RU2542475C2 - Method for preparing diagnosticum for detecting hepatitis c antigen in reaction of volumetric agglomeration (rva) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical virology. What is presented is a method for preparing a diagnosticum for detecting hepatitis C antigen in reaction of volumetric agglomeration (RVA).

EFFECT: thanks to higher specificity of the reaction and reduced length of blood analysis, the invention can be successfully used in the laboratory diagnostics for the direct detection of hepatitis C virus in human blood.

3 tbl, 3 ex

Description

A method for producing a diagnosticum for detecting hepatitis C antigen in a bulk agglomeration (RAO) reaction.

The present invention relates to medical virology and can be used in laboratory diagnostics for direct detection of hepatitis C virus in human blood.

Currently, there is a problem of detecting hepatitis C virus (HCV) in the early stages of human infection. The main widely used diagnostic method for detecting HCV in the blood serum of infected patients is polymerase chain reaction (PCR).

However, the PCR method, as a rule, gives negative results the first 6 months after infection with HCV. In addition, PCR is an expensive method that requires highly qualified specialists, expensive equipment, primers, reagents, and therefore it has significant restrictions on the use of blood samples for mass screening.

A known test system in the form of a set of antigens for the detection of antibodies to human hepatitis C virus (see US Pat. RU No. 2262704, IPC G01N 33/576, 20.10.2005), which consists in the fact that the proposed kit includes a number of recombinant antigens obtained on the basis of amino acid sequences of the most immunoreactive epitopes of hepatitis C virus proteins of different genotypes immobilized on the surface of a solid-phase carrier.

A disadvantage of the known test system is the complexity of its reproduction and the duration of the study, which makes it impossible to use it for screening studies.

For the prototype, a method for the accelerated diagnosis of hepatitis C was selected, based on the detection of hepatitis C virus hemagglutinins and antibodies to them in the RGA and RTGA in the blood (see Pat. RU No. 2194993, IPC G01N 33/569, G01N 33/576, 20.12.2000 g .), which provides for the detection of HCV antigens on the ability of the latter to agglutinate red blood cells of geese or pigeons in a hemagglutination reaction (RGA), while the results are considered positive if the titers of HCV antigens in the RGA exceed 1: 20-1: 40.

The disadvantages of this method:

firstly, RGA is a non-specific erythrocyte agglutination reaction that does not allow us to confirm the presence of HCV antigens in the blood of people, and not other antigens with the ability to agglutinate goose erythrocytes (arbovirus, rubella virus antigens, etc.);

secondly, to identify the HCV antigens found in the RGA, it is necessary to formulate a more complex test - the hemagglutination inhibition reaction (RTGA) using antibodies to HCV antigens;

thirdly, the establishment of rtga may be difficult due to the presence of HCV-specific antigen-antibody complexes in the blood serum of HCV infected people.

Thus, RGA and RTGA for the detection of HCV antigens require the presence of formalized goose red blood cells, the method of obtaining which from a 6-month-old male goose would not lead to their ability to agglutinate in the presence of antigen; in addition, the method does not differ in standard and obvious results in view of the fact that the receptors for hemagglutinins of the virus on red blood cells vary from the blood type of the bird. Therefore, this method cannot be widely implemented in practical public health.

The purpose of the invention was to develop a new diagnosticum, allowing to carry out screening studies of blood samples in a short time to detect hepatitis C antigen in standard mode and characterized by good visibility.

This goal is achieved by the fact that in the known method of obtaining a diagnosticum for the detection of hepatitis C antigen in the bulk agglomeration (RAO) reaction, including immunization of rabbits to obtain blood serum with the release of antibodies to HCV antigens and immobilization of the latter on a polymer carrier, followed by RAO, characterized in that blood serum is obtained by phased immunization of rabbits with Virus hepatitis C strain, GKV-963 at a dose of 5 · 10 ⁶ LD ₅₀ , MIC (100) in a volume of 0.5 ml and is isolated by the Duberta method from the obtained immune serum anti-HCV, the latter are covalently bound to polymer microspheres; for this, 100 mg of the polymer carrier is washed twice in 0.1 M phosphate-buffered solution of pH 7.0 and poured into 10 ml containers and centrifuged at 3000 rpm for 10 min then the supernatant is decontaminated and the precipitate is suspended in 1.5 ml of 0.1 M phosphate buffer solution with the addition of 200 μg / ml anti-HCV from rabbit serum, stirred, left to contact for 2 hours at 20 ° C and 16-18 hours at 7 ° C, after which 2 ml of 1.0% solution is added to the resulting suspension gelatinous thief in a 0.9% sodium chloride solution and left with constant stirring for 2 hours at a temperature of 20 ° C, then the resulting diagnosticum suspension is centrifuged and washed three times with a 0.9% sodium chloride solution at 3000 rpm for 5 minutes, obtained the precipitate is resuspended in 8 ml of a 0.1% gelatine solution in a 0.9% sodium chloride solution, after which the specific activity of the obtained preparation is determined, which is then poured into ampoules and lyophilized.

In this case, the phased immunization of rabbits is carried out in four stages, at the first within 3 days, the strain D-1, GKV-963 is administered intravenously in an amount of 0.5 ml, at 8, 9 and 10 in Freud's complete adjuvant in the ratio 1: 1 and in a volume of 0.5 ml is injected subcutaneously into the withers of the animal, and on days 15, 16 and 17, the same preparation is re-administered in 0.5 ml and on day 22 also in Freud's complete adjuvant in a ratio of 1: 1 and in an amount of 1, 0 ml is injected into the withers of the animal and only on day 30, the animal is bled, followed by isolation of anti-HCV antibodies from the serum.

In addition, the carrier is prepared by dissolving 1.5 g of polyvinyl pyrrolidone and 75 ml of acrolein in 250 ml of distilled water, then a solution of 0.3 safronin in 20 ml of 0.1 M sodium chlorine is added with stirring to a solution cooled to 12 ° C, after to stop raising the temperature, cooling is removed, the reaction mixture is stirred for 1 hour, then 2 g of potassium persulfate are added and the solution is purged with nitrogen and the polymerization is carried out for another 2 hours at a temperature of 70 ° C, after which the resulting microspheres with a diameter of 1.1 μm are washed with water by precipitation in a centrifuge.

At the same time, the test serum is prepared for RAO, for which 0.1 ml of the latter is diluted in 0.9 ml of a 0.9% sodium chloride solution, after which it is heated for 30 minutes in a water bath at a temperature of 56 ° C and left at 20 ° C for 10 min, after which studies are carried out in the radioactive waste.

The method is as follows.

To carry out the method requires source material:

- strain Virus hepatitis C, D-1, ST-963, which is deposited and stored in the State virus collection of the Research Institute of Virology. DI. Ivanovo RAMS;

Initially, antibodies are obtained from hepatitis serum. For

This is carried out immunization of rabbits in four stages:

Stage I - for 3 days, rabbits are injected intravenously in a volume of 0.5 ml with a strain of Virus hepatitis C, D-1, GKV-963, at a dose of 5 · 10 ⁶ LD ₅₀ , MIC (100);

Stage II - on 8, 9 and 10 days after the first immunization, the drug (strain D-1, GVK-963) is suspended in Freud's complete adjuvant in a ratio of 1: 1 and injected subcutaneously into the withers of the animal in a volume of 0.5 ml;

Stage III - on the 14th, 15th and 16th day, intravenous administration of the drug in an amount of 0.5 ml is repeated;

Stage IV - on day 20, strain D-1 (ST-963) is suspended in Freud's complete adjuvant in a ratio of 1: 1 and injected subcutaneously into the withers of the animal in 1.0 ml.

Exsanguination of animals was performed on day 30 from the start of immunization.

Hepatitis C virus antibodies (HCV) are isolated from the obtained immune serum by Duberta.

The next step in obtaining a diagnosticum is to immobilize anti-HCV on polymeric carriers. As a carrier, spherical polymer particles with a diameter of 1.0 mm containing 1.3 mM / g of aldehyde groups are used.

The carrier was obtained by polymerization of acrolein in an aqueous alkaline medium in the presence of a radical initiator, potassium persulfate. For this, 1.5 g of polyvinyl pyrrolidone and 75 ml of acrolein are dissolved in 250 ml of distilled water. To a solution cooled to 12 ° C, a solution of 0.3 safranin in 20 ml of 0.1 M NaOH is added with stirring. After stopping the rise in temperature, the cooling was removed, the reaction mixture was stirred for 1 hour, then 2 g of potassium persulfate was added to it. The solution was purged with nitrogen and the polymerization was carried out for another 2 hours at a temperature of 70 ° C. The microspheres are washed with water by centrifugal precipitation.

Anti-HCV is covalently bound to polymer microspheres. For this, 100 mg of carrier (1 ml of a suspension of microspheres) is placed in a centrifuge cup and washed twice with 0.1 M phosphate buffer solution, pH 7.0 is poured into 10 ml containers and centrifuged at 3000 rpm for 10 minutes. After decontamination of the supernatant, the precipitate of the carrier is suspended in 1.5 ml of 0.1 M phosphate buffer solution containing 200 μg / ml anti-HCV immunoglobulins and, while stirring, left to contact for 2 hours at a temperature of 20 ° C. Then leave for contact at a temperature of 7 ° C for 16-18 hours

After that, 2 ml of a 1.0% gelatose solution in a 0.9% sodium chloride solution was added to the resulting suspension and left under constant stirring for 2 hours at a temperature of (20 ± 2) ° C. The diagnosticum suspension thus obtained is centrifuged and washed three times with a 0.9% sodium chloride solution at 3000 rpm for 5 minutes. The diagnosticum precipitate is resuspended in 8 ml of a 0.1% gelatose solution in a 0.9% sodium chloride solution.

After that, determine the specific activity and specificity of the suspension of the obtained diagnosticum, which is then poured into ampoules and lyophilized.

The use of the obtained diagnosticum is confirmed by examples 1,2,3 (see tables).

A series of studies of blood serum of people suspected of hepatitis C in the agglomeration of volumetric (RAO) using the proposed diagnosticum.

The diagnostic titer of the drug is 1: 20-1: 40.

To set up RAO, the test sera are first prepared as follows: 0.1 ml of the test serum and 0.9 ml of 0.9% sodium chloride solution are taken. The diluted sera are heated for (30 ± 1) min in a water bath or inactivator at a temperature of (56 = 1) ° C. After this, the sera are left at (20 ± 2) ° C for 10 min and then examined in the RAW.

Then, 50 μl of 1% solution of normal rabbit serum (NCC) is poured into the U-wells of the immunological reaction plate. 50 μl of test serum are added to the first well of the series and titrated to 12 wells. 25 μl of a suspension of the diagnosticum antibodies immobilized on microspheres of anti-HCV are added to each well, after which the contents of the wells are stirred by swaying for 1 minute and left at 20 ° C for 2.5 hours.

To control the diagnosticum for spontaneous agglomeration in 2 wells, only NKS and diagnosticum are introduced. After 2.5 hours, the reaction is monitored.

For a positive result (+) take a bright pink agglomerate, evenly lining the entire bottom of the hole ("probe"). A positive result indicates the presence of HCV in the test serum. The calculation of results is based on the calculation of HCV antigen titers. The results are considered positive if the titer of the HCV antigen in the radioactive waste exceeds 1: 20-1: 40. In the case of a negative result (-), the particles roll to the bottom in the form of a dense “buttons” deposit or in the form of a small ring. Upon receipt of a negative result, it can be assumed that hepatitis C virus was not detected in the test serum.

Example 1

We examined 18 blood sera of people suspected of having hepatitis C by two methods: polymerase chain reaction (RT PCR) and in the serological reaction of RAO with hepatitis C immunoglobulin polymer diagnostics (see Table 1).

Table 1 No. p / p RAO PCR one 1: 160 + 2 1:80 + 3 1: 160 + four 1:40 + 5 1: 640 + 6 1: 640 + 7 1:40 + 8 1: 640 + 9 1: 1280 + 10 1: 320 + eleven 1: 320 + 12 1:40 + 13 1: 160 + fourteen 1: 1280 + fifteen 1: 640 + 16 1: 1280 + 17 1: 160 + eighteen 1:20 +

From table 1 it is seen that all the serums according to PCR contained HCV RNA and were also positive in the RAW, and the titers of the HCV antigen significantly varied, indicating the possibility of differentiating the stages of the disease with hepatitis C.

Example 2

It differs from Example 1 in that donor sera were taken as the studied sera (see Table 2).

table 2 No. p / p RAO PCR one 0 0 2 0 0 3 0 0 four 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 10 0 0

The data in table 2 indicate that in the blood serum of the studied group of donors, hepatitis C virus antigens were not detected by either RT PCR or RAO. This indicates the absence of false positive results when using the diagnosticum of immunoglobulin polymer hepatate C.

Example 3

It differs from examples 1 and 2 in that the sera of patients with viral infections and the control sera of patients with hepatitis B (from the ELISA assay kit) were taken as the studied sera.

Table 3 No. p / p serum of patients (diagnosis) RAO PCR one flu 1:80 0 2 flu 0 0 3 flu 0 0 four orvi 0 0 5 Tsmvi 0 0 6 herpes 0 0 7 Tsmvi 0 0 8 hepatitis B virus control 0 0 9 hepatitis B virus control 0 0 10 hepatitis B virus control 0 0

Table 3 shows that in one of the sera of patients with influenza infection, titers for HCV were recorded and HCV RNA was not detected. In this case, two explanations of the observed phenomenon are possible. Firstly: the result is false positive and not anti-HCV antibodies are present in the patient’s blood, but there is an increased level of C-reactive protein, which causes spontaneous agglutination of the diagnosticum microspheres. Or, the detected concentration of HCV in the blood of a patient with influenza infection indicates the early stages of hepatitis C disease, which makes it possible to establish a diagnosis of infection in cases where HCV RNA cannot yet be detected by PCR.

The use of the present invention allows, due to the obtained diagnosticum, to conduct screening studies of a large number of people suspicious of hepatitis C disease in standard mode and with good visibility without expensive equipment, primers and reagents, which will significantly reduce the cost of conducting single or mass studies.

Claims (1)

A method for producing a diagnosticum for detecting hepatitis C antigen in a bulk agglomeration (RAO) reaction, comprising the following steps:
a) receive blood serum by immunization of rabbits with a viral preparation of the strain Virus hepatitis C, D1, GKV-963 at a dose of 5 · 10 ⁶ LD ₅₀ , MIC (100) in four stages:
I - within 3 days, the said drug is administered intravenously in a volume of 0.5 ml;
II - on 8, 9 and 10 days after the first immunization, the preparation of the strain Virus hepatitis C, D1 is suspended in Freud's complete adjuvant in a ratio of 1: 1 and in a volume of 0.5 ml and injected subcutaneously into the withers of the animal;
III - on the 14th, 15th and 16th day, the intravenous administration of the drug of the strain Virus hepatitis C, D1 is repeated in an amount of 0.5 ml;
Stage IV - on day 20, the viral preparation of the strain Virus hepatitis C, D1 is suspended in Freud's complete adjuvant in a ratio of 1: 1 and in the amount of 1.0 ml is injected subcutaneously into the withers of the animal, then bleeding animals is carried out on the 30th day from the start of immunization;
b) isolate hepatitis C virus antibodies (HCV) from the obtained immune serum by Duberta;
c) a pre-polymer carrier is prepared which is obtained by dissolving 1.5 g of polyvinyl pyrrolidone and 75 ml of acrolein in 250 ml of distilled water, then a solution of 0.3 safronin in 20 ml of 0.1 M is added to the solution cooled to 12 ° C with stirring sodium chlorine, after stopping the rise in temperature, cooling is removed, the reaction mixture is stirred for 1 hour, then 2 g of potassium persulfate are added and the solution is purged with nitrogen and the polymerization is carried out for another 2 hours at a temperature of 70 ° C, after which the resulting microspheres with a diameter of 1.1 μm are washed by centrifuge sedimentation;
d) hepatitis C anti-HCV is immobilized onto a polymer carrier, which is covalently bound to the polymer microspheres of the carrier; for this, 100 mg of the latter are washed twice in 0.1 M phosphate-buffered solution of pH 7.0 and poured into a 10 ml container and centrifuged at 3000 rpm for 10 min, then the supernatant is decontaminated, and the precipitate is suspended in 1.5 ml of 0.1 M phosphate buffer solution, with the addition of 200 μg / ml anti-HCV from rabbit serum, stirred, left to contact for 2 hours at a temperature of 20 ° C;
d) a diagnosticum suspension is obtained, which is centrifuged and washed three times with a 0.9% sodium chloride solution at 3000 rpm for 5 minutes, the resulting precipitate is re-suspended in 8 ml of a 0.1% gelatine solution in a 0.9% sodium chloride solution, determined the specific activity of the resulting preparation, which is then poured into ampoules and lyophilized;
f) carry out preliminary preparation of the test serum for radioactive waste, for this they take serum in the amount of 0.1 ml, dilute in 0.9 ml of 0.9% sodium chloride solution, after which it is heated for 30 minutes in a water bath at a temperature of 56 ° C and left at 20 ° C for 10 min, after which conduct research in the radioactive waste;
g) carry out a volumetric agglomeration reaction in tablets by adding 50 μl of 1% normal rabbit serum, 50 μl of suspension of the test culture and 25 μl of diagnosticum to the wells, followed by stirring for 1 minute and contacting at a temperature of 20 ° C for 2.5 hours.