RU2605621C1 - METHOD FOR PREPARING A DIAGNOSTICUM FOR QUANTITATIVE DETERMINING THERAPEUTIC RECOMBINANT α-INTERFERON IN BLOOD SERUM OF PATIENTS WITH VIRAL INFECTIONS - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immunology, and can be used for preparing diagnosticum for determining concentration of therapeutic recombinant αinterferon in blood serum of patients with viral infections. For this purpose method comprises obtaining experimental hyperimmune rabbit serum by immunizing rabbits with weight of 2.5 to 3 kg using the preparation "Altevir" in a dose of 100 mcg according to protein in an inguinal lymph node, 3 injections every 14 days, then in 14 days after the last injection of serum is being prepared from blood with subsequent determining an antibody titre; immunoglobulins are being selected from rabbit serums, for this purpose, 4 ml of the produced serum is being mixed with 2 ml of buffered saline, placed in centrifuge cups, put on ice, and serum is being poured into them at temperature of 0 °C, then buffered saline is being added, stired, methanol water is being poured, lowering the temperature to -5 °C, then, the cup with a mixture is being put in the refrigerator for 30-40 minutes, and then centrifuged at 2,000 rpm for 20 minutes at zero temperature, produced supernatant is being drained, and the precipitate is being suspended in ½ of the initial volume of the serum, amount of protein is being determined by the Lowry method, obtaining sensitin; polymer carrier is being prepared, using anionic monomer polymerisation - acrylic aldehyde in aqueous alkaline medium to produce monodisperse spherical particles with diameter of 1.2±0.1 mcm, which are being stained with safranine, then the carrier is being washed with distilled water at 100 °C and treated with tannin; sensitin is being immobilized on the carrier, for this purpose, 100 mg of microsphere suspension is being placed in the centrifugal cup, suspended in 5 ml of the buffered saline and centrifuged at 3,000 rpm for 10 minutes at 20 °C, washed carrier is suspended in 2 ml of buffered saline and while constant stirring is being connected with sensitin in amount of 1.2 mg of protein, left for contact for 2 hours at room temperature, further immobilization is bing performed at 4-5 °C for 16-18 hours; free aldehyde groups are being blocked by adding 2 ml of 0.5 % gelatose solution to the suspension of the carrier with sensitin in buffered saline at pH equal to 7.1, left for 2 hours while stirring by a mixer at room temperature; obtained diagnosticum is being washed three times with buffered saline solution at pH equal to 7.1, preliminary centrifuging at 3,000 rpm for 10 minutes, final sediment is being suspended in 8 ml of 0.1 % gelatose solution on buffered saline at pH equal to 7.1; preparation sensitivity is being determined, for this purpose, 50 mcl of normal rabbit serum are being introduced in the board holes, then, 50 mcl "Altevir" are introduced with protein content of 60 mcl/ml in the first hole of the first row and twice titrated to 22-th hole, inclusive, 50 mcl of fluid are being removed from the last 22-th hole, 23-rd and 24-th holes are being controlled by spontaneous diagnosticum agglutination, 25 mcl of diagnosticum suspension are being introduced in all holes of two rows in 2.5 hours of incubation at room temperature results of reaction are being visually analyzed, latter hole, namely 19-th, which shows a positive result - pink agglomerate, dilution of which is equal to 23.5 protein pg/mL, that corresponds to the sensitivity of the diagnostic preparation, and liquid antigen preparation activity and specificity are being checked in a reaction of volumetric agglomeration (RVA); diagnosticum lyophilizing is performed by suspending preparation in 20 ml of 3 % gelatose-saccharose medium, then the suspension is being poured into the ampoule of 1 ml with subsequent drying at room temperature for a day.

EFFECT: use of this diagnosticum enables to determine the concentration of therapeutic recombinant αinterferon in blood serum of patients with viral infections, that enables assessing the clinical effectiveness.

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Description

The present invention relates to the field of biotechnology and medical virology and can be used in laboratory clinical practice for monitoring interferon therapy with recombinant α-interferons in the dynamics of treatment in order to determine the optimal effective dose and duration of therapy.

Currently, the main method of treatment of many viral diseases is the standard complex antiviral therapy with recombinant α-interferons from various manufacturers. The choice of dose of therapeutic drugs and the duration of the prescribed course of treatment are empirical. Recombinant α-interferon preparations are very expensive, treatment for a long time - six or more months, is accompanied by severe side effects, while not always being effective. Therefore, to select an individual effective dose and duration of interferon therapy, it is necessary to determine the concentration of α-interferon used in the patient's serum at various stages of treatment.

To date, in medical practice there are no methods and preparations for determining the concentration of therapeutic recombinant α-interferons in the sera of patients. Usually, the concentration of intrinsic endogenous human α-interferon produced by immunocompetent cells - leukocytes is determined. For this, an enzyme-linked immunosorbent assay using enzyme-linked immunosorbent assay systems of both foreign and domestic production.

Known test systems "Human IFN-alfa Platinum ELISA" (affimetrix eBIOSCIENCE, USA, "Human IFN alpha ELISA Kit (Multi-Subtype)" (Thermo Scientific, USA), "VeriKine Human Interferon alpha ELISA Kit" (eBIOSCIENCE, USA) However, these test systems are expensive and not always available, in addition, most diagnostic rooms are designed for research purposes and cannot be used for diagnostic purposes.

The prototype is a test system for the quantitative determination of intrinsic human α-interferon "A set of reagents for the enzyme immunoassay for determining the concentration of alpha-interferon" Alpha-interferon-IFA-Best "" (CJSC Vector-Best, Russia). The essence of the method is the use of a “sandwich” enzyme-linked immunosorbent assay, where specific components of the system are monoclonal antibodies to leukocyte α-interferon, adsorbed on the surface of the wells of a polystyrene tablet, conjugate of monoclonal antibodies with horseradish peroxidase and calibration samples containing leukocyte α-interferon. The concentration of α-interferon in the studied sera is determined using the calibration curve, based on the concentration and optical density of the calibration samples.

A disadvantage of the known test system is that it is able to determine its own endogenous α-interferon and cannot determine the amount of therapeutic recombinant α-interferon in the blood serum of patients, which is reflected in the selection of effective doses in clinical practice.

The technical task of the invention is the creation of a diagnostic drug capable of detecting and determining the amount of therapeutic recombinant α-interferon in the blood serum of patients with viral infections during treatment.

The problem is achieved in that the method of obtaining a diagnosticum for determining therapeutic recombinant α-interferon in the blood serum of patients with viral infections includes the following stages:

a) experimental hyperimmune rabbit serums are obtained by immunization of rabbits weighing from 2.5 to 3 kg with the introduction of the drug "Altevir" at a dose of 100 μg of protein in the inguinal lymph node in the amount of 3 injections with an interval of 14 days, then after 14 days after the last injection, rabbits are bled, and serum is prepared from the blood, followed by determination of antibody titer;

от исходного объема сыворотки, определяют количество белка по методу Лоури, получая в результате сенситин;b) isolate immunoglobulins from rabbit serums, for this purpose, physiological saline is prepared, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate at pH 7.2, then an aqueous solution of methanol is prepared, for this 6 ml of methanol is mixed with 8 ml of distilled water, the mixture is cooled then the obtained serum in the amount of 4 ml is combined with 2 ml of buffered saline solution, placed in centrifuge glasses, put on ice and the serum is poured into them at a temperature of 0 ° C, then a buffer is added physiological saline solution, mix, pour methanol water, while lowering the temperature to -5 ° C, after a glass of the mixture is placed in the refrigerator for 30-40 minutes, and then centrifuged at 2000 rpm for 20 minutes at zero temperature, obtained the supernatant is drained and the sediment is suspended in

from the initial volume of serum, the amount of protein is determined by the Lowry method, resulting in sensitin;

c) a polymer carrier is prepared using the anionic polymerization of a monomer — acrylic aldehyde in an aqueous alkaline medium to obtain monodisperse spherical particles with a diameter of 1.2 ± 0.1 microns, which are stained with safranin, after which the carrier is washed with distilled water at 100 ° C and treated with tannin;

g) carry out the immobilization of sensitin on a carrier, for this a suspension of microspheres in an amount of 100 mg is placed in a centrifuge cup, suspended in 5 ml of buffered saline and centrifuged at 3000 rpm for 10 min at a temperature of 20 ° C, the carrier washed in this way is suspended in 2 ml of buffered saline and with constant stirring on a magnetic stirrer, connect with sensitin in an amount of 1.2 mg of protein, leave for contact for 2 hours at room temperature, while further immobi ization is conducted at a temperature of 4-5 ° C for 16-18 hours;

e) block free aldehyde groups by adding to a suspension of a carrier with sensitin 2 ml of a 0.5% gelatose solution in buffered saline at pH 7.1, leave for 2 hours while stirring on an electric stirrer at room temperature;

f) the diagnosticum obtained is washed three times with buffered saline at pH 7.1, previously centrifuged at 3000 rpm for 10 minutes, the final precipitate is suspended in 8 ml of a 0.1% gelatine solution in buffered saline at pH 7.1;

g) determine the sensitivity of the drug using α-interferon Altevir, for this, 50 μl of normal rabbit serum is added to the wells of the tablet, then 50 μl of Altevir with a protein content of 60 μl / ml is added to the first well of the first row and titrated twice to 22 wells, including 50 μl of fluid removed from the last 22nd hole, the 23rd and 24th wells serve as a control for spontaneous agglutination of the diagnosticum, while 25 μl of the diagnosticum suspension is added to all wells of two rows, after 2.5 hours incubation at room temperature the reaction results visually, with the last hole, namely the 19th, giving a positive result, a pink agglomerate, the dilution of which is 23.5 pg of protein / ml, which corresponds to the sensitivity of the diagnostic drug, and the activity and specificity of the liquid antigenic drug is checked in the agglomeration reaction bulk (RW);

h) lyophilization of the diagnosticum is carried out by suspending the preparation in 20 ml of a 3% gelatin-sucrose medium, then the suspension is poured into 1 ml ampoules, followed by drying at room temperature for one day.

In this case, the volumetric agglomeration reaction is carried out in tablets by adding 50 μl of 1% normal rabbit serum to the wells, then 50 μl of the test serum is added to the first well of each row and titrated twice to the end of the row, 50 μl is removed from the last well and the diagnosticum is 25 μl is added to each well of the row and left at room temperature, the results of the RAW reaction are taken into account after 2-2.5 hours, a positive result gives a uniform agglomerate lining the bottom of the well, with clearly defined edges, and in negative cases x and the negative control forms a ring or spot in the center of the wells.

In addition, the concentration of therapeutic recombinant α-interferon in the blood serum of patients is determined; for this, the agglomeration reaction is performed according to the RAO method, but the patient’s serum is titrated twice in the wells of the dilution fluid NKS tablet, then 25 μl of the immunoglobulin diagnostic drug is added, with the last well with a positive reaction fixes the value of the last dilution of the test serum, therefore, the concentration of the drug α-interferon is calculated by the formula

K = P · C

where K is the desired concentration of the drug α-interferon;

P is the last dilution of the test serum in which a positive result was recorded;

C is the sensitivity of the diagnosticum in pg / ml.

The method is as follows.

At the first stage, a diagnostic preparation is prepared according to the following technology:

a) Experimental hyperimmune rabbit sera are obtained using the pharmacological preparation Altevir (3 million ME).

To select immunizing doses, determine the protein concentration according to Lowry. Then, male Californian rabbits weighing 2.5 to 3 kg are selected for immunization. Immunization is carried out by introducing drugs at a dose of 100 μg of protein in the inguinal lymph node - 3 injections with an interval of 14 days. 14 days after the last injection, total rabbits are bled, followed by serum from this blood. After determining the titer of antibodies, the resulting hyperimmune serum is Packed and stored frozen at a temperature of 30 ° C.

b) Isolate immunoglobulins from rabbit hyperimmune sera using the methanol method. Before isolation of the immunoglobulins, a physiological saline solution, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate, is preliminarily prepared. Then an aqueous solution of methanol is prepared, for this 6 ml of methanol is mixed with 8 ml of distilled water. The resulting mixture was cooled.

от исходного объема сыворотки, после чего определяют количество белка по методу Лоури, получая таким образом сенситин.4 ml rabbit serum is combined with 2 ml of buffered saline solution, placed in centrifuge glasses and cooled on ice. Glasses are placed on ice and serum is poured into them at a temperature of 0 ° C, then buffered saline is added and mixed well. After that, shaking the glass, add methanol water, gradually lowering the temperature to -5 ° C. A glass with the mixture is placed in the refrigerator for 30-40 minutes. After that, centrifuged for 20 minutes in the cold at 0 ° C 2000 rpm. The supernatant is drained and the sediment is suspended in

from the initial volume of serum, after which the amount of protein is determined by the Lowry method, thereby obtaining sensitin.

c) Prepare a polymer carrier by the method of anionic polymerization of a monomer - acrylic aldehyde in an aqueous alkaline medium. The obtained monodisperse particles of a spherical shape with a diameter of 1.2 ± 0.1 microns are stained with safranin. Then the carrier is washed with distilled water at 100 ° C and treated with tannin to ensure protein binding.

g) Immobilization of sensitin on the carrier.

A suspension of microspheres in an amount of 100 mg is placed in a centrifuge cup, suspended in 5 ml of a buffered saline solution and centrifuged at 3000 rpm for 10 min at a temperature of 20 ° C, so the washed carrier is suspended in 2 ml of a buffered saline solution with constant stirring on a magnetic stirrer Magnetic Stirer MSH 300 (BioSan, Latvia) is combined with sensitin, the latter in the amount of 1.2 mg of protein, left for contact for 2 hours at room temperature. Then, immobilization is carried out at a temperature of 4-5 ° C for 16-18 hours.

d) Carry out the blocking of free aldehyde groups. To a suspension of a carrier with sensitin add 2 ml of a 0.5% gelatose solution in buffered saline at pH 7.1 and leave it under constant stirring on a magnetic stirrer for 2 hours at room temperature.

As a result of stabilization of gelatosis, it allows blocking reaction groups on particles that did not contact a specific immunoglobulin solution during immobilization.

e) Washing the diagnosticum. After incubation with a 0.5% gelatine solution, the precipitate is centrifuged at 3000 rpm for 10 minutes and washed three times with buffered saline at pH 7.1. The final precipitate is suspended in 8 ml of a 0.1% gelatine solution in buffered saline at pH 7.1. The activity and specificity of a liquid antigenic diagnostic drug is checked in the bulk agglomeration reaction (RAW).

g) Determining the sensitivity of the drug using the commercial therapeutic α-interferon Altevir.

50 μl of normal rabbit serum are added to the two rows (A and B) of the wells of the 96-well immunological reaction plate, then 50 μl of Altevir with a protein content of 60 μl / ml are added to the first well of the first row (A) and titrated twice 22 holes inclusive. 50 μl of liquid was removed from the last well. Thus, in each subsequent well, the protein content was half as much as in the previous one. The last two holes (23rd and 24th) served as a control for spontaneous agglutination of the diagnosticum. 25 μl of a suspension of a diagnostic preparation was added to all wells of two rows. The result of the reaction was taken into account visually after 2.5 hours of incubation at room temperature. The last well in which a positive result was recorded — a pink agglomerate that evenly lines the bottom of the well — was the 19th, dilution of which corresponded to 23.5 pg of protein / ml. The control wells showed a negative result - a point in the center of the hole.

Thus, the sensitivity of the diagnostic drug is 23.5 pg / ml.

h) Lyophilization of the diagnosticum.

The final step in obtaining a diagnosticum is lyophilization, which is carried out by suspending the drug in 20 ml of a 3% gelatin-sucrose medium, then the suspension is poured into 1 ml ampoules, followed by drying at room temperature.

The contents of the ampoules are frozen by alcohol cooling at a temperature of -45 ± 5 ° C for 10 ± 2 minutes. The frozen diagnosticum is placed in a low-temperature cabinet for deep freezing at -50 ° C and kept for 17 hours, after which the ampoules with frozen diagnosticum are placed in the chamber of the vacuum dryer.

The second stage of the method consists in the methodology for setting the reaction of RAO and determining the concentration of recombinant α-interferon in medical preparations.

Before RAO, normal rabbit serum of NKS and the studied preparations of recombinant α-interferon are diluted 1:10 with buffered saline (pH 7.1). The sera of patients with viral hepatitis receiving interferon therapy are used whole.

For staging RAW, 50 μl of NKS are added to each well of the corresponding row of a 96-well plate for immunological reactions. Then, 50 μl of the test drug (or serum) is added to the first well of each row and titrated twice to the end of the row. From the last well, 50 μl of liquid is removed. Diagnosticum in working dilution is added in a volume of 25 μl to each well of the row and left at room temperature.

To test the diagnosticum for spontaneous agglutination, 50 μl of normal rabbit serum and 25 μl of diluted diagnosticum are added to any two wells.

The reaction results are taken into account visually after 2-2.5 hours.

For a positive result, a colored pink agglomerate that lines the entire bottom of the well evenly, or an agglomerate that lines the bottom of the well with clearly defined edges, is taken. In negative cases and in the negative control, a compact ring or “dot” is formed in the center of the well.

Determination of the concentration of recombinant α-interferon in commercial preparations.

Example 1

In 12 wells of one row of a 96-well plate for immunological reactions, 50 μl of diluent liquid — NKS — was added.

50 μl of a commercial preparation of recombinant α-interferon (Pegintron) was added to the first well of the series and titrated twice to 10 wells, the last two wells served as a negative control. The result of the reaction was taken into account visually after 2.5 hours of incubation at room temperature. The last hole, in which a positive result was recorded - a pink agglomerate lining the entire bottom of the hole, corresponded to a dilution of 1: 2560. The concentration of interferon in this drug is:

K = P · C, where P = 2560, C = 23.5 pg / ml,

K = 2560 · 23.5 = 60.1 ng / ml,

where K is the desired concentration of the drug in the serum of the patient;

2560 - the value of the last dilution of serum, in which a positive result was recorded;

23.5 pg / ml - the sensitivity of the diagnostic drug.

The result obtained corresponds to the content of the interferon protein in the Pegintron preparation stated in the instructions attached to the preparation.

Example 2

Differs from example 1 in that they investigate the commercial preparation of the recombinant α-interferon "Pegasys".

The volumetric agglomeration reaction is set up analogously to Example 1, but in the wells of the plate with the diluting liquid NKS, the recombinant Pegasys interferon is twice titrated and then 25 μl of the immunoglobulin diagnostic preparation is added. The last well, in which a positive reaction was recorded, corresponded to a dilution of 1: 640.

The concentration of recombinant interferon is:

K = 640 · 23.5 = 15 ng / ml.

where K is the desired concentration of the drug in the serum of the patient;

640 - the value of the last dilution of serum, in which a positive result was recorded;

23.5 pg / ml - the sensitivity of the diagnostic drug.

Example 3

Differs from examples 1, 2 in that they study a commercial preparation of endogenous leukocyte α-interferon person.

The volumetric agglomeration reaction is set up analogously to examples 1, 2, but in the wells of the plate, endogenous human α-interferon is twice titrated and 25 μl of diagnostic immunoglobulin preparation is added.

After 2.5 hours of incubation at room temperature, a negative reaction was recorded in all wells — a point in the center of the well.

These examples show that the proposed diagnostic drug makes it possible to determine the concentration of various commercial preparations of recombinant α-interferon in the bulk agglomeration reaction and does not react with intrinsic human endogenous α-interferon.

Example 4

It differs from other examples in that the test substance is the serum of a patient who received interferon therapy for a month with the preparation of the recombinant α-interferon Altevir.

In the wells of the first row of a 96-well plate for immunological reactions, twice-integral serum of a patient who received treatment with Altevir for a month was triturated. Then, 25 μl of immunoglobulin diagnostic preparation was added to the serum. After 2.5 hours of incubation at room temperature, the results of the reaction were visually taken into account. The last hole, in which a positive result was recorded, corresponded to a dilution of 1: 8.

Determination of the concentration of the drug in serum:

K = P · C, where P = 8, C = 23.5 pg / ml,

K = 8 · 23.5 = 188 pg / ml,

where K is the desired concentration of the drug in the serum of the patient;

8 - value of the last dilution of serum in which a positive result was recorded;

23.5 pg / ml - the sensitivity of the diagnostic drug.

Example 5

It differs from Example 4 in that the test substance is the serum of a patient who received interferon therapy with the recombinant Pegasys α-interferon drug for 3 months.

The agglomeration reaction volumetric is set similarly to the previous examples. In the wells of one row of a 96-well plate for immunological reactions, the whole serum of a patient who was treated with Pegasys recombinant α-interferon for 2 months was titrated twice. 25 μl of suspension of a diagnostic immunoglobulin preparation was added. After 2.5 hours of incubation at room temperature, the results of the reaction were visually taken into account. The last well, in which a positive result was recorded, corresponded to a dilution of 1: 4. The concentration of the drug in the serum of the patient will be:

K = 4 · 23.5 pg / ml = 94 pg / ml,

where K is the desired concentration of the drug in the serum of the patient;

4 - value of the last dilution of serum in which a positive result was recorded;

23.5 pg / ml - the sensitivity of the diagnostic drug.

Example 6

It differs from the previous ones in that the test substance is the serum of a patient who has been treated with the therapeutic drug of the recombinant α-interferon “Roferon” for 6 months.

In the wells of one row of a 96-well polystyrene tablet for immunological reactions, the whole serum of a patient who received Roferon interferon therapy for 6 months was titrated twice. Then 25 μl of a polymer immunoglobulin diagnostic preparation was added to these wells. The result of the reaction was taken into account after 2.5 hours of incubation at room temperature. The last hole, in which a positive result was recorded - a pink agglomerate, evenly lining the bottom of the hole, corresponded to a dilution of 1: 2.

The concentration of therapeutic interferon in the patient's serum in this case will be:

K = 2 · 23.5 = 47 pg / ml,

where K is the desired concentration of the drug in the serum of the patient;

4 - value of the last dilution of serum in which a positive result was recorded;

23.5 pg / ml - the sensitivity of the diagnostic drug.

Example 7

It differs from previous examples in that the test substance is the serum of a patient who has not received treatment with recombinant α-interferon preparations, but has a high level of intrinsic leukocyte α-interferon.

In the wells of the polystyrene tablet, the whole serum of the patient was titrated twice. Then, 25 μl of polymer immunoglobulin diagnosticum was added and after 2.5 hours of incubation at room temperature, the results were visually taken into account.

A negative result was recorded in all wells — a point in the center of the hole.

This suggests that in the serum of this patient was not found therapeutic drug α-interferon.

Thus, it can be seen from the examples that the Rostov-on-Don Anti-Plague Institute developed by the Rospotrebnadzor specialists of the Rospotrebnadzor immunoglobulin α-interferon diagnosticum based on antibodies to one recombinant α-interferon Altevir can also correctly determine other therapeutic preparations of recombinant α-interferon nor does it give a reaction with endogenous human leukocyte α-interferon. The drug has high sensitivity and specificity.

Using the proposed diagnosticum allows using it to determine the concentration of therapeutic recombinant α-interferon in the sera of patients at different stages of treatment differentially from their own endogenous leukocyte α-interferon. This gives the doctor the opportunity to analyze the effectiveness of the use of the drug, as well as to choose an individual dose and treatment regimen.

The proposed method is single-stage, easy to set up and account for the results, does not require additional specific reagents, expensive equipment and specially trained personnel.

Claims (3)

1. A method of obtaining a diagnosticum for determining therapeutic recombinant α-interferon in the blood serum of patients with viral infections, comprising the following stages:
a) experimental hyperimmune rabbit serums are obtained by immunization of rabbits weighing from 2.5 to 3 kg with the introduction of the drug "Altevir" at a dose of 100 μg of protein in the inguinal lymph node in the amount of 3 injections with an interval of 14 days, then after 14 days after the last injection, rabbits are bled, and serum is prepared from the blood, followed by determination of antibody titer;
b) isolate immunoglobulins from rabbit serums, for this purpose, physiological saline is prepared, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate at pH 7.2, then an aqueous solution of methanol is prepared, for this 6 ml of methanol is mixed with 8 ml of distilled water, the mixture is cooled then the obtained serum in the amount of 4 ml is combined with 2 ml of buffered saline solution, placed in centrifuge glasses, put on ice and the serum is poured into them at a temperature of 0 ° C, then a buffer is added The physiological saline solution is stirred, methanol water is poured, while lowering the temperature to -5 ° C, after the beaker with the mixture is placed in the refrigerator for 30-40 minutes, and then centrifuged at 2000 rpm for 20 minutes at zero temperature, obtained the supernatant is drained and the sediment is suspended in

from the initial volume of serum, the amount of protein is determined by the Lowry method, resulting in sensitin;
c) a polymer carrier is prepared using the method of anionic polymerization of a monomer — acrylic aldehyde in an aqueous alkaline medium to obtain monodisperse spherical particles with a diameter of 1.2 ± 0.1 μm, which are stained with safranin, after which the carrier is washed with distilled water at 100 ° C and treated with tannin;
g) carry out the immobilization of sensitin on a carrier, for this a suspension of microspheres in an amount of 100 mg is placed in a centrifuge cup, suspended in 5 ml of buffered saline and centrifuged at 3000 rpm for 10 min at a temperature of 20 ° C, the carrier washed in this way is suspended in 2 ml of buffered saline and with constant stirring on a magnetic stirrer, connect with sensitin in an amount of 1.2 mg of protein, leave for contact for 2 hours at room temperature, while further immobilization iju carried out at a temperature of 4-5 ° C for 16-18 hours;
e) block free aldehyde groups by adding to the suspension of the carrier with sensitin 2 ml of a 0.5% gelatose solution in buffered saline at pH 7.1, leave for 2 hours while stirring on an electric stirrer at room temperature;
f) the diagnosticum obtained is washed three times with buffered saline at pH 7.1, previously centrifuged at 3000 rpm for 10 minutes, the final precipitate is suspended in 8 ml of a 0.1% gelatine solution in buffered saline at pH 7.1;
g) determine the sensitivity of the drug using α-interferon Altevir, for this, 50 μl of normal rabbit serum is added to the wells of the tablet, then 50 μl of Altevir with a protein content of 60 μl / ml is added to the first well of the first row and titrated twice to 22 wells, including 50 μl of fluid removed from the last 22nd hole, the 23rd and 24th wells serve as a control for spontaneous agglutination of the diagnosticum, while 25 μl of the diagnosticum suspension is added to all wells of two rows, after 2.5 h incubation at room temperature the reaction ultates visually, while the last well, namely the 19th, giving a positive result is a pink agglomerate, the dilution of which is 23.5 pg of protein / ml, which corresponds to the sensitivity of the diagnostic drug, and the activity and specificity of the liquid antigenic drug is checked in the agglomeration reaction bulk (RW);
h) lyophilization of the diagnosticum is carried out by suspending the preparation in 20 ml of a 3% gelatin-sucrose medium, then the suspension is poured into 1 ml ampoules, followed by drying at room temperature for one day. 2. The method according to p. 1, characterized in that the volumetric agglomeration reaction is carried out in tablets by adding 50 μl of 1% normal rabbit serum to the wells, then 50 μl of the test serum is added to the first well of each row and titrated twice to the end of the row, from the last 50 μl wells are removed, and a diagnosticum in a volume of 25 μl is added to each well of the row and left at room temperature, the results of the RAW reaction are taken into account after 2-2.5 hours, a positive result gives a uniform agglomerate lining the bottom of the hole, with clearly defined edges , and in negative cases and negative control, a ring or a dot is formed in the center of the hole. 3. The method according to p. 2, characterized in that the concentration of therapeutic recombinant α-interferon in the blood serum of the patients is determined; for this, the agglomeration reaction is performed according to the RAO method, but the patient’s serum is titrated twice in the wells of the dilution fluid NSC, then 25 μl of an immunoglobulin diagnostic drug, while the last well with a positive reaction fixes the value of the last dilution of the test serum, so the concentration of the α-interferon drug is calculated by the formula
K = P · C
where K is the desired concentration of the drug α-interferon;
P is the last dilution of the test serum in which a positive result was recorded;
C is the sensitivity of the diagnosticum in pg / ml.