EA027858B1 - Agent for diagnostics of bovine leukaemia and method of its application - Google Patents

Abstract

The group of inventions relates to veterinary medicine and can be used for diagnostics of bovine leukaemia. An agent for diagnostics of bovine leukaemia comprises a water soluble protein fraction of the cell line of porcine embryonic kidney, contaminated with oncornaviruses C and D, and obtained by culturing under anaerobic conditions, having a molecular weight of from 75 to 82 kD in an amount of 0.49-0.52 wt.% and characterized by the presence of peaks at the wavelength of 214 nm in the ultraviolet spectral range, and additionally comprises phosphate-buffered saline. The method consists in sampling animal blood, preparing suspension of citrated blood of 0.25-0.5% concentration, mixing the suspension with the agent for diagnostics with the ratio of the agent and the suspension of 1:4-6, maintaining the mixture at a temperature of 4-8°C for 12-15 hours, and recording the results of the reaction by the nature of the interaction of the citrated blood suspension with the agent for diagnostics, wherein the presence of leukaemia is determined based on the positive reaction of the citrated blood suspension with the agent. The use of the inventions enables to expand the range of diagnostic products, to generate new approaches to the problem of diagnostics of bovine leukaemia, to diagnose bovine leukaemia quickly and cost-effectively while maintaining efficiency of diagnostic tests.

Description

The invention relates to veterinary medicine, in particular to veterinary oncology, and can be used to diagnose cattle leukemia. The invention extends the arsenal of means of the claimed purpose.

Known tools for the diagnosis of leukemia in cattle and methods for diagnosing a disease using these tools.

In particular, a serological diagnostic kit is known that contains a leukemia virus antigen, a leukemia virus antigen diluent, a specific precipitating serum, an agar salt mixture, an agar salt diluent (see L1p: //№№№.110.gu/ue1_b1adp).

The kit is intended for the formulation of the immunodiffusion reaction, which is carried out in an agar gel in order to detect antibodies against the leukemia virus glycoprotein antigen.

However, the immunodiffusion reaction (RID) does not allow the detection of virus-bearing animals whose antibody titer is insufficient to identify them.

A method for diagnosing cattle leukemia (Bovine) leukemia is used to solve the problem of identifying animal virus carriers using the RID reaction using this kit. This method involves the use of an inactivated bovine leukemia vaccine, which is administered three times with an interval of 14 days at a dose of 2 ml (see patent RF № 2264628 according to class IPC Ο01Ν 33/53, publ. 11/20/2005). The method allows to identify animal virus carriers among seronegative cows and heifers older than five months of age. This method consists in a repeated serological examination in the RID after provoking an immune response by three times the introduction of an inactivated vaccine.

However, any provocation of the immune response leads to the development of a significant amount of antibodies, which can lead to irreversible changes in the animal organism and, ultimately, their death.

Also known is a kit for the diagnosis of leukemia in cattle, designed for enzyme-linked immunosorbent assay (see ibid: 1Sp: // \ y \ y \ u.yukgi / u1_Fa8P). The kit contains 11 components:

component No. 1 - a polystyrene 96-well whole or stripped plate with a glycoprotein antigen (dr51) VLU immobilized in the wells;

component No. 2 - cattle blood serum containing antibodies to other 51 VLU (positive control);

component No. 3 - cattle blood serum that does not contain antibodies to other 51 VLU (negative control);

component No. 4 - milk containing antibodies to other 51 VLU (positive control); component No. 5 - milk not containing antibodies to other 51 VLU (negative control); component No. 6 - antispecific conjugate (monoclonal antibodies to 1§O of cattle labeled with peroxidase);

component No. 7 - concentrate (x 20) of buffer solution for dilution of control and test samples of blood serum and conjugate;

component No. 8 - concentrate (x 20) of washing buffer solution;

component No. 9 - substrate solution (H202);

component No. 10 - chromogen solution (TMB);

component No. 11 - stop solution (0.2 M sulfuric acid solution).

The kit is designed to detect antibodies to cattle leukemia virus in blood serum and cattle milk by enzyme-linked immunosorbent assay (ELISA) (see Guidelines for the diagnosis of cattle leukemia. Ministry of Agriculture of the Russian Federation, Department of Veterinary Medicine No. 13-72 / 2130 of 23.08. 00).

There is also a method for the diagnosis of leukemia in cattle using enzyme-linked immunosorbent assay (see RF patent No. 2425377, class IPC Ο01Ν 33/487, publ. 07/27/2011), which consists in preliminary before ELISA incubation of a sample of skim milk or blood serum, which increases the sensitivity of ELISA diagnostics up to 20%.

The enzyme immunoassay method has significant advantages over RID: higher sensitivity and specificity, the ability to automate the process and reduce analysis time.

However, the kit and the method of conducting ELISA with its help does not allow to detect antibodies that can be neutralized by the leukemia virus in the blood serum and milk of infected animals.

Known means and methods for diagnosing cattle leukemia by setting up a polymerase chain reaction (PCR), which is carried out in cases where animals are not detected by serological methods, for example, when the infection proceeds atypically without the formation of antibodies.

In particular, there is a known method for the diagnosis of cattle leukemia (see RF patent No. 2445370, class IPC S12C 1/68, publ. March 20, 2012), which consists in using oligonucleotide primers of a specific structure to detect a fragment of the bovine leukemia virus cattle.

However, PCR is a very expensive method. Moreover, the effectiveness of PCR

- 1,027858 is from 58.3 to 72.6% (see, for example, N. Dvoeglazov. Evaluation of the effectiveness of various methods for diagnosing cattle leukemia virus infection // Materials of the dissertation, - Novosibirsk. - 2009, published on the website of the Scientific Center : //teFsa1-F55.sot/ye1eg1pag1ua/o15epka-eGGek11Upo511-ga/NsNpuNte1obou-b1adpo8Yk1-1praseek18Yu1g8a-1euku7akgirpododa1odo8ko1a#1Kh773ar0.

Closest to the claimed is a tool for the diagnosis of leukemia in cattle, containing a protein fraction of the leukemia virus (glycoprotein antigen §p51), used for enzyme-linked immunosorbent assay (see NCR: //iii.b. ok.gi / ye1_Fadp).

However, this protein component is used only in the kit for enzyme immunoassay. The set itself is multicomponent, and the diagnostic method using this set is lengthy and time-consuming to implement.

The invention is aimed at solving the problem of expanding the range of diagnostic drugs and the formation of new approaches to the problem of diagnosing cattle leukemia.

The technical result achieved in this case consists in creating a simple and inexpensive means for the diagnosis of cattle leukemia, which allows quickly and with minimal cost to diagnose cattle leukemia while maintaining the effectiveness of diagnostic studies.

The specified technical result in terms of the tool itself is achieved by the fact that the tool for the diagnosis of leukemia in cattle containing a water-soluble protein fraction, according to the invention, as a water-soluble protein fraction contains a protein fraction of the cell line of the kidney of a pig embryo contaminated with oncornaviruses C and I and obtained by cultivation under anaerobic conditions, having a molecular weight of 75 to 82 kDa and characterized by the presence of peaks at a wavelength of 214 nm in the ultraviolet region of the spectrum , and additionally contains a phosphate-saline buffer solution in the following ratio of components, wt.%: water-soluble protein fraction of the cell line of the kidney of the embryo of the SPEV pig, contaminated with oncornaviruses C and I, having a molecular weight of 75-82 kDa - 0.49-0.52% ; phosphate-saline buffer solution - the rest.

The specified technical result in terms of the method of using this agent is achieved by the fact that the method consists in taking the blood of an animal, preparing a suspension of citrated blood of 0.25-0.5% concentration, mixing the suspension with a diagnostic tool with a ratio of the agent to suspension of 1: 4-6 keeping the mixture at a temperature of 4-8 ° C for 12-15 hours and taking into account the result of the reaction by the nature of the interaction of a suspension of citrate blood with a diagnostic tool, while the presence of leukemia is judged with a positive reaction of a suspension of citrated blood with a tool , which is a water-soluble protein fraction of the kidney cell line of a pig embryo contaminated with oncornaviruses C and I and obtained by cultivation under anaerobic conditions, having a molecular weight of 75 to 82 kDa and characterized by the presence of peaks at a wavelength of 214 nm in the ultraviolet region of the spectrum, and phosphate saline buffer solution in the following ratio of components, wt.%: protein fraction - 0.49-0.52 wt.% and phosphate-saline buffer solution - the rest.

These signs are essential and sufficient to obtain the desired technical result.

Known to the authors, the sources of patent and scientific and technical information do not describe means for the diagnosis of leukemia in cattle based on a water-soluble protein fraction of the cell line of the kidney of a pig embryo contaminated with oncornaviruses C and I and obtained by cultivation under anaerobic conditions, having a molecular weight of 75 to 82 CD and characterized by the presence of peaks at a wavelength of 214 nm, which additionally contains a phosphate-saline buffer solution at certain ratios of components.

Also unknown are methods for diagnosing cattle leukemia based on the agglutinating ability of the interaction of two components: the first is a diagnosticum made on the basis of a water-soluble protein component from the cell line of a kidney of a pig embryo contaminated with oncornoviruses C and D (and not an erythrocyte diagnosticum, as is customary in the formulation agglutination reactions), and the blood of a sick animal (a suspension of citrated blood of a certain concentration) acts as the second component.

Studies of the effectiveness of existing methods for diagnosing cattle leukemia (RID, ELISA, and PCR) have shown that none of these methods can fully detect all virus-bearing animals (see, for example, 1Sp: // \ y \ y \ u. RapaF. gi / 465284).

The efficiency of RID is 76.1%, ELISA - 83.3%, PCR - from 58.3 to 72.6%. Currently, to achieve the best diagnostic effect, researchers propose the combined use of serological and molecular-molecular methods, which increases the time and cost of diagnostic studies.

It is known to use the supernatant obtained by culturing SPEV cells for rapid diagnosis of human oncological diseases (see RF patent No. 2383891, class IPC 01-33 / 49, publ. 03/10/2010).

However, the use of this method, in which the supernatant is used as a reagent

- 2 027858 liquid obtained by culturing SPEV cells for the diagnosis of cattle leukemia had no effect: there was no reaction of interaction between washed red blood cells and suspension of citrated blood with supernatant.

The supernatant is a multicomponent structure in which lipids, polysaccharides, and protein are present.

That is why the task was set to identify such a protein fraction that would interact (enter into an agglutination reaction) with the blood of a sick animal (erythrocytes - washed and unwashed).

In the course of the studies, the results of the interaction of a 0.49-0.52% phosphate-saline buffer solution of a protein fraction with a molecular weight of 75-82 kDa isolated from the supernatant of the SPEV cell line contaminated with oncornoviruses C and D, only with non-washed red blood cells (t .e. with a suspension of citrate blood, and a certain concentration). Moreover, the obtained results coincided with the results of blood tests of the same animals in RID, ELISA and PCR.

Thus, the authors found a new approach to the diagnosis of cattle leukemia by using the reaction of the suspension of citrated blood (non-washed red blood cells) of infected animals from the protein fraction from the SPEV cell line during diagnosis.

Those. along with existing methods for diagnosing cattle leukemia (by conducting serological and molecular-molecular studies), the authors proposed a new approach to solving the problem of diagnosing cattle leukemia - determining the infection of animals with leukemia virus by the agglutinating ability of citrate blood red blood cells with a protein component of a certain molecular weight isolated from the supernatant of the cell fluid line SPEV contaminated with oncornoviruses C and D.

The foregoing allows us to conclude that the proposed solution meets the criterion of inventive step.

The invention is illustrated in FIG. 1, which presents the result of high pressure liquid chromatography (HPLC).

A cattle leukemia diagnostic agent is prepared as follows.

The cell culture SPEV contaminated with oncornoviruses C and D is seeded on a growth medium and cultured at 37 ° C for 3-4 days until the formation of a monolayer.

Then the growth medium is drained and Versene solution with trypsin is added, which is drained after 3-5 minutes and growth medium is added again.

Disintegrated cells are counted and the number of cells in 1 ml of medium is adjusted to 100-120 thousand.

This cell suspension is placed in sterile tubes and anaerobic conditions are created (the ratio of the nutrient medium to the air phase is 1: 5).

The tubes are placed in a thermostat and cultivation is also carried out at 37 ° C for 3-4 days until a monolayer is formed in these tubes.

The growth medium is drained and a support medium is added to each tube.

Place the tubes in a thermostat at 37 ° C. After 16-18 hours, they are examined and those tubes are taken in which the destruction of the monolayer occurred.

Tubes with a destroyed monolayer are once frozen and thawed, and the remaining tubes are discarded. The result is a suspension of thawed destroyed cells SPEV contaminated with oncornaviruses C and D.

To exclude artifacts from nonspecific destruction of the monolayer of cells and the accumulation of a larger volume of suspension to obtain a diagnostic tool, the suspension obtained from thawed destroyed cells of SPEV is added to the next batch of tubes with a monolayer and supporting medium grown under the conditions described above, based on 20% suspension and 80% supporting environment with a monolayer.

After a day, tubes with a destroyed monolayer of cells are taken. The procedure is repeated several times until 90% of the number of destroyed cells in one batch of tubes appears.

The suspension of cells with the destroyed monolayer is frozen and thawed 2-3 times, centrifuged for 30 min at a speed of 3000 rpm and the supernatant is taken.

Then the supernatant is precipitated with saline (for example, ammonium sulfate) in an amount of 60% of saturation, centrifuged at 4500 rpm for 20 minutes The supernatant is removed, and the sediment is redissolved in a minimum volume of 0.01 M phosphate-buffered saline at ρ 7.4. It is dialyzed using a dialysis membrane with a pore diameter of 3500 Da in a 0.01 M solution of phosphate-salt buffer at pH 7.4 for 24 hours. After that, the protein concentration is determined by conventional methods.

To determine the molecular weight of the obtained protein, high pressure liquid chromatography (HPLC) was performed, in particular, using a Stayer liquid chromatograph using a A214 spectrophotometric detector (wavelength 214 nm). For separation, a Vu8er-8ES-8 2000 5 micron 145A, 300x7.8 mm column was used. 0.01 M phosphate-buffered saline was used as eluent; the flow rate was 1 ml per minute.

Bovine serum albumin with a molecular weight of 64-70 kDa, lactoferrin with a molecular weight of 75-80 kDa and papain with a molecular weight of 20 kDa were used as standards.

Example 1 preparation of means for the diagnosis of cattle leukemia

A cell culture of SPEV contaminated with oncornoviruses C and D is taken at a concentration of 100 120 thousand cells in 1 ml of growth medium, the culture is inoculated on mattresses with a volume of 50 ml per growth medium Eagle with the addition of 10% allogeneic serum in an amount of 10 ml and cultured at 37 ° C for 3-4 days until the formation of a monolayer.

Then the growth medium is drained and Versene solution with trypsin in the ratio of 1: 1 in the amount of 3-5 ml is added, which is drained after 3-5 minutes and the growth medium is added again. Needle with 10% allogeneic serum.

The mattresses are shaken to remove the monolayer, the disintegrated cells are counted and the number of cells in 1 ml of medium is adjusted (by adding the same growth medium) to a concentration of 100-120 thousand cells in 1 ml of medium.

This suspension of cells in an amount of 1 ml is placed in 10 sterile Florinsky tubes with a volume of 5 ml, 1 ml each and anaerobic conditions are created (the ratio of suspension to air phase is 1: 5).

The tubes are placed in a thermostat and cultivation is also carried out at 37 ° C for 3-4 days until the formation of a monolayer.

The growth medium is drained and 1 ml of Needle support medium is added to each tube. Tubes are placed in a thermostat at 37 ° С, after 16-18 hours they are examined and those tubes in which the monolayer was destroyed - 2 tubes are taken. The remaining 8 tubes are discarded.

Selected 2 tubes with a destroyed monolayer are once frozen and thawed, as a result of which a suspension of 2 ml of suspension from thawing destroyed cells of SPEV contaminated with oncornaviruses C and D is obtained.

The resulting suspension from thawed, destroyed SPEV cells is introduced into the next batch of 10 tubes with a monolayer and a support medium grown under the conditions described above, at the rate of 0.2 ml of suspension and 0.8 ml of medium.

After 16-18 hours, the tubes with the destroyed monolayer of cells are again taken. The procedure for accumulating a larger volume of the suspension is repeated several times until 90% of the number of destroyed cells in one batch of tubes appears.

Finally, 120 tubes are taken, in each of which 0.2 ml of suspension and 0.8 ml of medium with a monolayer are added. As a result, 100 ml of suspension are obtained.

The suspension of cells with the destroyed monolayer is frozen and thawed 2-3 times, then centrifuged for 30 min at a speed of 3000 rpm and the supernatant is taken.

Take 100 ml of the supernatant, add 42 g of ammonium sulfate to precipitate the protein, centrifuged at 4500 rpm for 20 minutes. The supernatant is removed, and the precipitate is redissolved in 1 ml of a 0.01 M solution of phosphate-saline buffer at a pH of 7.4. Dialyzed using a dialysis membrane with a pore diameter of 3500 Da in a 0.01 M solution of phosphate-saline buffer at pH 7.4 for 24 hours, after which the protein concentration was determined by the Lowry method on a 81a1Pax 3300 biochemical analyzer. It was 5 mg / ml.

To justify the boundary values of protein concentration, a series of experiments was carried out similarly to that described in example 1. In all cases, the protein concentration was in the range from 4.9 to 5.2 mg / ml.

The results of HPLC chromatography showed (see Fig. 1) that the main proteins (peaks 1 and 2) had a molecular weight of 75 to 82 kDa and accounted for 93% of the total weight of the proteins entering the supernatant.

In the table. 1 presents the result of liquid chromatography.

Table 1

No. p / p Time min Height, aphids Square, tai * sec Attitude each peak to total peak area, A,% Molecular mass of peaks one. 11.36 58.44 5029.95 93.18 75-82 kDa 2. 16.04 0,03 0.02 0.00 15-20 kDa 3. 21.44 4.38 367.99 6.82 10 kDa and less

The method of application of the funds is as follows.

In tubes with a stabilizer (citric acid sodium or heparin), 2-5 ml of blood from one animal is taken.

- 4 027858

0.25-0.5% suspension in physiological saline is prepared from citrated blood.

The reaction is carried out on special plates with holes. Rows of holes are filled with 0.25-0.5% suspensions of citrated blood. One of the rows of wells with different concentrations of suspensions of citrated blood is filled with saline in each well (control), and in other rows with the same different concentrations of suspensions, a means for diagnosing cattle leukemia obtained according to Example 1 of the present invention is added in the same amount as saline solution.

At the same time, the ratio of the agent to suspension (as well as saline to suspension) is selected from the condition: one part of the agent and four to six parts of the suspension.

These mixtures are kept at a temperature of 4-8 ° C for 12-15 hours, after which the reaction is taken into account.

For the positive result of the reaction, the presence of a precipitate in the form of an umbrella with uneven edges over the entire surface of the hole is taken.

The presence of a precipitate in the form of a button or a point on the bottom of the hole is taken as a negative reaction result.

The suspension concentration of citrated blood (0.25-0.5%), the ratio of the administered drug to citrated blood (1: 4-6), as well as the temperature regime (4-8 ° C), at which the mixture is kept for 12-15 h, were selected experimentally.

So, at a concentration below 0.25% and the amount of the diagnostic agent less than one part, the reaction with citrate blood was practically absent, and at a concentration above 0.5% and the amount of the agent more than one part the reaction was not expressed, it was difficult to take into account.

The temperature regime at which the agent interacted with suspensions of citrate blood was also selected experimentally. 3 modes were tested: at room temperature (22 ° C), at 37 ° C and 4-8 ° C. At room temperature and at 37 ° C, the reactions were poorly expressed and difficult to take into account.

An example implementation of method 2

In tubes with a stabilizer (citric acid sodium or heparin), 2-5 ml of blood from one animal is taken.

0.25-0.5% suspension in physiological saline is prepared from citrated blood. Put the reaction on the plates with the wells.

The rows of wells are filled with 0.25-0.5% suspensions of citrated blood, which are taken in an amount of 0.5 ml in each well: a number of holes are filled with a suspension of 0.25% concentration, the other with 0.5% concentration, etc.

One of the rows of wells with different concentrations of suspensions of citrate blood is filled with saline in an amount of 0.1 ml in each well (control), and the agent for the diagnosis of cattle leukemia obtained in Example 1 of the present invention is also added to other rows with the same concentrations of suspensions in the amount of 0.1 ml.

The ratio of funds to suspension in this example is 1: 5.

All mixtures are maintained at a temperature of 4-8 ° C for 12-15, after which the reaction is taken into account.

The result of the reaction is positive (the presence of sediment in the form of an umbrella over the entire surface of the hole was observed).

Similarly to that described in Example 2, another 50 blood samples of animals were examined, of which 32 samples were positive and 18 negative.

Additionally, an experiment was carried out, similar to that described in example 2, but with washed red blood cells prepared by the conventional method from animal citrate blood. The method was carried out at the same concentrations and modes as in example 2.

50 samples were also investigated. There were no positive results when using washed red blood cells. All results were negative and did not coincide with well-known methods.

A comparative assessment was made of the effectiveness of the proposed method for the diagnosis of leukemia in cattle and tests RID, ELISA, PCR for the diagnosis of leukemia in cattle.

Studies were conducted in one of the dysfunctional farms on cows of 3 years of age. In the experiment, 10 cows infected with cattle leukemia virus were selected, which simultaneously reacted positively in RID, ELISA and PCR, and 10 animals that did not react simultaneously in any of these reactions.

All studies were performed in duplicate in order to confirm the coincidence of the results of the study and identify possible errors. The results are presented in table. 2.

- 5,027858

table 2

Groups investigated animals The name of the reaction and the nature of the reaction Reed IFA PCR The claimed the way Group 1 (10 goals) + + + + Group 2 (10 goals) - - - -

From the table. 2 it follows that the claimed method can effectively detect infected animals and coincides with the results of all generally accepted methods for the diagnosis of cattle leukemia, which proves the specificity of the claimed funds.

Thus, the claimed invention solves the problem of expanding the range of diagnostic drugs for leukemia in cattle. The tool allows you to quickly and at minimal cost to diagnose cattle leukemia while maintaining the effectiveness of diagnostic studies.

Claims (2)

1. The tool for the diagnosis of leukemia in cattle containing a water-soluble protein fraction of the cell line of the kidney of a pig embryo (SPEV), contaminated with oncornaviruses C and Ό and obtained by cultivation under anaerobic conditions, and this fraction has a molecular weight of 75 to 82 kD and is characterized by the presence peaks at a wavelength of 214 nm in the ultraviolet region of the spectrum, and phosphate-saline buffer solution in the following ratio of components, wt.%: protein fraction SPEV - 0.49-0.52%; phosphate-saline buffer solution - the rest. 2. A method for the diagnosis of leukemia in cattle, including blood sampling, preparation of 0.25-0.5% suspension of citrated blood, mixing the suspension with the agent according to claim 1, with the ratio of the agent to suspension 1: 4-6, keeping at a temperature of 4- 8 ° C for 12-15 hours and determining the nature of the interaction of a suspension of citrate blood with the drug, and the presence of leukemia is judged by the presence of sediment in the form of an umbrella with uneven edges.