Immunological analysis of human medullation and diagnosis of multiple sclerosis with it

 

The invention relates to medicine, in particular to immunology. The method provides high accuracy of immunological measurement of the content of human medullation in the blood and the diagnosis of multiple sclerosis. Carry out steps (a) and (b), the step (a) is a step in the destruction of leukocytes in the blood sample of aqueous liquid (i) and (ii) or a mixture of aqueous liquids (i) and (ii) where (i) an aqueous liquid having an osmotic pressure of 250 mOsm/kg H2About or less, or an aqueous liquid having an osmotic pressure of 310 mOsm/kg H2About or more; (ii) aqueous liquid comprising hemolysate; step (b) immunological determination of the number of human madellaine released in a specified sample of blood from the blood, destroyed on the stage (a), using antibodies against human madellaine. Using the above described method is carried out diagnosis of multiple sclerosis. 2 C. and 16 h.p. f-crystals, 5 tab., 5 Il.

The present invention relates to a method of immunologically measuring human medullation in the blood and the method for the diagnosis of multiple sclerosis. In more detail, it relates to a method of immunologically measuring Chelopechene content medullation in granulocytes of the blood, and to a method for the diagnosis of multiple sclerosis using content medullation in the blood.

DESCRIPTION of PRIOR art Medellin, which is a kind of serine proteases, found in granulocytes, etc. and is considered to play an important role in the protective mechanism, including the manifestation of inflammation, particularly chronic inflammation. The number medullation in granulocytes increased at the stage of progression of several chronic inflammatory diseases and normal in remission. However, in patients suffering from multiple sclerosis, it was noted that the number madellaine increases considerably for a few days before the beginning of the progression of the disease and is normalized to the onset of remission. Multiple sclerosis is characterized by local demyelinizing damage in the white matter of the Central nervous system and gliosis. This is a severe chronic inflammatory disease that progresses with repeated remissions and exacerbations, and in many cases leads to death in 10-15 years. The cause of multiple sclerosis is still not clear, but believe that this disease is a type of autoimmune disease, in which suboleski quite complex and are currently carried out using magnetic resonance imaging (MRI) or other similar methods. However, this method, as MRI, requires bulky equipment and highly qualified specialists to work with him, and is also expensive. In addition, the method of analysis of bone marrow fluid is associated with causing the patient considerable pain.

In light of these circumstances is currently developing simple diagnostic method by which this may be done diagnosis, understanding of the disease and prognostic assessment. In the study devoted to the methods of measuring the activity medullation in granulocytes of the blood, and in conjunction with the development of immunological method of measurement by which this activity can be easily measured, suggested the diagnosis of multiple sclerosis content medullation in granulocytes of the blood. For example, for the immunological determination medullation in the blood has been proposed a method of measuring madellaine using polyclonal antibodies (Y. AOKI et al. Enzyme immunoassay of medullasin in peripheral blood, Clin. Chim. Acta, 15; 178(2), 193-204 (1988)).

However, there was observed a phenomenon in which, if the measurement was carried out after dilution of the blood sample water credo to fully wipe madellaine, available in granulocyte, from the granulocyte, the reproducibility of the measured values was poor, which led to fluctuations in the measured values. Accordingly there is a need to develop a method of immunologically measuring the amount of human medullation in blood with a good reproducibility of the results.

As for the question about whether the diagnosis of multiple sclerosis based on the number medullation in the blood, such judgment requires verification of a significant amount of clinical data. However, until now, this type of data does not exist, and moreover, it is difficult to make an accurate diagnosis because of the difficulty of obtaining precisely measured quantities medullation in granulocytes of the blood due to the large scatter of the measured values. Accordingly, the diagnosis of multiple sclerosis based on the number madellaine blood was difficult.

A BRIEF DESCRIPTION of the nature of the INVENTION Accordingly, the authors of the present invention conducted extensive research to solve the problems described above. As a result, they found that human medullation in the blood can be accurately measured with good reproducibility by immunological measurement of people who tov by processing a blood sample of the aqueous liquid, including hemolysate, or water liquid having a specific osmotic pressure that is different from the osmotic pressure of human blood.

In addition, after the development of this method for accurate measurement of human madellaine with good reproducibility of the results, the authors of the present invention noticed that the size and change in the measured content of human medullation in a sample of blood is closely associated with early multiple sclerosis, the extent of its distribution in the body, etc. on the basis of these findings was carried out by the present invention.

The first feature of the present invention relates to a method of immunologically measuring the content of human medullation in the blood, which is characterized by the following steps (a) and (b): (a) a stage of destruction of leukocytes in the blood sample by processing a specified sample of blood following aqueous liquid (i) or (ii) or a mixture of aqueous liquids (i) and (ii) (i) an aqueous liquid having an osmotic pressure of 250 mOsm/kgH2About or less, or an aqueous liquid having an osmotic pressure of 310 mOsm/kgH2About or more; (ii) an aqueous liquid, including GE and out of cells, destroyed on the stage (a), using antibodies against human madellaine.

The second feature of the present invention relates to a method of diagnosing multiple sclerosis, which is characterized by the following steps (a), (b) and (C): (a) a stage of destruction of leukocytes in the blood sample by processing a specified sample of blood following aqueous liquid (i) or (ii) or a mixture of aqueous liquids (i) and (ii) (i) an aqueous liquid having an osmotic pressure of 250 mOsm/kgH2About or less, or an aqueous liquid having an osmotic pressure of 310 mOsm/kgH2About or more; (ii) an aqueous liquid, comprising hemolysate;
(b) immunological measurement of human madellaine released into the blood sample from the blood, destroyed on the stage (a), using antibodies against human madellaine;
(c) monitoring the size and/or changes in the content of human medullation in the blood, measured in step (b).

BRIEF DESCRIPTION of DRAWINGS
Figure 1 is a calibration curve for measuring human madellaine built by drawing on a graph of absorption, as measured by enzyme immunoassay described the th for the measurement of human madellaine, built by drawing on a graph of absorption, as measured by enzyme immunoassay described in example 3, as a function of the concentration of antigen.

Figure 3 shows the values of human medullation in blood samples (μg/108granulocytes), plotted on a graph separately for healthy people, patients suffering from multiple sclerosis, and patients suffering from neurological diseases noninflammatory nature.

Figure 4 shows the values of human medullation in blood samples (μg/108granulocytes), plotted on a graph separately for patients, male and female, suffering from multiple sclerosis.

Figure 5 shows the values of human medullation in blood samples (μg/108granulocytes), plotted on a graph separately for patients of different age groups suffering from multiple sclerosis.

DETAILED DESCRIPTION of PREFERRED embodiments of the PRESENT INVENTION
In this document, the invention will be described in more detail.

Preferred embodiments of the present invention include (1) and (2), below.

(1) first, we have created a method of immunologically measuring the content of ceania specified sample of blood following aqueous liquid (i) or (ii) or a mixture of aqueous liquids (i) and (ii)
(i) an aqueous liquid having an osmotic pressure of 250 mOsm/kgH2About or less, or an aqueous liquid having an osmotic 310 mOsm/kgH2About or more;
(ii) an aqueous liquid, comprising hemolysate;
(b) the step of capturing human madellaine labeled immune complex by contacting the blood sample containing the specified human Medellin released from leukocytes, ruined in the step (a) with the antibody against human madellaine immobilized on an insoluble carrier in the presence of labeled antibodies against human madellaine, with the formation of the sandwich complex by reaction antigen-antibody;
(c) the step of measuring the activity of labeled material in the complex obtained in step (b).

(2) there is also a method for the diagnosis of multiple sclerosis, including:
(a) a stage of destruction of leukocytes in the blood sample by dilution of a specified sample of blood following aqueous liquid (i) or (ii) or a mixture of aqueous liquids (i) and (ii)
(i) an aqueous liquid having an osmotic pressure of 250 mOsm/kgH2About or less, or an aqueous liquid having an osmotic pressure of 310 mOsm/kg

A major part of human medullation in the blood sample to be measured of the present invention, is inside of granulocytes, which are one component of leukocytes present in the blood, and the complete destruction of granulocytes to release all madellaine outward toward the cell membrane before measurement is, therefore, a necessary requirement for accurate resultative fluctuations and can be obtained only data with poor reproducibility.

Mechanical methods, methods using ultrasonic waves and methods using repeated freezing and thawing can be used as a means complete destruction of leukocytes in the blood sample. However, the authors of the present invention after conducting extensive research has established that this method is particularly effective as a practical method, which allows to obtain high-precision measurement and can be done relatively simply compared with the above methods, is the following method.

(1) first, the method of processing a blood sample of aqueous liquid having an osmotic pressure than the osmotic pressure of blood;
(2) secondly, the method of processing a blood sample of the aqueous liquid comprising the hemolysate, which is a pharmaceutical means by which the cell membrane of granulocytes can be destroyed under gentle conditions.

The osmotic pressure of the blood is in the range from about 280 to 290 mOsm/kgH2Oh, and it is therefore difficult to completely destroy the granulocytes in the blood using, for example, aqueous liquid having an osmotic pressure is in a person's blood can be achieved by dilution of the blood aqueous liquid, having an osmotic pressure of less than 250 mOsm/kgH2On or aqueous liquid having an osmotic pressure of more than 310 mOsm/kgH2O.

The aqueous liquid of this kind, which can be used include pure water, which may include water-soluble organic solvents, and aqueous solutions and buffer solutions, which are a water liquid having a very high concentration or a very low concentration of dissolved substances consisting of water-soluble substances, such as inorganic salts, organic acid salts, sugars, sugar alcohols, amino acids and proteins that have an osmotic pressure, which can completely destroy the granulocytes. Specifically, sodium chloride, phosphates of sodium, etc. are the preferred salts of inorganic acids, and sodium acetate, sodium citrate, etc., are the preferred salts of organic acids. In addition, glucose, sorbitol, etc. are preferred sugars and sugar alcohols. Aqueous solutions having an extremely high concentration described above dissolved substances containing 0.05 mol.% or more, preferably of 0.1 mol.% or more of the solute, contain 0,005 mol.% or less, preferably of 0.001 mol.% or less. The amount of aqueous fluid exceeds 50-100000 times the amount of the blood sample by volume, preferably 100-10000 times and particularly preferably 500-2000 times.

In addition, the method of processing a blood sample of an aqueous medium comprising the above-mentioned hemolysate, is also preferred. Cationic surfactants, such as salts of higher fatty acids, alkylarylsulfonates, alkyl sulphonates and alkyl sulfonic acid esters; anionic surfactants such as salts of alkylpyridine, salts of alkyltrimethylammonium, alkylphenoxy-ethylenamine; nonionic surfactants, such as alkylphenol esters of polyoxyethylene, alkyl ethers and polyoxyethylene esters of fatty acids and polyoxyethylenesorbitan; amphoteric surfactants, such as alkylbetaine; natural surfactants such as saponin, lecithin and cholic acid; and biological components, such as compliments and snake venom, bee venom and an enzyme such as proteinase, are non-limiting specific examples of hemolysates. These hemolysate can be used in occhialino 0.005 to 1 wt.%. This aqueous liquid medium may, for example, be a water or a mixed environment, including the ox and the water-soluble organic solvent. The amount of aqueous fluid exceeds 50-100000 times, preferably 100-10000 times and particularly preferably 500-2000 times the amount of blood sample volume.

Immunological method for measuring human madellaine performed on dilute aqueous fluid sample of blood obtained by processing the blood samples described above aqueous liquid (i) liquid or water (ii), in which granulocytes completely destroyed. This method includes the stage of the immune response, during which the sample for measurement is in contact with the antibody against human medullation in the presence of labeled antigen or antibody to capture human medullation in the form of labeled immune complex through reaction antigen-antibody; and the stage of discovery, during which obtained in this way, the immune complex is measured using a labeled material present in the molecule. For the reaction of antigen-antibody during the stage of the immune response can be used any way.

Non-limiting examples of ways that you can COI blood, designed for measuring, after his capture antibody immobilized on an insoluble carrier;
(2) method two antibodies used antibody of animal origin that is different from the antibodies immobilized on the insoluble carrier in the sandwich method, in which a second antibody labeled with respect to this antibody, then interacts with the formed sandwich complex;
(3) competitive method in which the antigen blood sample intended for measurement interacts with the antibody immobilized on an insoluble carrier, in the presence of antigen, labeled with the enzyme horseradish peroxidase;
(4) the agglutination-precipitation, in which a blood sample, comprising the antigen used for measurement, processed, labeled antibody that interacts with it a specific way, causing agglutination-precipitation, and then detected using labeled material in the immune complex, which is separated by the separation in the centrifuge; and
(5) the Biotin-aydinbey way in which labeled avidin interacts with the antibody labeled with Biotin.

In the case of insoluble carrier in the method of immunologically measuring the human connection, such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, dextran cross and polysaccharides, as well as glass, metals, magnetic particles, and combinations thereof. The insoluble carrier may for example be used in a variety of forms, such as trays, beads, fibers, rods, discs, blood vessels, cells, microplates and tubes. For fixing antigens or antibodies to such insoluble carriers can be used any way. For example, you can use the methods in physical adsorption, the methods of covalent binding and methods of ion binding.

In the method of immunologically measuring human medullation of the present invention can be used antibodies of any class of immunoglobulins, but preferred is the use of antibodies of the IgG class. You can use it as monoclonal antibodies and polyclonal antibodies, but preferred are monoclonal antibodies. They can be used, for example, in the form of antibodies or fragments, such as F(ab')2and Fab. These antibodies can be obtained from various sources, but preferred is the use of antibodies derived from mice, rats, CR is uminescence substances and radioactive substances, etc., as a label for the measurement at the stage of detection of labeled immune complex human madellaine captured in this way. Non-limiting examples include enzymes such as peroxidase, alkaline phosphatase and-D-galactosidase; fluorescent substances such as isocyanates of fluorescein and ficobiliproteins; fluorescent substances such as luminol, dioxetane and salt acridine; and radioactive substances, such as125I131I111In and99mTS.

If the label is an enzyme, to measure its activity is used, the substrate and, if necessary, a coloring agent, a fluorescent agent or a fluorescent agent. In the case when the enzyme is peroxidase, the substrate may be used hydrogen peroxide, etc. as a coloring agent is ammonium salt of 2,2'-asindi[3-ethylbenzthiazolinesulfonic acid] (ABTS), 5-aminosalicylic acid, o-phenylenediamine, 4-aminoantipyrine, 3,3',5,5'-tetramethylbenzidine, etc. as a fluorescent agent - 4-hydroxyphenylarsonic acid, 3-(4-hydroxyphenyl)propionic acid, etc., and as a fluorescent agent - luminally, lucigenin, what if the enzyme is used as alkaline phosphatase, the substrate can be used 4-nitrophenylphosphate, 4-methylumbelliferone, cortisol-21-phosphate and so on; in the case when the enzyme is used-D-galactosidase, the substrate can be used 2-nitrophenyl--D-galactoside, 4-methylumbelliferyl-D-galactoside, 3-(2'-spiroadamantane)-4-methoxy-4(3"--D-galactosylceramide)-1,2-dioxetane (AMPGD), etc.

Preferred polyclonal antibody that can be used in the method of immunologically measuring human medullation of the present invention, is a material identified as or antibody-based test component of anticraving serum against madellaine person, obtained by immunization of an animal according to the conventional method using human madellaine as antigen. For example, preferably used goat polyclonal antibodies against human medullation and rabbit polyclonal antibodies against human madellaine. Monoclonal antibodies that can be used in the present invention, and santillo against human madellaine, which can be used in the method of immunologically measuring human medullation of the present invention, was obtained by culturing the hybridomas in culture medium; these hybridoma was obtained by fusion of myeloma cells and cells producing antibodies isolated from an animal immunized with human medullation extracted from granulocytes, which were isolated from the blood of healthy individuals, and through the allocation of monoclonal antibodies from culture or by intraperitoneal the introduction of a hybrid animal, the proliferation of hybrid in ascitic fluid and selection of monoclonal antibodies from ascitic fluid.

Hybridoma producing monoclonal antibody against human madellaine can be obtained using the method of cell fusion. That is, the desired hybridoma producing a monoclonal antibody can be obtained by selection of cells producing antibodies from animals immunized with human medullation, merge these cells producing antibodies with myeloma cells, selective proliferation of the obtained hybridomas, screening hybridomas that produce antibodies obtained from hybridomas and cloning otobject nodes, B-lymphocytes, etc., that are obtained from an animal immunized with human medullation, or cells or compositions containing human madellaine. Examples of animals that can immunize are mice, rats, rabbits, goats, sheep and horses. Immunization can be performed, for example, by subcutaneous, intramuscular or intraperitoneal introduction human madellaine animal in a dose from about 1 μg to 1 mg 1-2 times per month 1-6 months. The collection of cells producing antibodies can be done in 2-4 days after the final immunization.

Myeloma cells can be obtained from mice, rats, etc., it is Preferable to obtain cells producing antibodies and the myeloma cells from the same species of animal.

To merge cells, you can use any method without limitation. For example, it can be made by mixing cells producing antibodies and myeloma cells in this environment, as modified by the method of Dulbecco Wednesday Needle (DMEM) in the presence of a catalyst for the merger, such as polyethylene glycol.

After merging cells hybridoma can be subjected to selection by breeding cells appropriately with DMEM, etc., centrifugation, what we are producers of antibodies are then subjected to screening using immunoassay using the culture supernatant and the selected hybridoma cultivate way limiting dilution getting hybridoma, producing a monoclonal antibody against human madellaine.

Monoclonal antibody can be obtained by culturing the obtained in the described way of hybridoma-producer of antibodies in a suitable culture medium or in the body of the animal, and by highlighting the monoclonal antibody from the culture. In order to obtain large quantities of monoclonal antibodies, preferred is a method in which hybridoma administered intraperitoneally administered to an animal of the same species as the donor myeloma cells; monoclonal antibody accumulates in ascitic fluid, and subsequently monoclonal antibody isolated from the ascitic fluid.

Selection of monoclonal antibodies from the culture or from the ascitic fluid can be performed using anion exchange chromatography or chromatography on a column of protein A, G, etc. or by fractionation with ammonium sulfate, which is usually used for the purification of IgG.

Thus obtained monoclonal antibodies against human madellaine exist in the form of four types, denoted 3F03, 3G03, E and 1G12 in accordance with the type of hybrid that was used to receive them. Each of these monokinetic with human medullation, which is the corresponding antigen. Accordingly, the above-described monoclonal antibodies against human madellaine are suitable for the method for immunological measurement of human medullation of the present invention.

Next, a method for the diagnosis of multiple sclerosis according to the present invention includes early diagnosis of multiple sclerosis and/or extent of its distribution in the organism on the basis of size and/or changes in the value of the content of human medullation in a blood sample obtained by the above-described immunological measurement of human madellaine. Specifically, you can use a technique in which the amount of human madellaine 108granulocytes is calculated by measuring the concentration of human medullation in the blood sample and measured the number of granulocytes in the same blood sample, and the beginning of multiple sclerosis is judged by comparing the size of this value with the border value (average value for healthy individuals + 2SD (standard deviation)), or about the changing degree of the disease from time judged on the basis of the comparison value changes over in the 5 were positive by value madellaine not less than the edge value, that was a high positive percentage of 75.8%. On the contrary, out of 80 patients suffering from various neurological diseases noninflammatory nature, the number of positive value madellaine not less than the edge value was equal to 13, which was low positive percentage of 16.3%. Thus, it has been observed that the method for the diagnosis of multiple sclerosis according to the present invention, the value of human medullation in the blood is a diagnostic method is extremely high reliability. In addition, the percentage of normal individuals (in other words, healthy people), in which the value madellaine would be positive, amounted to 0% (see table 5 and figure 3). It was also found that the level values medullation in patients with multiple sclerosis is not different in men and women (see figure 4) and did not differ depending on age (see figure 5).

EXAMPLES
Hereinafter the present invention will be described more specifically by way of examples and reference examples. The present invention is in no way limited to these examples. The percentages given in the examples are weight percentages.

Reference example 1
Obtaining a purified man is popular weight 200000-300000) in physiological solution were mixed in the ratio of blood : an aqueous dextran solution = 2:1 and the resulting mixture was gently stirred with a glass rod, after which it was left to stand at 4-8oC for approximately 1 hour. Dropped into the sediment the erythrocytes were removed and the obtained nodosa-colonic fluid was centrifuged at 15,000 rpm, after which the collected precipitate to obtain leukocytes. In order to get the leukocytes were added extracting buffer containing 1 mm disodium salt ethylenediaminetetraacetic acid (EDTA) and 1 mm p-mercury-chlorbenzoyl acid (RSPS) in 1 M potassium phosphate buffer (RKV) with a pH of 7.0, and the mixture was incubated with stirring at 37oC for 20 minutes. Then the resulting mixture was affected by ultrasound for 15 seconds for the complete destruction of the cells and then incubated at 37oC for 20 minutes and centrifuged at 4oWith 12,000 rpm for 10 minutes. The supernatant was collected and deliberately against distilled water. Dropped into the sediment residue was subjected to the above-described operations several times to perform the extraction. The obtained extracted liquid was placed on a gel column with CM-separate, balanced 50 mm RVC (pH 6.0) and the column was washed with the same buffer. The adsorbed substances were then suirable 1 M RKV (pH 6.0) and suirvey solution Deliziosa is theft, using membranes from collodion to obtain 1.5 mg of purified human madellaine.

Reference example 2
Obtaining monoclonal antibodies against human madellaine
(1) development of hybridomas by fusion of cells producing antibodies and myeloma cells
Human Medellin extracted from human granulocytes and purified in reference example 1 was emulsiable in complete Freund's adjuvant and injected subcutaneously to BALB/C mice aged 7 weeks at a dose of 50 μg per mouse. After 4 weeks, mice additionally were immunized in the same way as in the first immunization. Seven days after immunization was confirmed by the increased level of antibodies in the blood. After 7 days as the last immunization administered intraperitoneally injected antigen in a dose of 50 μg per mouse. On the other hand, cells of mouse myeloma P3-X63-Ag8-U1 (P3U1) were passively in modified according to the method of Dulbecco environment Needle (DMEM) containing 20% fetal bovine serum. Three days after the last immunization merged splenic cells from mice were collected and P3U1 cells using polyethylene glycol 4000 were placed in wells of 96-well microtiter tablet. After the operation merge cells, the medium was replaced with DMEM containing 100 μm gipoksantina, 0.4 µm of aminopterin, and 16 μm thymidine (among the e 2-3 weeks.

(2) Screening of hybridomas producing antibodies against human madellaine
The antibody titers in the culture fluids hybridomas were determined by ELISA (enzyme-linked immunosorbent assay), exercising, thus screening. Namely, the human Medellin was adsorbing on the walls of the microtiter tablet for ELISA and wall blocked with a 2% solution of bovine serum albumin (BSA) in 10 mm saline solution with phosphate buffer (PBS) (pH 7.4). Fifty microlitres cultural liquid hybrid was added to each well and left to stand for 1 hour. After removal of the hybridoma culture was washed wells. 100 μl of 2 μg/ml solution in each well was added peroxidase labeled antimurine IgG-Fc antibody goat in PBS, the mixture was left to stand for reaction for 1 hour at 37oC. After removal of the solution of enzyme labeled antibodies and washing of the wells was added 200 μl of 0.1 M solution of the nitrate phosphate buffer (pH 4,6) containing 0.05% of ABTS and 0,0034% of hydrogen peroxide, to obtain a coloring that permit the selection of hybridomas producing antibodies against human madellaine.

(3) Cloning of cells producing antibodies and receiving monoc the Gali cloning method of limiting dilution, resulting ultimately got 4 kinds of monoclonal hybridomas. These hybridoma separately administered intraperitoneally injected to BALB/C mice that had previously received the Wharf, and hybridoma were grown to obtain ascitic fluid containing monoclonal antibody. Then to the resulting ascitic fluid was added a 50% saturated solution of ammonium sulfate to precipitate antibodies. The precipitate was separated and dissolved in PBS. The resulting solution deliberately against 50 mm buffer solution of Tris-hydrochloric acid (pH 7,3) containing 3 M NaCl. Then it was placed on CL4B column a-separate and protein (commercially available from Pharmacia). The adsorbed antibody was suirable 0.1 M buffer solution of glycine-Hcl (pH 5.0) and suirvey the solution was neutralized, after which the antibody was purified by obtaining monoclonal antibodies 4 species, 3F03, 3G03, E and 1G12.

(4) Properties of monoclonal antibodies Western blotting
The antigen corresponding to the monoclonal antibody, immobilizovana using the method of Western blotting.

First Medellin from human granulocytes were subjected to electrophoresis in SDS-polyacrylamide gel. Protein was transferred from a block of gel on a piece of nitrocellulose for the methane, 192 mm glycine and 20% methanol, added to the electrolyte buffer solution. Then each track on a piece of nitrocellulose was cut off and one of the sheets subjected to the staining of protein with amide black, and the other sheet was subjected to ELISA analysis as follows. Namely, after blocking sheet 2% BSA/PRS as a primary antibody was added mouse monoclonal antibody against human medullation, and then as a secondary antibody was added peroxidase labeled antipyrine IqG-Fc-specific antibody goat and left to stand for reaction. After washing the sheet to obtain the colouring was added to the substrate solution containing 0.04% of 3,3'-diaminobenzidine and 0,0034% hydrogen peroxide in PBS. This confirmed that all four mouse monoclonal antibodies against human madellaine recognize Medellin derived from human granulocytes.

Analysis of inhibition
Human Medellin immobilized on the walls of the microtiter tablet for ELISA, interacted with biotinylated first antibody in the presence of its second antibody then reacts peroxidase conjugated with Avidya, is generowania. For any combination of monoclonal antibodies, the number entered in the interaction of the biotinylated antibodies was not changed. Thus, it was confirmed that the 4 monoclonal antibodies recognize epitopes (binding sites of the antigen), which differ from each other.

Example 1
Obtaining calibration curves for measuring human madellaine
(1) preparation of granules with immobilized them a monoclonal antibody
After thorough washing of the pellets of polystyrene (diameter 6 mm) were immersed for one day and night at a temperature of 4oC in PBS (pH 7.4) solution containing 10 μg/ml mouse monoclonal antibodies against human madellaine (E). Then they were washed in PBS and subjected to blocking by placing a 1% aqueous solution of BSA at a temperature of 4oWith 1 day and night to get the beads with immobilized them a monoclonal antibody.

(2) Obtaining peroxidase labeled monoclonal antibodies
To 1.0 mg/ml solution of mouse monoclonal antibodies against human madellaine (E) in PBS solution was added 0.1 ml of 10 mg/ml solution of N-(m-maleimidomethyl acid)-N-operations ester (MBS) in dimethylformamide and the mixture octaves carried on a column with Sephadex G-25 and was carried out by gel chromatography using a 0.1 mm solution of phosphate buffer (pH 6.0) for the Department associated with maleimido monoclonal antibodies from unreacted MBS.

Meanwhile, a solution of ethanol with a concentration of N-Succinimidyl-3-(2-pyridylthio)propionate (SPDP) 10 mg/ml) was added to a solution of PBS with a concentration of peroxidase lozhechnitsy seaside (HRP) 1.0 mg/ml as the enzyme peroxidase, and they worked together at a temperature of 25oC for 30 minutes. Then the solution of the reaction mixture was transferred to a column of Sephadex G-25 and was carried out by the penetration of the gel using 10 mm acetate buffer solution (pH 4.5). Fractions containing HRP linked by a disulfide groups pyridium, collected and concentrated approximately ten times using a bag of collodion while cooling with ice. Then added 1 ml of 0.1 M saline solution with acetate buffer (pH 4.5) containing 0.1 M of dithiothreitol, after which it was stirred for 30 minutes at a temperature of 25oWith to restore the disulfide groups pyridyl built into the molecule of HRP. This solution of the reaction mixture was then subjected to gel permeation chromatography using a column of Sephadex G-25 and received the fraction containing etiolirovannuyu HRP.

Then associated with maleimido monoclonal antibody and etiolirovannuyu HRP were mixed, and the mixture was concentrated to a concentration of 4 mg/I, and then was carried out by gel permeation chromatography on a column of Ultrogel AcA44 (production SEPRACOR) and received monoclonal antibody labeled with enzyme peroxidase.

(3) Enzyme-linked immunosorbent sandwich analysis of human madellaine
One pellet with immobilized therein mouse monoclonal antibody against human madellaine (3F03), 50 μl of a solution of PBS with 2% BSA containing purified human medullation (standard calibration material) in a concentration of 0.1, 10, 100, or 200 ng/ml, and 350 μl of a solution of PBS with 2% BSA were mixed and the mixture is incubated at 37oC for 30 minutes.

Then, after the extraction of solution from the test tube, the pellet was washed with saline, and then the tube was filled with 400 µl of a solution of PBS with 2% BSA and labeled HBP mouse monoclonal antibody against human madellaine (E) at a concentration of 0.2 μg/ml and then incubated at temperature 37oC for 30 minutes. The solution from the test tube was removed by suction, and then washed with a physiological solution. 400 μl of 0.1 M buffer solution of phosphoric acid (pH of 4.6), containing 0,0034% hydrogen peroxide and 0.05% ABTS then added to each tube, after which they were incubated at 37oWith over 30 minoritasnya enzymatic reaction. Then using the spectrophotometer measured the absorption of the resulting solution at a wavelength of 420 nm. By drawing on a graph of the measured absorption depending on the concentration of the standard calibration material was prepared calibration curve with a good dependence on the concentration, as shown in figure 1.

Example 2
Measurement medullation in clinical samples using enzyme-linked immunosorbent assay
The frozen samples of blood taken from a normal individual (healthy person), in a patient suffering from multiple sclerosis, was thawed at room temperature, and 10 µl of each sample were added to 2 ml of distilled water (osmotic pressure = 0 mOsm/kgH2Oh and adequately mixed using a Voltex mixer to obtain solutions of the samples. Then 10 μl of these solutions were added to test tubes and diluted by adding 390 μl of PBS solution (pH 7.4) with 2% BSA. Then to each tube was added one pellet with immobilized therein mouse monoclonal antibody against human madellaine (3F03) and incubated at temperature 37oC for 30 minutes. After removal of the solution from the tubes by sucking them washed physiologists who elem against human madellaine (E) at a concentration of 0.2 μg/ml, after which they were incubated at temperature 37oC for 30 minutes. Then was carried out by washing, the enzymatic reaction and the interruption of the enzymatic reaction using the same operations that were used to obtain the calibration curves described earlier. Then using the spectrophotometer measured the absorption at a wavelength of 420 nm and determined the concentration of human medullation on the calibration curve. The steps of measuring, starting with a dilution of the sample was repeated 5 times for each sample to study the reproducibility of these measurements. In the result, it was confirmed that the concentration of human madellaine measured in blood samples showed very good reproducibility, as shown in table 1.

Comparative example 1
Measurement medullation in clinical samples using enzyme-linked immunosorbent assay
Blood samples, which were respectively taken from a normal individual and in a patient suffering from multiple sclerosis, and frozen for storage, thawed by their return to conditions with room temperature. Took 10 ál of each sample was added to 2 ml PBS (pH 7.4) (osmotic pressure = 290 mOsm/kg

Comparative example 2
Measurement medullation in a clinical sample using enzyme immunoassay
Blood samples, which were respectively taken from a normal individual and in a patient suffering from multiple sclerosis, and frozen for storage, thawed by their return to conditions with room temperature. Took 10 ál of each sample was added to 2 ml PBS (pH 7.4) and mixed to homogeneity to obtain the sample solution. Then 10 μl of these solutions were added to test tubes and diluted by adding 40 μl of PBS solution (pH 7.4) with 2% BSA. Then to each tube was added one pellet with immobilized therein mouse monoclonal antibody against human madellaine (3F03) and 350 μl of a solution of PBS with 2% BSA and HRP labeled mouse monoclonal antibody against human madellaine (E) at a concentration of 0.2 μg/ml, after which they were incubated at temperature 37oC for 30 minutes. Then carried out the washing, the enzymatic reaction and the interruption of the reaction is by using a spectrophotometer measured the absorption at a wavelength of 420 nm and determined the concentration of human medullation on the calibration curve. The steps of measuring, starting with a dilution of the sample was repeated 5 times for each sample to study the reproducibility of these measurements. In the result, it was confirmed that the concentration of human madellaine measured in blood samples, have provided data, the reproducibility of which could not be described as good, as shown in table 4.

Example 4
The calculation of the value of human medullation in the blood sample and diagnosis
Blood samples, which were respectively taken from a normal individual, in a patient suffering from multiple sclerosis, in a patient suffering from a neurological disorder noninflammatory nature, and frozen for storage, thawed by their return to conditions with room temperature. Took 10 ál of each sample was added to 2 ml of distilled water (osmotic pressure = 0 mOsm/kgH2Oh and adequately mixed using a Voltex mixer to obtain solutions of the samples. Then 10 μl of these solutions were added to test tubes and diluted by adding 390 μl of PBS solution (pH 7.4) with 2% BSA. Then to each tube was added one pellet with immobilized therein mouse monoclonal antibody is of the solution from the tubes by sucking them were washed in saline solution. Then the tubes were filled with 400 µl of a solution of PBS with 2% BSA and labeled HBP mouse monoclonal antibody against human madellaine (E) at a concentration of 0.2 μg/ml, after which they were incubated at temperature 37oC for 30 minutes. Then carried out the washing, the enzymatic reaction and termination reactions using the same operations that were used to obtain the calibration curves described earlier. Then using the spectrophotometer measured the absorption at a wavelength of 420 nm, and determined the concentration of human medullation on the calibration curve.

The significance of the human madellaine (μg/108granulocytes), showing the number madellaine 108granulocytes were calculated from the concentration of human medullation and the number of granulocytes measured in each blood sample, and presented in figure 3.

Figure 3 shows a comparison of the values respectively medullation in normal individuals, patients with multiple sclerosis and patients suffering from neurological diseases noninflammatory nature. From this figure the following results were obtained.

Patients with multiple sclerosis: 355117 (n=112)
Patientindividual: 21334 (n=25)
These results were compared with the boundary value (281 µg/108granulocytes) and classified as positive or negative. The number of each option and the percentage of positive presented in table 5.

On the basis of the above results made the observation that the diagnosis of multiple sclerosis by value medullation in the blood is a diagnostic method with high reliability. Besides, after the classification levels values medullation in patients with multiple sclerosis on the basis of sex (see figure 4), and age (see figure 5) were obtained the following results:
Men/women
- Patients with multiple sclerosis
Women: - 351107 (n=78)
Men: - 367143 (n=34)
Normal individuals: 21434 (n=24)
Border size: 281 (μg/103granulocytes)
Age
Patients with multiple sclerosis
10-19 - 421154 (n=9)
20-29 - 32994 (n=26)
30-39 - 357104 (n=30)
40-49 - 330157 (n=17)
50 and older - 375125 (n=21)
Normal individuals: 213

The EFFECT of the PRESENT INVENTION
Using the above-described invention may immunological accurately and with good reproducibility to measure the content of human medullation in the blood sample. In addition, early diagnosis of multiple sclerosis or extent of its distribution in the body or condition of the disease can be carried out using a diagnostic blood test for chronic inflammatory diseases, especially multiple sclerosis, using the measured values of the content of human medullation in the blood.


Claims

1. Method of immunologically measuring the content of human medullation in the blood, characterized in that it comprises the following steps: (a) a stage of destruction of leukocytes in the blood sample of aqueous liquid (i) and (ii) or a mixture of aqueous liquids (i) and (ii) where (i) an aqueous liquid having an osmotic pressure of 250 mOsm/kg H2About or less, or an aqueous liquid having an osmotic pressure of 310 mOsm/kg H2About or more; (ii) an aqueous liquid, comprising hemolysate; (b) immunological determination of the number of human madellaine released in a specified sample of blood from leukocytes, Raymarine content of human madellaine blood on p. 1, characterized in that the aqueous liquid (i) is a buffer solution and/or distilled water, which may include water-soluble organic solvent.

3. Method of immunologically measuring the content of human medullation in the blood under item 1, characterized in that the aqueous liquid (i) is an aqueous solution containing a water-soluble substance selected from the group consisting of salts of inorganic acids, salts of organic acids, sugars, sugar alcohols, amino acids and proteins.

4. Method of immunologically measuring the content of human medullation in the blood under item 1, characterized in that the amount specified aqueous liquid (i) exceeds the blood sample volume in 50-100000 time.

5. Method of immunologically measuring the content of human medullation in the blood under item 1, characterized in that the aqueous liquid (ii) is an aqueous solution of surface-active substances.

6. Method of immunologically measuring the content of human madellaine blood on p. 5, characterized in that the aqueous liquid (ii) is an aqueous solution of at least one type GE is tov, salts alkylsulfates, salt alkylpyridine, salts of alkyltrimethylammonium, alkylphenyl esters of polyoxyethylene, alkyl ethers of polyoxyethylene, fatty acid esters and polyoxyethylenesorbitan and alkylbetaine.

7. Method of immunologically measuring the content of human medullation in the blood under item 1, characterized in that the amount specified aqueous liquid (ii) exceeds the blood sample volume in 50-100000 time.

8. Method of immunologically measuring the content of human medullation in the blood under item 1, characterized in that the step (b) immunological determination of the content of human medullation in a specified sample of blood involves the contacting of the blood sample containing the specified human Medellin released from leukocytes, ruined in the step (a) with the antibody against human madellaine immobilized on the insoluble carrier, in the presence of labeled antibodies against human madellaine, for the formation of the sandwich complex and capture human madellaine labeled immune complex by the reaction of antigen-antibody, and then determining the amount of labeled material in the specified sector.

9. How immune the tap (b) involves the formation of a sandwich structure specified human medullation in a specified sample of blood between the antibody against human madellaine, immobilized on an insoluble carrier and a labeled antibody against human madellaine, with the formation of the complex by the reaction of antigen-antibody, and determining the amount of label in the specified property.

10. Method of immunologically measuring the content of human medullation in the blood under item 8, characterized in that at least one of these antibodies against human madellaine is a monoclonal antibody against human madellaine.

11. A method for diagnosing multiple sclerosis, characterized in that it comprises the following steps: (a) a stage of destruction of leukocytes in the blood sample by contacting a specified sample of blood with the following aqueous liquids (i) and (ii) or a mixture of aqueous liquids (i) and (ii) where (i) an aqueous liquid having an osmotic pressure of 250 mOsm/kg H2About or less, or an aqueous liquid having an osmotic pressure of 310 mOsm/kg H2About or more; (ii) an aqueous liquid, comprising hemolysate; (b) immunological determination of the number of human madellaine released in a specified sample of blood from the blood, destroyed on the stage (a), using antibodies against human madellaine; (C) comparing the size and/or area is the average value for healthy individuals + WITH (standard deviation).

12. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that at least one of these antibodies against human madellaine is a monoclonal antibody against human madellaine.

13. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that the aqueous liquid (i) is a buffer solution and/or distilled water, which may include water-soluble organic solvent.

14. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that the amount specified aqueous liquid (i) exceeds the blood sample volume in 50-100000 time.

15. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that the aqueous liquid (ii) is an aqueous solution of surface-active substances.

16. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that the amount specified aqueous liquid (ii) exceeds the blood sample volume in 50-100000 time.

17. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that the step (b) immunological determination of the content of human medullation in a specified sample of blood vkluchau, destroyed at the step (a) with the antibody against human madellaine immobilized on the insoluble carrier, in the presence of labeled antibodies against human madellaine, for the formation of the sandwich complex and capture human madellaine labeled immune complex by the reaction of antigen-antibody, and then determining the amount of label in the specified property.

18. A method for the diagnosis of multiple sclerosis on p. 11, characterized in that the step (b) includes the formation of a sandwich structure specified human medullation in a specified sample of blood between the antibody against human madellaine immobilized on an insoluble carrier and a labeled antibody against human madellaine, with the formation of the complex by the reaction of antigen-antibody, and determining the amount of label in the specified property.

Priority items:
03.02.2000 and 21.04.2000 - p. 1;
03.02.2000 - PP.2-4,8;
21.04.2000 - PP.5-7,9,10;
03.02.2000 - PP.11-18.

 

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SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.

EFFECT: improved assay method.

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