RU2377962C1 - Method for diagnostics of cattle leucosis - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary biotechnology. The method includes performance of staged immobilisation of leucosis virus antigen on a solid media as a result of its specific immunological bonding with absorbed mouse monoclonal antibodies against leucosis virus glycoproteid antigen, introduction and incubation of reference and analysed samples of cattle blood serum or milk, introduction and incubation of antigens to cattle immunoglobulins labelled with peroxidase, introduction of fermentative reaction substrate and account for the reaction results based on the value of optical density of the samples analysed. The leucosis virus antigen immobilisation proceeds in three stages. The first stage stage consists in non-specific absorption of mouse monoclonal antibodies to sheep 1H8 globulins that do not react with cattle immunoglobulins The second stage consists in specific immunological bonding of sheep monoclonal antibodies to 8C12 cattle leucosis virus glycoproteid antigen with the antibodies specified The third stage consists in specific immunological bonding of leucosis virus glycoproteid antigen with the sheep monoclonal antibodies to the antigen in question and detection of antibodies to leucosis virus in cattle blood serum or milk with addition of conjugate with peroxidase of mouse monoclonal antibodies against 5A10 cattle globulins that do not react with sheep immunoglobulins.

EFFECT: proposed method is characterised by high sensitivity and specificity.

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Description

The present invention relates to the field of veterinary biotechnology, in particular to the development of methods for the diagnosis of animal leukemia, and can be used in veterinary medicine.

The cattle leukemia virus causes a malignant lymphoproliferative disease in cattle - leukemia, which ranks first in the structure of cattle infectious pathology in the Russian Federation. The infectious process is characterized by the absence of viremia with the simultaneous production of antibodies to viral proteins, among which antibodies to the viral envelope glycoprotein (gp51) predominate.

Diagnosis of leukemia includes a complex of pathomorphological, histological, hematological, virological, molecular biological and serological research methods, of which the latter are most widely used. The sensitivity of serological methods is especially high in those cases when they are aimed at detecting antibodies to the glycoprotein gp51.

High sensitivity and specificity in the detection of antibodies to the leukemia virus have methods based on the principle of enzyme-linked immunosorbent assay (ELISA).

A known method for the diagnosis of bovine leukemia by determining antibodies to cattle leukemia virus by indirect enzyme-linked immunosorbent assay. Its implementation includes:

- sensitization of wells for microtiter plates with purified cattle leukemia virus antigen;

- the introduction and incubation of control and analyzed samples;

- the introduction and incubation of species-specific antibodies labeled with an enzyme; after each stage of the analysis, the wells of the plates are washed from unbound reagents;

- introduction of the substrate of the enzymatic reaction.

The reaction results are taken into account by the optical density of the analyzed samples (1).

Purification of the antigen that is part of the test system is a complex and time-consuming process. A leukemia virus antigen is obtained from a culture fluid of transplantable cell cultures, in particular a kidney cell culture of a sheep embryo chronically infected with leukemia virus. The cultivation of virus-producing cultures is carried out on growth nutrient media, supplemented most often with 10-20% of the blood serum of cow embryos, or less often, calves, lambs, adult cows, horses, etc. The virus production is extremely low. A multi-step procedure for purifying a virus or its antigens requires expensive sophisticated equipment. The antigen preparation, even obtained in the laboratory, is contaminated with impurities of cellular and serum origin, which leads to a decrease in the specificity of the analysis. In addition, purification leads to the loss of surface glycoprotein by the virions and, as a result, to a decrease in the sensitivity of the analysis.

The use of monoclonal antibodies to immobilize the antigen on the plastic of the titration tablet avoids the antigen purification procedure and at the same time significantly increases the specificity and sensitivity of the analysis compared to test systems in which sensitization of the solid phase is carried out by direct adsorption of purified cattle leukemia virus.

There is also a method for the diagnosis of leukemia by enzyme immunoassay using monoclonal antibodies to a glycoprotein antigen, characterized by high specificity and sensitivity. The method consists in sequential incubation in the wells of the tablet components of the test system and the analyzed material according to the following scheme:

- sensitization of the surface of the wells of the tablet for titration with a specific antigen, which is carried out in two stages:

a) the first stage - nonspecific adsorption of monoclonal antibodies of the mouse against the glycoprotein antigen of cattle on the surface of the holes of the panels for microtiter;

b) the second stage - specific immunological binding of the crude glycoprotein antigen gp51 of bovine leukemia virus with adsorbed monoclonal antibodies;

- the introduction and incubation of control samples and analyzed blood serum of cattle (preferred dilution of the latter - 1:60);

- the introduction and incubation of the conjugate of antibodies or Fab fragments of antibodies against cattle globulins with the enzyme; after each stage of the analysis, the wells of the plates are washed from unbound reagents;

- adding a substrate mixture.

The reaction results are taken into account according to the optical density of the analyzed samples.

When conducting comparative studies of 678 blood serum of cattle by the methods of RDP and ELISA, it was revealed: RDP + / ELISA + 119; RDP- / IFA-552; IFA + / RDP-7; IFA- / RDP + 0 (2). Prototype.

The sensitivity and specificity of this method allows you to identify virus carriers that produce antibodies in titers that are not detected in the diffusion precipitation reaction, and use both individual and precast samples of blood serum and cattle milk as research material. This allows you to increase the effectiveness of anti-leukemia measures and reduce their time.

However, the production of monoclonal antibodies to the glycoprotein antigen of cattle leukemia virus encounters a number of obstacles. Thus, immunization of mice that are not susceptible to the cattle leukemia virus species is ineffective. The mouse responds to immunization with insufficiently intense formation of antibody-producing cells; in addition, the glycoprotein antigen content of purified virion preparations is very low. Immunization of mice with a purified glycoprotein antigen is more effective, however, the cleaning procedure itself is quite time-consuming with a low yield.

The objective of our research was to develop a sensitive and specific method for the diagnosis of cattle leukemia by ELISA using monoclonal antibodies to the gp51 glycoprotein antigen obtained by interspecific hybridization using a sensitive laboratory model.

We have developed a method for the diagnosis of cattle leukemia by determining the virus carrier by indirect enzyme-linked immunosorbent assay using monoclonal antibodies to the cattle leukemia virus glycoprotein antigen.

The essence of the proposed method is as follows.

Leukemia virus antigen is immobilized on the surface of a solid-phase carrier in 3 stages, the first of which is nonspecific adsorption of mouse monoclonal antibodies to sheep globulins that do not react with cattle immunoglobulins, the second is specific immunological binding of sheep with monoclonal antibodies to these antibodies to glycoprotein antigen and leukemia virus antigen and the third is the specific immunological binding of the leukemia virus glycoprotein antigen with sheep monoclonal antibodies to this antigen. Next, test material is introduced and incubated, peroxidase-labeled mouse monoclonal antibodies against cattle globulins that do not react with sheep immunoglobulins are introduced and incubated. After each reaction step, unbound reagents are washed. The substrate of the enzymatic reaction is added to detect bound antibodies and the presence of antibodies is determined by the optical density of the test samples compared to the control.

To implement the method, a test system is used, which includes:

- cattle leukemia virus antigen sensitized microtiter plate;

- Peroxidase-labeled monoclonal antibodies against cattle globulins;

- control positive and negative samples of blood serum and milk.

In addition, a substrate mixture of hydrogen peroxide and chromogen (proton donor), diluents of the reaction components and a solution for washing the tablets are used.

Getting test system components

For the manufacture of an antigen-sensitized leukemia virus solid-state carrier, 1H8 monoclonal antibodies against cattle IgG globulin, 8C12 monoclonal antibodies to cattle leukemia virus glycoprotein antigen and cattle leukemia virus antigen are used.

Monoclonal antibodies to sheep IgG produce cells of the strain of the hybridoma cell line 1H8, deposited in 2007 in the "Specialized collection of transplantable somatic cultures of agricultural and commercial animals" (CJS RASHN) under No. 73.

The cells of the strain at a dose of 2-10 × 10 ⁶ cells / mouse in 0.5 ml of phosphate-buffered saline are injected into the abdominal cavity of linear BALB / c mice, which were previously injected with 0.3 ml of pristan in 7 days. On day 10-14, ascitic fluid is obtained, the titer of specific monoclonal antibodies to sheep IgG in which in ELISA was 1: 640-1: 5120. The preparation of monoclonal antibodies is obtained from ascitic fluid by three-time precipitation with a solution of ammonium sulfate 50% saturation followed by dialysis against phosphate-buffered saline, pH 7.2-7.4, and the protein concentration and antibody activity are determined.

Monoclonal antibodies to the glycoprotein antigen gp51 of bovine leukemia virus produce cells of the strain of interspecific hybrid cell line 8C12, deposited in 2007 in the Union of Agricultural Sciences under 72 under.

Cells of strain 8C12 are cultivated in glass mattresses at a temperature of 37 ° C in a nutrient medium consisting of RPMI-1640 medium, 10% blood serum of cow embryos and antibiotics (penicillin and streptomycin of 500,000 units / l). The optimal planting density is 2 × 10 cells / ml of medium. 3-5 days after the formation of the suspension, the culture fluid is partially removed and the required amount of fresh nutrient medium is added. The titer of antibodies to the VLCC glycoprotein antigen in the culture fluid is determined by enzyme immunoassay with purified VLCC antigen and species-specific rabbit antibodies against sheep globulins labeled with peroxidase. In the culture fluid, it was 1: 32-1: 64. The culture fluid is clarified by centrifugation at 5-10 thousand rpm for 20-30 minutes at 4 ° C.

The cattle leukemia virus antigen is produced by a chronically infected transplanted FLK-BLV cell culture (3).

The production of cattle leukemia virus antigen is carried out according to one of the following methods.

Cultivation of the FLK-BLV culture is carried out in a stationary mode in glass mattresses at a temperature of 37 ° C in a nutrient medium consisting of basal needle medium or MEM with a double concentration of amino acids and vitamins, supplemented with 10 volume percent blood serum of cow embryos. The optimal cell density is 1-2 × 10 ⁵ cells / ml medium. On the 6-7th day of cultivation, the culture fluid is collected, clarified by centrifugation at 5-10 thousand rpm for 20-30 min at 4 ° C and used to make a solid phase carrier sensitized by VLCC antigen.

The culture of the FLK-BLV culture in roller mode is carried out in glass bottles with a capacity of 5 l on a medium containing 45 volume percent of Eagle’s medium, 45 volume percent of lactalbumin hydrolyzate and 10 volume percent of serum of cow embryos at a temperature of 37 ° C. The optimal cell density is 1-2 × 10 ⁵ cells per 1 ml of medium. A monolayer is formed within 3-4 days. On the 8-11th day of cultivation, the culture fluid is collected, clarified by filtration and used for the manufacture of an antigen-sensitized leukemia virus solid-state carrier.

The cultivation of the culture of FLK-BLV in fermenters for monolayer cultivation of Girogen G300 and G600 cells (Netar, Switzerland) is carried out according to the "Method for the production of cattle leukemia virus antigen" (RF patent No. 2020960, MKI ⁵ AK61K 39/12 10/15/94. ), using a growth medium containing Eagle’s medium, FGMS, horse or reindeer blood serum and vitamin-salt supplements (5). The inoculum cell concentration was 4 × 10 cells / ml of growth medium. The culture is grown at 37 ° C for 7-10 days without changing the environment. When a cell monolayer is formed, the culture fluid is drained. The resulting culture fluid was clarified by filtration and used as a source of leukemia virus antigen for the manufacture of a sensitized solid-phase carrier for enzyme-linked immunosorbent assay.

The control of antigen specificity and activity is carried out in a diffusion precipitation (RDP) reaction after concentration of the culture fluid by a factor of 10-100. The antigen should form a precipitation band with the control standard antiserum identical to the precipitation band formed by the control standard antigen and the control standard antiserum. The activity of the glycoprotein antigen should not be lower than 1: 16-1: 64.

The production of a solid phase carrier sensitized by a glycoprotein antigen is carried out in three stages.

Stage 1. The preparation of 1H8 monoclonal antibodies to IgG immunoglobulin sheep is diluted with phosphate-buffered saline pH 7.2-7.4 to a protein concentration of 5 μg / ml and introduced into 96-well microtiter plates 100 μl per well. Incubated for 16 hours at 2-8 ° C, washed from unbound antibodies with a washing solution.

2 stage. Pipette 100 μl of culture fluid containing 8C12 monoclonal antibodies into each well, incubate for 1.5 hours at 37 ° C, wash off unbound antibodies with a wash solution.

3 stage. Pipette 100 μl of culture fluid containing cattle leukemia virus into each well, incubate for 1.5 hours at 37 ° C, and wash off the unbound antigen with a wash solution. Dry the tablet in air at room temperature (18-22 ° C) for 16 hours. Packed hermetically in laminated foil or polyethylene, removing excess air.

Production of control sera and milk

The control positive serum and milk are blood serum and milk of a cow infected with cattle leukemia virus. The control positive serum was titrated according to the national standard of sensitivity of the enzyme-linked immunosorbent assay to detect antibodies to the leukemia virus in biological fluids of cattle. The control negative blood serum and milk are prefabricated blood serum and milk obtained from at least 10 cows, free from the virus-induced leukemia infection, from a successful leukemia farm.

The production of peroxidase-labeled monoclonal antibodies against immunoglobulin IgG in cattle was performed using a strain of hybridoma cell line 5A10, deposited in 2007 at the Union of Agriculturalists of the Russian Academy of Agricultural Sciences under No. 71. Cells of strain line 5A10 at a dose of 2-10 × 10 ⁶ cells per mouse in 0.5 ml of phosphate-buffered saline are introduced into the abdominal cavity of linear BALB / c mice, which were previously injected with 0.3 ml of pristan in 7 days. An ascitic fluid with monoclonal antibody activity in ELISA of 1: 640-1: 5120 was obtained on days 10-14, from which antibodies were then isolated by three-fold precipitation with a solution of ammonium sulfate 50% of saturation, followed by dialysis against 0.01 M sodium carbonate buffer, pH 9.5. The antibody content in the preparation (for protein) was 10-12 mg / ml. The binding of antibodies to horseradish root peroxidase is carried out by the method developed by Wilson, Nakane, after activation of the enzyme with sodium periodate (4). The resulting conjugate is titrated by enzyme immunoassay. The activity of the obtained conjugate in an enzyme-linked immunosorbent assay for the detection of antibodies to cattle leukemia virus was 1: 5000-1: 30,000.

Enzyme-linked immunosorbent assay to detect antibodies to cattle leukemia virus is as follows.

0.1 ml of diluent for samples and conjugate, 0.004 ml of positive and control samples of blood serum and / or milk are added to microtiter plates sensitized with the glycoprotein antigen of cattle leukemia virus, the contents of the wells are thoroughly mixed, the plate is covered and incubated with 37 ° C for 1.5 hours. Then the contents of the wells are removed and the wells are washed three times with a washing solution, adding 0.3 ml to each well. Then, 0.1 ml of a working solution of the conjugate of monoclonal antibodies 5A10 against cattle peroxidase IgG immunoglobulins is added to each well, the plate is covered and incubated for 1.5 hours at 37 ° C. The contents of the wells are removed and washed four times with wash solution. Then, 0.1 ml of a substrate mixture containing chromogen and hydrogen peroxide is added to each well and left at a temperature of 18-22 ° C for 5-10 minutes in a place protected from direct sunlight. Measure the optical density of the solution in the wells. A test is considered reliable if:

- the optical density of the positive control exceeds 0.5;

- the ratio between the optical density of the positive and negative control P / N is not less than 2.

The reaction is considered positive if the optical density of the solution in the well of the test sample exceeds the optical density of the solution in the hole of the negative control (P / N) 2 times or more.

The implementation of the diagnostic method is reflected in specific examples.

Example 1. Production of a test system and assessment of the reliability of the test for determining antibodies to bovine leukemia virus in blood serum. Production of a test system and analysis with control sera and monitoring the level of background reaction of the test system (“blank sample” with serum diluent and conjugate) is carried out as described above. Zeroing the photometer is carried out against an empty tablet of the same series that was used to make the test system.

The measurement results are shown in table 1.

The optical density values obtained as a result of the measurements are: for a blank sample - less than 0.05 (0.02-0.04); for a negative control serum of 0.100-0.130; for a positive control serum ≥0.5.

Table 1
Reliability assessment of a test system using a leukemia virus antigen obtained in various ways The method of producing antigen Absorbance values P / N ratio Background negative control positive control FLK-BLV, stationary, medium Needle MEM, DMEM, 10% serum of cow embryos <0.05 0.118 0.648 5.5 FLK-BLV, roller mode, 45% of the medium MEM needle, 45% GLA, 10% serum of cow embryos <0.05 0.104 0.894 8.5 FLK-BLV, Girogen Fermenters, MEM Needle, FGMS, 10% Horse Serum <0.05 0.125 0.536 4.3

For all tested leukemia virus antigen preparations, the test was characterized by a low background optical density and was highly reliable (P / N> 2).

Example 2. Evaluation of the reliability of the test system in the study of milk A test system for detecting carriers of cattle leukemia virus by indirect enzyme immunoassay was prepared as described above using leukemia virus antigen made by roller culture of the FLK-BLV cell line. As the research material, the milk of an infected cow was used. The control was milk from a cow free of cattle leukemia virus. At the same time, a control positive serum sample was titrated. The results of the study are shown in table 2.

table 2
Reliability of using a test system to detect antibodies to cattle leukemia virus in milk research Breeding control positive samples Optical density P / N ratio Positive test Negative sample A) blood serum 1: 100 1,077 0.107 10 1: 200 0.564 0.124 4,5 1: 400 0.362 0.104 3,5 1: 800 0.143 0.130 ≤2 B) milk 1: 500 0.704 0,099 7 1: 1000 0.339 0,089 four 1: 2000 0.211 0,088 2 1: 4000 0.162 0.123 ≤2

The antibody titer in milk was 1: 2000. The results obtained indicate the applicability and high reliability of the proposed method for the diagnosis of leukemia in cattle using milk as a material for research.

Example 3. Comparison of the sensitivity and specificity of serological methods for the diagnosis of leukemia - RDP and ELISA

A test system for detecting cattle leukemia virus carriers by indirect enzyme-linked immunosorbent assay was prepared as described above using a leukemia virus antigen made by stationary cultivation of the FLK-BLV cell line. In 2006, a parallel study of 185 blood serum of cattle from a leukemia dysfunctional farm No. 1 (closed type) was carried out in this test system and the diffusion precipitation reaction. The results of the study are shown in table 3.

The coincidence of the results (RDP + / ELISA + and RDP- / ELISA-) was noted in 94.6% of cases, mismatch (RDP + / ELISA- and RDP- / ELISA +) in 5.4% of cases. An ELISA revealed 9 (4.9% of the number of investigated) positive samples negative in the RDP. One sample (0.5% of the number studied), positive for RDP, gave a negative result in ELISA. As can be seen from the table, the proposed method is more sensitive compared to the RDP.

Example 4. The sensitivity and specificity of enzyme-linked immunosorbent assay using monoclonal antibodies for the study of blood serum. A test system for detecting carriers of bovine leukemia virus by indirect enzyme-linked immunosorbent assay was prepared as described above. As the antigen of the leukemia virus, a preparation obtained in the roller culture mode of the FLK-BLV cell line was used. In 2007, they conducted a parallel study of 662 cattle blood serum from a leukemia-poor household No. 2 (closed type) in this test system and the diffusion precipitation reaction.

The coincidence of results (RDP + / ELISA +; RDP- / ELISA-) was noted in 560 or 84.6%, mismatch (RDP + / ELISA-; RDP- / ELISA +) in 15.4% of cases. Only 3 positive samples according to the results of the RDP reacted negatively in ELISA (0.5%). In ELISA, 190 (28.7%) positive samples were detected, while in the RPD - only 94 (14.2%), i.e. 14.5% or twice as much.

Table 4 shows that the sensitivity of the enzyme-linked immunosorbent assay was 2 times superior to RDP with a glycoprotein antigen and was not inferior to it in specificity.

Example 5. The sensitivity and specificity of the method for the diagnosis of leukemia in cattle by enzyme immunoassay in comparison with RDP and polymerase chain reaction (PCR)

A test system for detecting cattle leukemia virus carriers by indirect enzyme immunoassay was prepared as described above. As a leukemia virus antigen, a preparation obtained by culturing a FLK-BLV cell line in Girogen type fermenters was used. A study was conducted of 116 heifers from the same farm No. 2 in 2007. The infection rate of the adult population of the farm exceeds 60%. The group of animals was formed from heifers negatively responding to the results of the RDP. 2 months after the formation of the group by the method of RDP, 4 seropositive animals were again detected (3.4% of the number examined). Negatively reacted in the RPD were studied by ELISA and an additional 5 positively responding animals were identified (4.5% of the number examined). Infection in the group, according to the results of serological studies (RDP + ELISA), amounted to 7.8%. Negatively reacting in two tests (RDP + ELISA) were studied by PCR and revealed 1 positively reacting heifer (0.9%).

These results indicate that, in sensitivity, the proposed method significantly exceeds the RDP and approaches a direct diagnostic test - polymerase chain reaction (PCR).

The above examples indicate the high specificity and sensitivity of the proposed method for the diagnosis of leukemia by identifying carriers of the causative agent of the disease by indirect enzyme-linked immunosorbent assay, regardless of the method for producing viral antigen.

Feasibility Study

The proposed method for the diagnosis of cattle leukemia induced by cattle leukemia virus by indirect enzyme-linked immunosorbent assay is characterized by the following useful properties:

- has high sensitivity and specificity due to the use of three monoclonal antibodies in the manufacture of the test system;

- is a rapid diagnostic method that allows you to get the results of the analysis within one working day;

- includes an objective assessment of results and lends itself to automation;

- allows you to use not only blood serum, but also milk as a material for research, which is economically beneficial and reduces the risk of spread of infection when taking blood.

The proposed method for the diagnosis of leukemia is tested with a positive result in the laboratory conditions of the laboratory of leukemology of the GNU All-Russian Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko and the Kursk Biofactory from 2006 to 2007

A method for the diagnosis of cattle leukemia by enzyme-linked immunosorbent assay will be used in veterinary medicine, which will increase the effectiveness of health measures, reduce the recovery time of livestock farms that are unsuccessful for leukemia, and ultimately dramatically reduce the incidence of cattle leukemia.

Information sources

1. J. Aim. Rech. Vet. 1978.- 9 (4). - 663-666.

2. J. J. Virol.Meth. - 1983 - v.6. - 19-29.

3. J. Bibliotheca Haematologica - 1976. - v. 43. - 255-259.

4. Prince "Immunofluorescence and related staining techniques" non ed. W Knapp. - Elsevier: North-Holland Biomedical Press. - 1978. - 215-221.

5. RF patent No. 2020960, MKI ⁵ AK61K 39/12; 1994

Claims (1)

A method for the diagnosis of bovine leukemia, comprising the step-by-step immobilization of a leukemia virus antigen on a solid carrier as a result of its specific immunological binding to adsorbed mouse monoclonal antibodies against the leukemia virus glycoprotein antigen, the introduction and incubation of control and analyzed samples of blood serum or milk of cattle and incubation of antibodies to cattle immunoglobulins labeled with peroxidase, introduction of a farm substrate entative reaction and taking into account the results of the reaction according to the optical density of the analyzed samples, characterized in that the leukemia antigen is immobilized in three stages, the first of which is nonspecific adsorption of mouse monoclonal antibodies to 1H8 sheep globulins that do not react with cattle immunoglobulins, the second - specific immunological binding with the indicated antibodies of monoclonal sheep antibodies to the glycoprotein antigen of bovine leukemia virus 8C12 and the third - specific ie immunological binding glycoprotein antigen leukemia with sheep monoclonal antibodies to this antigen and identification of antibodies to the virus in the blood serum of leukemia and milk cattle conjugate addition of peroxidase-mouse monoclonal antibodies against bovine globulin 5A10, not reacting with immunoglobulins sheep.