CS248247B1 - Method of selecting hemorrhagic fever virus strains with renal syndrome by immunoenzymatic method - Google Patents

Abstract

The purpose of the solution is to select strains of hemorrhagic fever with renal syndrome by immunoenzymatic method. The stated purpose is achieved by applying a clear test material to a film of immune gamma globulin containing antibodies to the virus of hemorrhagic fever with renal syndrome, after washing and drying, anti-immunoglobulin labeled with the enzyme peroxidase or phosphatase is applied, after repeated washing, 1,4-diaminobenzene or 4-nitrophenyl phosphate is added, the reaction is stopped after 30 to 45 min and 1 to 1.01 mol. i-1 sulfuric acid solution and the color is evaluated visually or photometrically. The method of selecting strains of hemorrhagic fever with renal syndrome has applications in healthcare.

Description

The purpose of the solution is to select hemorrhagic fever strains with renal syndrome by immunoenzymatic method.

This is accomplished by applying a clear test material to an immuno-gamaglobulin-containing film of antibodies to the hemorrhagic fever virus with renal syndrome, washing and drying, adding peroxidase-labeled anti-immunoglobulin, or phosphatase after a thorough wash. diaminobenzene or 4-nitrophenylphosphate, the reaction is stopped after 30 to 45 min and 1 to 1.01 mol. i ^-1 sulfuric acid and the color is evaluated visually or photometrically.

The method of selecting haemorrhagic fever strains with renal syndrome is of use in healthcare.

The invention relates to a method for selecting strains of haemorrhagic burner virus with renal syndrome (HFRS) of the bad type immunoenzymatic (EIA) method.

Hemorrhagic burner with renal syndrome is a serious public health problem in the world, especially in Asia and Europe [C. Gajdusek: WHO Report (1982)]. To date, attempts have been made to capture the virus or the virus antigen of a hemorrhagic burner with renal lung syndrome by indirect immunofluorescence [H. W. Lee, P.W. Lee, I.J. Back, K. Tsuchiya, R. S. Foulke: Am. J. Trop. Med. Hyg. 30, 477 (1981); Mr Brummer-Korvenkontio, Mr Vaheri, Mr Hovi,

Von H. Bonsdorf, J. Vuorimies, T. Manni, K. Penttinen, N. O. ker-Blom, J. Lahdevirta: J. Infect, Dis. 141, 131 (1980); I. N. Gavrilovsikaya, N. S. Apekina. Yu. A. Myasnikov, A.

D. Bernshtein, E.V. Ryltseva, E.A. Gorbachkova, A. D. Chumakov: Arch. Virol. 75, 313 (1983); M. Grešíková, J. Rajcian, M. Seikey, M. Brumimer-Korvenkontio, O. Kozuch, M. Labuda, R. Turek, P. Weismann, J. Nosek, J. Lysy: Acta virol. 28, 416 (1984)], or on a human carcinomatous lung cell line (A 549) as well as a stabilized) green monkey kidney cell line (VERO clone F6 cells) [G. R. French: Abstract V. Int. Symp. Virology (1981); Kitamura T., M. Chlharu, K. Toshihiko, S. Kazuyoshi, A. Jiro, S. Sadashi, T. Hisao, A. Yoriyuki, I. Kiyoshi: JPN J. Med. Sci. Biol. 36, 17 (1983)].

The disadvantage of the methodologies used so far is the high workload. The methodology for the preparation of ultrathin sections from rodent lung and subsequent antigen demonstration by indirect immunofluorescence is striking. A similar methodology for culturing suspensions from small rodent lungs on cells is laborious, since it is only after a few blind passages that hemorbagic fever antigen with renal syndrome can be detected in the cytoplasm of hunks by indirect immunofluorescence.

SUMMARY OF THE INVENTION The present invention is based on the application of clear test materials to the immune gamma globulin film containing the antibodies to the hemorrhagic fever virus with renal syndrome after washing and drying, and after washing and drying an anti-immunoglobulin labeled with peroxidase or phosphatase enzyme is applied. after careful washing, 1,4-diaminobenzene or 4-nitrophenylphosphate is added, the reaction is stopped for 30 to 45 min with 1 to 1.01 mol. I ^'1 sulfuric acid and the color is evaluated visually or photometrically.

Immunochemical reactions, i. j. the reactions of antigens with antibodies are very often used to demonstrate biologically active substances because of their high specificity and sensitivity. Enzymes are an important antigen and antibody master. These have been used to label antibodies to detect antigens in histological preparations and to visualize weak precipitation lines in agar gel immunodiffusion techniques.

As it has been shown, enzyme conjugates are very closely related to histological and cytological examinations, because they have a great advantage over classical immunofluorescence not only in higher sensitivity, stability of preparations and usability in electron microscopy, but also in the ability to observe tissue cellular structures simultaneously. thus the exact localization of the antigen to be sought.

After the preparation of the first enzyme conjugates, so-called " the enzyme enzyme immunoassay EIA method, which evaluates the enzyme activity, typically by decomposition of a suitable substrate to provide colored reaction products.

Because the association constants of the antigen-antibody complex are optimally in the order of ^10-12 moles. 1 ^_1 the sensitivity by EIA immunoenzymatic order ^_15 to 10 mol. 1 ^_1 . Immunoenzymatic methods meet modern requirements for evaluation, accuracy and reproducibility.

The advantage of the method of selection of haemorrhagic fever virus strains with renal syndrome by immunoenzymatic method consists in saving material: media, sera and cells.

Another advantage is that the proposed solution is time-saving, reduces labor and risk from the point of view of safety at work.

Example 1

Specific 1 'human gamaglobulin containing antibodies to hemorrhagic fever with renal syndrome is applied to the polystyrene microplates in 0.05 ml portions. After adsorption of gammaiglobulin to the hard support for 18 hours (overnight) at 22 ° C, the wells are washed 3 times with 0.01 ml. 1 ^-1 phosphate buffer pH 7.2 with 0.5 wt. polyoxyethylene 20 sorbitant monolaurate (Tween 20) having a molecular weight of 1200.

U.P

Phosphate buffer composition:

Phosphate buffered saline (pH 7.2) was prepared. Solution A: 8 g of sodium chloride, 0.20 g of potassium chloride, 2.89 sodium hydrogen phosphate, 0.20 g of potassium dihydrogen phosphate are dissolved in redistilled water and the solution is made up to 800 ml with redistilled water. Solution B: Dissolve 0.1 g of calcium chloride in 100 ml of redistilled water. Solution C: Dissolve 0.1 g of magnesium chloride hexahydrate in 100 ml of distilled water.

Solutions A, B and C are mixed and then 0.5 wt. polyoxyethylene 20 'sorbitant nionolaurate. After washing and drying the plates, clarified suspensions from lung rodents, 0.05 ml each are added. After the adsorption of the antigen at 4 ° C for 18 h, the wells are washed again with phosphate buffer containing 0.5 wt. polyoxyethylene 20 sorbitant monolaurate. After drying, peroxidase-labeled antiglobulin is added. After washing three times, 1,4-diamino-benzene is added. The reaction is stopped after 30 minutes of 1 mole. I ^-1 sulfuric acid solution. The results are read on a photometer (Microelisa MR 590 j at 210 nm or visually.

Positively responding samples are selected for culture on a stabilized green monkey kidney cell line (VERO clone E6 cells). These stabilized cell lines are deposited at the Institute of Virology, Mlýnská dolina 1, 817 03 Bratislava.

Example 2

The procedure is as in Example 1 except that the blood of a diseased human or clarified organs (lung, kidney post mortem) is used. An immunoglobulin film containing antibodies to renal syndrome haemorrhagic fever virus is coated with clear test material and then after drying the applied preimytí and antlimunoglobulín, peroxidase-labeled enzyme, after re-washing, was added 1,4-diaminobenzene, the reaction is stopped after 30 minutes with a 1 mol. I ^-1 sulfuric acid and the color is assessed visually-f or- otometricky the 210 nm.

Example 3

The procedure is as in Example 1 except that the urine of a sick person adjusted to neutral pH is used. A patient's immunomodule is applied to a film of immunomammaglobulin with antibodies to hemorrhagic fever with renal syndrome. After adsorption at 4 ° C for 18 hours, the wells are washed 3 times with phosphate buffer at 0.5% w / w. polyoxyethylene 20 sorbitant monolaurate. After drying, peroxidase-labeled anti-immunoglobulin is added. 1,4-diamino-benzene (orthophenylenediamine) is added three times with phosphate buffer. The reaction is stopped after 30 minutes with 1 µl. I ^'1 sulfuric acid. The results are read on a photometer at 210 nm or visually. Positively responding samples are selected for culture on a stabilized green monkey kidney cell line (VERO clone E6 cells).

Example 4

The procedure is as in Example 1 except that an alkaline phosphatase-labeled antiglobulin is used. After washing for 3 times, the disodium salt of 4-nitrophenyl phosphate is added. The reaction was allowed to proceed in the mandrel and then stopped after 1.01 minutes with 1.01 mole. 1 ^_1 sulfuric acid. The results are read on a photometer at 490 wavelength light or visually. Positively responding samples are selected for culture on a stabilized green monkey kidney cell line (VERO clone E6 cells).

The method of selection of haemorrhagic fever strains with renal syndrome can find wide application in health care to isolate haemorrhagic fever strains with renal syndrome, t-iteration and identification in said virus strains.

Claims (4)

248247 With small rodents of 0.05 ml. After antigen uptake at 4 ° C during 18 hours, the wells were washed gently with a phosphate buffer of 0.5% by weight. polyoxyethylene 20 sorbitan monolaurate. After drying, anti-globulin, labeled with enzyme peroxidation, is added. After washing three times, 1,4-idiaminobenzene is added. The reaction is stopped after 30 minutes of 1 mole. I-1 sulfuric acid solutions. The results are read by a photometer (Miro Croisel MR 590) at a wavelength of 210 nm or visually. Positive reactive samples are selected by culturing on a stabilized green monkey kidney cell line (VERO clone E6 cells). The cell-stabilized Tiet-o cell deposited in the Virological Institute of the Slovak Academy of Sciences, Mlýnská dolina 1, 817 03 Bratislava. Example 2 The procedure is as in Example 1, with the difference that the blood of a diseased human, or clarified organs (, lung, kidney) is used postmortem. A clear test material is then applied to the immune gamma globulin film containing the antibodies to the hemorrhagic fever virus with renal sananesia syndrome, and then the peroxydase-labeled anti-immunoglobulin is added to the wash and dried, and 1,4-diamino-benzene is added after washing again; the reaction is stopped after 30 min s1 mol. The sulfuric acid solution was evaluated by visual inspection or photomicrography at 210 nm. Example 3 The procedure is as in Example 1, except that urine of a sick person, adjusted to a neutral pH, is used. For a film of immunoglobulin gamma globulin with antibodies to haemorrhagic fever with renal sananesia imo patient. After adsorption at room temperature 4 ° C for 18 hours, the wells are washed 3 times with phosphate buffer at 0.5% w / w. polyoxyethylene 20 sorbitan monolaurate. After drying, the peroxydase-labeled anti-imino-immunoglobulin is added. After washing three times with phosphate buffer, 1,4-diamino-benzene (orthophenylenediamine) is added. The reaction was stopped after 30 minutes with 1 ml. Acid acid solution. The results are read on a photometer at 210 nm or visually. The positively responding samples are selected for cultivation on a stabilized green monkey kidney cell line (VERA clone E6 cells). EXAMPLE 4 The procedure of Example 1 was followed except that antiglobulin, labeled with an alkaline phosphatase enzyme, was used. After washing three times, 4-nitrophenyl phosphate disodium salt is added. The reaction was allowed to run in a mandrel and then stopped at 1.01 mole after 45 minutes. 11 with sulfuric acid solution. The results are read on a photometric wavelength photometer 490 or visually. The positively reacting samples are selected to be cultured on a stabilized cell line from green monkey beads (VERO clone E6 cells). A method for selecting strains of hemorrhagic ear with renal syndrome can be widely used in the medical field to isolate strains of hemorrhagic fever with recurrent syndrome, titration, and identification of said strains of viruses. SUBJECT - A method for selecting haemorrhagic fever virus strains with renal syndrome by immuno-enzymatic method, characterized in that clear test materials are applied to a film of immune-gamma globulin containing antibodies to haemorrhagic renal syndrome and then washed after washing. and drying the invention with an anti-immunoglobulin labeled with enzyme peroxidase, or phosphatase after cautious washing, add 1,4-diamino-benzene, or 4-nitrophenylphosphate, stop the reaction for 30 to 40 minutes with 1 to 1.01 moles. The sulfuric acid solution " I " is colored visually or photometrically.