 7D12.

The main properties of monoAT 7D12 are:

- belongs to the IgGI class:

- binds to PV:

- does not compete for binding to PV with monoAT 2H2 and 53Z.

The ability of the above monoATs to bind to PV and the lack of competition between them for binding to this antigen (see Example 1) allowed the use of all three antibodies to create an ELISA system for determining PV. Antibody 5C3 was used as immobilizing, and biotin-labeled antibodies 2H2 and 7D12 as detecting antibodies. The use of a mixture of two antibodies (2H2 and 7D12) for the detection of immobilized PV increased the sensitivity of ELISA (see example 2). The proposed method allows to determine the PV in the plasma of patients with von Willebrand disease in order to diagnose this pathology (see example 3).

To obtain the above monoAT used strains of hybrid cultured animal cells Mus. Musculus 5C3, 2H2 and 7D12, respectively.

All strains are the products of the fusion of mouse X-63.Ag8.653 mouse myeloma cells (subclone P ₃ O ₁ and mouse spleen cells immunized with purified PV. Mice were immunized according to the following scheme: 25 μg PV intraperitoneally in Freund's complete adjuvant: after 2 weeks - 25 μg of intraperitoneal PV in Freund’s adjuvant, 2 μg of intraperitoneal PV in 2 weeks (3 days before hybridization), 5 μg of intravenous PV merging and cloning was carried out according to the method of Köhler G. and Milstein C. Nature, 1975, 256: 495-7]. The fusion was carried out using polyethylene glycol P EG-1500, and the selection of hybrid cells using a selective GAT medium (hypoxanthine - aminopterin - thymidine).

Strains are characterized by the following properties.

Strain of hybrid cultured animal cells Mus. Musculus 5C3.

Line 5C3 is the product of the fusion of X.63.Ag8.653 myeloma cells (subclone P ₃ O ₁ ) and BALB / c mouse spleen cells immunized with PV. The merger was carried out using PEG-1500 polyethylene glycol. Selection of hybrid cells was carried out using a selective medium GAT (hypoxanthine - aminopterin - thymidine). The strain passed 3 cloning, positive clones of at least 100%. Currently, after the last cloning, the strain has passed 3 passages.

STANDARD CULTIVATION CONDITIONS: RPMI-1640 medium with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 u / ml penicillin and 100 μg / ml streptomycin.

CULTURAL PROPERTIES: Culture bottles can be used to grow the strain. 1 x 10 ⁶ cells are seeded in a 25 cm ² vial in 5 ml of medium. Passage is performed when the culture density reaches 1 × 10 ⁶ cells per 1 ml of medium. BALB / c mice are suitable for growing cells in the abdomen of mice. 10 days before the injection of the strain, 0.5 ml of pristan was intraperitoneally administered to the mice. The strain is injected intraperitoneally at 1-5 × 10 ⁶ cells per mouse. Ascitic fluid is collected after 10-12 days with a maximum volume of ascites.

KARIOLOGICAL CHARACTERISTIC OF THE STRAIN: Modal number of chromosomes 66. No marker chromosomes were detected.

CONTAMINATIONS: Bacteria and fungi in the culture were not detected during prolonged observation. Mycoplasma test is negative.

USEFUL PRODUCT BIOSYNTHESIS: In vitro secretion of 5C3 monoAT is 5-10 μg / ml, in ascites fluid 2-5 mg / ml when determined by binding to PV immobilized on plastic (see below - selection of monoAT).

Cryopreservation: The cells of the strain are resuspended in fetal bovine serum containing 10% dimethyl sulfoxide at a concentration of 3 × 10 ⁶ cells in 1 ml, poured into plastic ampoules at 4 ° C, placed in a low-temperature refrigerator at -70 ° C for 24 hours and then transferred to a liquid nitrogen. Cells are quickly thawed at 37 ° C, diluted in 10 ml of serum-free medium, pelleted by centrifugation, resuspended in 5 ml of the same medium containing 20% serum, and transferred to a culture bottle. Cell viability, determined by the inclusion of trypan blue, is more than 80%.

The strain is deposited in the RKKK collection - No. RKKK (P) 684D.

Strain of hybrid cultured animal cells Mus. Musculus 2H2.

Line 2H2 is the product of the fusion of P X.63.Ag8.653 cells (subclone P ₃ O ₁ and BALB / c mouse spleen cells immunized with PV. Fusion was performed using PEG-1500 polyethylene glycol. Selection of hybrid cells was performed using GAT selective medium ( hypoxanthine - aminopterin - thymidine). The strain passed 3 cloning, positive clones at least 100%. Currently, after the last cloning, the strain passed 3 passage.

STANDARD CULTIVATION CONDITIONS: RPMI-1640 medium with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 u / ml penicillin and 100 μg / ml streptomycin.

CULTURAL PROPERTIES: Culture bottles can be used to grow the strain. 1 x 10 ⁶ cells are seeded in a 25 cm ² vial in 5 ml of medium. Passage is performed when the culture density reaches 1 × 10 ⁶ cells per 1 ml of medium. BALB / c mice are suitable for growing cells in the abdomen of mice. 10 days before the injection of the strain, 0.5 ml of pristan was intraperitoneally administered to the mice. The strain is injected intraperitoneally at 1-5 × 10 ⁶ cells per mouse. Ascitic fluid is collected after 10-12 days with a maximum volume of ascites.

KARIOLOGICAL CHARACTERISTIC OF THE STRAIN: Modal number of chromosomes 66. No marker chromosomes were detected.

CONTAMINATIONS: Bacteria and fungi in the culture were not detected during prolonged observation. Mycoplasma test is negative.

BIOSYNTHESIS OF A USEFUL PRODUCT: In vitro secretion of monoAT 2H2 is 5-10 μg / ml, in ascites fluid 2-5 mg / ml as determined by binding to PV immobilized on plastic (see below - selection of monoAT).

Cryopreservation: The cells of the strain are resuspended in fetal bovine serum containing 10% dimethyl sulfoxide at a concentration of 3 × 10 ⁶ cells in 1 ml, poured into plastic ampoules at 4 ° C, placed in a low-temperature refrigerator at -70 ° C for 24 hours and then transferred to a liquid nitrogen. Cells are quickly thawed at 37 ° C, diluted in 10 ml of serum-free medium, pelleted by centrifugation, resuspended in 5 ml of the same medium containing 20% serum, and transferred to a culture bottle. Cell viability, determined by the inclusion of trypan blue, is more than 80%.

The strain is deposited in the RKKK collection - No. RKKK (P) 683 D.

Strain of hybrid cultured animal cells Mus. Musculus 7D12.

Line 7D12 is the product of the fusion of X-63.Ag8.653 myeloma cells (subclone P ₃ O ₁ and BALB / c mouse spleen cells immunized with PV. Fusion was performed using PEG-1500 polyethylene glycol. Hybrid cells were selected using GAT selective medium ( hypoxanthine - aminopterin - thymidine). The strain passed 3 cloning, positive clones at least 100%. Currently, after the last cloning, the strain passed 3 passage.

STANDARD CULTIVATION CONDITIONS: RPMI-1640 medium with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 u / ml penicillin and 100 μg / ml streptomycin.

CULTURAL PROPERTIES: Culture bottles can be used to grow the strain. 1 x 10 ⁶ cells are seeded in a 25 cm ² vial in 5 ml of medium. Passage is performed when the culture density reaches 1 × 10 ⁶ cells per 1 ml of medium. BALB / c mice are suitable for growing cells in the abdomen of mice. 10 days before the injection of the strain, 0.5 ml of pristan was intraperitoneally administered to the mice. The strain is injected intraperitoneally at 1-5 × 10 ⁶ cells per mouse. Ascitic fluid is collected after 10-12 days with a maximum volume of ascites.

KARIOLOGICAL CHARACTERISTIC OF THE STRAIN: Modal number of chromosomes 66. No marker chromosomes were detected.

CONTAMINATIONS: Bacteria and fungi in the culture were not detected during prolonged observation. Mycoplasma test is negative.

USEFUL PRODUCT BIOSYNTHESIS: In vitro monoAT 7D12 secretion is 5-10 μg / ml, in ascites fluid 3-6 mg / ml as determined by binding to PV immobilized on plastic (see below - selection of monoAT).

Cryopreservation: The cells of the strain are resuspended in fetal bovine serum containing 10% dimethyl sulfoxide at a concentration of 3 × 10 ⁶ cells in 1 ml, poured into plastic ampoules at 4 ° C, placed in a low-temperature refrigerator at -70 ° C for 24 hours and then transferred to a liquid nitrogen. Cells are quickly thawed at 37 ° C, diluted in 10 ml of serum-free medium, pelleted by centrifugation, resuspended in 5 ml of the same medium containing 20% serum, and transferred to a culture bottle. Cell viability, determined by the inclusion of trypan blue, is more than 80%.

The strain is deposited in the RKKK collection - No. RKKK (P) 685D.

Specific monoAT binding to PV was selected using ELISA. For this, the bottom of 96-well polystyrene plates was coated with purified PV — 5 μg / ml PV in phosphate-buffered saline (PBS), pH 7.4, 100 μl per well, 1 hour at 37 ° C. The non-plastic binding protein was washed with FSB containing 0.05% Tween 20 (FSB / Tween), and nonspecific binding sites were blocked with 1% bovine serum albumin (BSA) in FSB / Tween - 150 μl per well, 1 hour at 37 ° C . Then, 50-100 μl of culture medium containing hybrid cell secretion products (monoAT) was added to the wells, incubated for 30 min at room temperature, washed with FSB / Tween wells, and peroxidase-labeled goat antibodies against mouse IgG were introduced into them (BioRad, United States) ( 100 μl per well in 1% BSA / FSB / Tween (diluted by the manufacturer) and incubated for 30 min at room temperature. The wells were washed with PBS / Tween and antibody binding was recorded using a chromogenic substrate (100 μg / ml orthophenylenediamine, 0.006% hydrogen peroxide, in citrate buffer, pH 4.5, 100 μl per well). The reaction was stopped by adding 50 μl of 50% sulfuric acid and the absorbance in the wells was recorded at a wavelength of 492 nm (A492).

To obtain preparative amounts of 5C3, 2H2 and 7D12 antibodies, cells of the corresponding producer strains were cultured in the abdominal cavity of BALB / c mice. 10 days before the injection of cells, mice are injected intraperitoneally with 0.5 ml of pristan. Cells are injected intraperitoneally at 1-5 × 10 ⁶ per mouse. Ascitic fluid is collected after 10-12 days with a maximum volume of ascites. Isolation of monoAT from ascites is carried out by precipitation with ammonium sulfate and subsequent purification using ion-exchange chromatography on ToyoPearl DEAE 650-M.

These strains and the monoATs produced by them are part of the claimed method, combined with it by a single inventive concept and are aimed at solving a single problem - the creation of ELISA for determining PV. This allows us to consider these inventions as components of a single group of inventions.

The essence of this group of inventions is illustrated by the following examples.

Example 1. Obtaining purified monoAT 5C3, 2H2 and 7D12, their binding to PV and the absence of competition between them for binding to antigen.

Obtaining preparative quantities of antibodies. Cells of strains 5C3, 2H2 and 7D12 were injected intraperitoneally to BALB / c mice in an amount of 5 × 10 ⁶ per 1 mouse. 10 days before the introduction of cells, mice were injected intraperitoneally with 0.5 ml of pristan. Ascitic fluid was taken 10-12 days after the introduction of cells with a maximum volume of ascites. Purification of antibodies from ascites fluid was carried out using salt deposition with ammonium sulfate and subsequent chromatography on ToyoPearl-DEAE 650-M (Tosoh Biosep, USA). One volume of a saturated solution of ammonium sulfate was added dropwise to one volume of ascites liquid and stirred for 1 hour at room temperature. The precipitate was precipitated at 10,000 g for 20 min, dissolved in 10 mM phosphate buffer, pH 8.1, and dialyzed against a 10-fold volume of the same buffer (3 shifts of 5 hours at 4 ° C). After dialysis and precipitation of insoluble material (10,000 g, 15 min, at 4 ° C), the antibody solution was applied to a DEAE-Toyo Soda-650 M column (not more than 10 mg per 1 ml of sorbent), the column was washed with the application buffer (2 column volumes ) and the antibodies were eluted with a linear gradient of a phosphate buffer of 10-150 mM, pH 8.1 (total volume of the eluent was 10 column volumes). Protein in fractions was determined by absorption at a wavelength of 280 nm. Fractions containing protein were tested for the presence of antibodies using ELISA using immobilized PV plastic (see above, selection of monoAT). Antibody fractions were pooled, dialyzed against a 10-fold volume of phosphate-buffered saline (PBS) containing 0.15 M NaCl, 10 mM sodium phosphate, pH 7.4 (3 shifts of 5 hours at 4 ° C). After dialysis, insoluble material was precipitated (10,000 g, 15 min at 4 ° C) and the protein content in the antibody solution was determined. Antibodies in aliquots of 1-2 ml were stored at -70 ° C.

Labeling of antibodies with biotin. Purified monoAT 2H2 and 7D12 were labeled with biotin using N-succinimidibiotin (Sigma, USA). Antibodies (2-3 mg in 1-2 ml of PBS) were dialyzed against a 100-fold volume of 100 mM carbonate buffer, pH 8.0 (2 shifts of 5 hours at 4 ° C). After dialysis, N-succinimidbiotin (freshly prepared solution in DMSO) was added to the antibody solution in the molar ratio of antibody: N-succinimidbiotin - 1:40 (the volume of the added N-succinimidbotin solution is not more than 1/20 of the volume of the antibody solution) and incubated for 40 min at 4 ° C and constant stirring). After incubation, the antibodies were dialyzed against a 100-fold volume of FSB, pH 7.4 (2 shifts of 5 hours at 4 ° C). Sodium azide (final concentration 0.05%) was added to biotin-labeled antibodies and stored at 4 ° C for 2-3 months.

Study of competition between antibodies 5C3, 2H2 and 7D12 for binding to PV. Unlabeled antibodies 5C3 and 2H2 at a concentration of 5 μg / ml in FSB, pH 7.4 were added to the wells of 96-well polystyrene plates (100 μl per well) and incubated for 1 hour at 37 ° C. Unsorbed antibodies were washed with FSB / Tween and blocked non-specific binding sites with 1% BSA / FSB / Tween (150 μl per well, 1 hour at 37 ° C). After that, healthy donor plasma pool was added to the wells (obtaining a pool - see below, Example 3) in various dilutions (Fig. 1A) or purified PV in various concentrations (Fig. 1B) in 1% BSA / FSB / Tween (100 μl per well), incubated for 30 min at room temperature and washed FSB / Tween unbound with antibodies. Then, biotin-labeled 2H2 antibody was added to wells with 5C3 sorbed antibody, and biotin-labeled 7D12 antibody was added to wells with 5C3 and 2H2 sorbed antibodies (biotin-labeled antibodies were added at a concentration of 10 μg / ml in 1% BSA in FSB / Tween, 100 μl per well), incubated for 30 min at room temperature, and unbound unbound labeled FSB / Tween antibodies were washed. After that, 100 μl of streptavidin peroxidase (IMTEK, Moscow) was added to the well, in 1% BSA / FSB / Tween diluted by the manufacturer (100 μl per well) were incubated for 30 min at room temperature and the wells were washed with FSB-Tween. Antibody binding was recorded using a chromogenic substrate as described above (see monoAT screening section). The results are shown in FIG. 1, where FIG. 1A shows the results obtained by titration of a plasma, and FIG. 1B - by titration of purified PV (in both FIG. 1–5C3 curves sorbed + 2H2-biotin, curves 2 - 5С3 adsorbed + 7D12-biotin, curves 3 - 2Н2 adsorbed + 7D12-biotin). As can be seen from the graphs, biotin-labeled antibody 7D12 effectively binds to PV, immobilized using both 5C3 and 2H2 antibodies, and biotin-labeled antibody 2H2, PV, immobilized using 7D12. Similar results were obtained using blood plasma as a source of PV and purified PV. These results indicate that all three antibodies do not compete with each other for binding to PV, i.e. directed against different epitopes in the antigen molecule.

Example 2. An enzyme-linked immunosorbent assay was carried out according to the procedure of Example 1, using 5C3 antibody for immobilization of PV, and for detection: (1) a mixture of two biotin-labeled antibodies, 2H2 (5 μg / ml) and 7D12 (5 μg / ml), (2) Biotin-labeled monoAT 2H2 (10 μg / ml), (3) Biotin-labeled monoAT 7D12 (10 μg / ml). A pool of healthy donor plasmas was used as the source of PV (preparation — see below, example 3). Figure 2 presents the results of determining the PV when using a mixture of two monoATs for detection (curve 1 -2H2-biotin (5 μg / ml) + 7D12-biotin (5 μg / ml)) or each antibody separately (curve 2 - 2H2- biotin (10 μg / ml) and curve 3 - 7D12-biotin (10 μg / ml)). As can be seen from this drawing, the determination of PV using two labeled antibodies increases the sensitivity of the method by approximately 2 times compared with the determination that was carried out using only one labeled antibody, despite the fact that the total concentration of antibodies was the same in all cases.

Thus, from the data presented it follows the possibility of using for the determination of PV both a mixture of biotin-labeled two monoAT, 2H2 and 7D12, and each of these antibodies separately (which in some cases is sufficient and at the same time economically more beneficial).

Example 3. Determination of PV in the plasma of healthy donors and patients with von Willebrand disease.

Plasma production from healthy donors and patients with von Willebrand disease. Blood from healthy donors and patients with von Willebrand disease (all patients were observed at the Hematology Research Center of the Russian Academy of Medical Sciences) was collected using 5% EDTA (pH 7.4) as an anticoagulant in a blood: anticoagulant ratio of 9: 1. To obtain plasma, the blood was centrifuged at 1500 g, 20 min at room temperature. Plasma was taken, which was re-centrifuged in a microcentrifuge at 10,000 g for 5 min at 4 ° C to completely remove platelets. Plasma was divided into 0.1-0.2 ml aliquots and frozen at -70 ° C. Immediately before determining the PV, the plasma was quickly thawed at 37 ° C in a water thermostat. Plasma obtained from 12 healthy donors was pooled and then aliquoted and frozen.

Determination of PV using ELISA. ELISA was carried out according to the method of example 1, using monoAT 5C3 for immobilization of antigen, and for detection a mixture of two antibodies labeled with biotin - 2H2 and 7D12. To construct the calibration curve used purified PV. The PV content was determined in a pool of healthy donor plasma, which was taken as 100%, and in the plasma of 4 patients with von Willebrand disease.

Measurement of the ristocytin-cofactor activity of the studied plasmas. Platelets of a healthy donor washed from plasma were obtained according to the procedure ([Vinogradov D.V. et al. Biochemistry, 1991, 56, p. 787-97) and suspended in a concentration of 5 × 10 ⁸ ml. 150 μl of washed platelets and 150 μl of plasma in various dilutions and then 1.5 mg / ml (final concentration) of ristocytin (Renam, Moscow) were added to an aggregometer cuvette (Biola, Moscow). Aggregation was recorded by the change in light transmission of the platelet suspension for 3 minutes at 37 ° C and with stirring at a speed of 800 rpm. Ristocytin-cofactor activity in each sample was evaluated by the maximum level of light transmission during the registration of aggregation. To construct the calibration curve, a pool of healthy donor plasmas in various dilutions was used, in which the ristocytin-cofactor activity was taken as 100%. Thus, the determination of ristocytin-cofactor activity in the plasma of 4 patients with von Willebrand disease, who also determined the content of PV by ELISA, was carried out. Comparison of the results of measuring PV using ELISA and ristocytin-cofactor activity in patients with von Willebrand disease is given in the table.

Results of EF measurement using ELISA and measurement of ristocytin-cofactor activity in patients with von Willebrand disease. Plasma sample FV (IFA) Ristocytin cofactor activity,% mcg / ml % Donor Plasma Pool 14.0 one hundred one hundred Patient M. (BV * 1 type) 7.7 55 fifty Patient F. (type 3 BV) 1,0 7 eighteen Patient L. (BV type 3) 2.1 fifteen 12 Patient O. (BV type 3) 0.14 one 13 * BV - von Willebrand disease.

From the above data it is seen that both the content of PV measured by ELISA and ristocytin-cofactor activity were reduced in all 4 patients with von Willebrand disease. In 2 patients with type 1 von Willebrand disease, the content of PV and ristocytin-cofactor activity were reduced by approximately 50% compared to a pool of healthy donor plasmas, and in 3 patients with type 3 von Willebrand disease these values were lower than 20%. It should be noted that in two patients (F. and O.), the concentration of PV was reduced to a greater extent than the ristocytin-coactor activity, which is possibly due to the appearance of large multimers of this protein in the bloodstream, as a result of which the decrease in the amount of PV can be combined with an increase in its functional activity [Shiffman F.D. Pathophysiology of blood, "Publishing House Binom" - "Nevsky dialect", M.-SPb., 2000, S. 233-238].

The obtained results indicate the possibility of using the developed ELISA for the diagnosis of von Willebrand disease and the feasibility of combining this method with the determination of ristocytin-cofactor activity.

Claims (9)

1. Enzyme-linked immunosorbent assay for determining von Willebrand factor, including sorption of antibodies to von Willebrand factor in the wells of a tablet, washing of non-absorbed antibodies with buffer solution, introducing into the wells an assayed sample containing von Willebrand factor, immobilizing von Willebrand factor, introducing an antibody-based detection reagent into samples, processing samples with a chromogenic substrate and measuring the results using spectroscopy, characterized in that a 5C3 monoclonal antibody is used as an immobilizing antibody, and as detection reagent mixture of biotin-labeled 2H2 and 7D12 monoclonal antibody. 2. The method according to claim 1, characterized in that the biotin-labeled antibody 2H2 is used as the detecting reagent. 3. The method according to claim 1, characterized in that the biotin-labeled antibody 7D12 is used as the detecting reagent. 4. Monoclonal antibody 5C3, characterized in that it is produced by a strain of cultured musibus L. 5C3 gibiridoma cultured cells, belongs to the IgG1 class, is directed against von Willebrand factor and does not compete for binding to von Willebrand factor with 2H2 and 7D12 antibodies. 5. Monoclonal antibody 2H2, characterized in that it is produced by a strain of cultured musibus L. 2H2 gibirid cultured cells, belongs to the IgG2b class, is directed against von Willebrand factor and does not compete for binding to von Willebrand factor with antibodies 5C3 and 7D12. 6. The monoclonal antibody 7D12, characterized in that it is produced by the strain of cultured musibus L. gibiridoma cells, belongs to the class IgG1, is directed against von Willebrand factor and does not compete for binding to von Willebrand factor with 5C3 and 2H2 antibodies. 7. The strain of hybrid cultured cells of Mus musculus L. 5C3, deposited in RKKK (No. RKKK (P) 684D), used as a producer of monoclonal antibodies 5C3 against von Willebrand factor. 8. The strain of hybrid cultured cells of Mus musculus L. 2H2, deposited in RKKK (No. RKKK (P) 683D), used as a producer of monoclonal antibodies 2H2 against von Willebrand factor. 9. The strain of hybrid cultured cells of Mus musculus L. 7D12, deposited in RKKK (No. RKKK (P) 685D), used as a producer of monoclonal antibodies 5C3 against von Willebrand factor.

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