WO2012128658A1

WO2012128658A1 - Method for producing a panel of sera with hbsag subtypes ad and ay for quality control in hepatitis в diagnostics

Info

Publication number: WO2012128658A1
Application number: PCT/RU2011/000317
Authority: WO
Inventors: Александр Николаевич KAHEB; Талгат Сальманович БАКИРОВ; Наталья Сангиреевна ЧЕРЕПАНОВА; Ирина Викторовна ЮДИНА; Михаил Викторович ЛОСЕВ; Ольга Николаевна НАДТОЧИЙ
Original assignee: Федеральное Бюджетное Учреждение Науки "Государственный Научный Центр Вирусологии И Биотехнологии "Beктop"
Priority date: 2011-03-22
Filing date: 2011-05-10
Publication date: 2012-09-27
Also published as: RU2463610C1

Abstract

The invention relates to bioengineering. The method for producing a panel of sera with certified low concentrations of the AD and ΑΥ subtypes of the HBs antigen, said panel being intended for quality control in hepatitis B diagnostics, comprises sampling donor sera which do not contain antibodies specific for primary markers of viral infections on the basis of data from the determination of the effect of HBsAg suppression and immunoenzyme analysis (IEA), producing dilute solutions (DS) on the basis of the sampled sera, certifying monopreparations of HBsAg subtypes AD and AY using the IEA procedure, titrating samples of said monopreparations in DS in a range of from 0.01 to 0.5 IU/ml in relation to international standards and performing statistical analysis of said samples with subsequent determination of the quantity of monopreparations of HBsAg of each subtype which is necessary for producing the required final antigen concentration, and producing a panel of sera by means of diluting monopreparations of HBsAg subtypes AD and AY in DS to final concentrations of 0.1, 0.05 and 0.01 IU/ml in accordance with the data from calibration curves.

Description

METHOD FOR MAKING A SERUM PANEL WITH HBSAG AD AND AY SUBTYPES FOR QUALITY CONTROL OF HEPATITIS DIAGNOSTICS IN

The invention relates to a manufacturing technology for reference panels of serums containing antigens of the most dangerous viral infections, in particular hepatitis B virus (HBV) and can be used in medicine and biotechnology.

Blood samples of donors in Russia must be examined for the presence of HIV 1 and 2 markers, hepatitis B and C, and syphilis. Approximately 1% of all samples are sent for re-confirmation to control laboratories. However, the effectiveness of the confirmation of the results depends on the qualifications of the clinical diagnostic laboratory staff, the quality of the test systems and the availability of standard drugs for the analyzed infectious markers in the control laboratories (VQC Pelispy Multi - Marker, run control. Central Laboratory of Red Cross, Netherlands, 1998) [1 ].

When using highly sensitive HBsAg screening tests, hepatitis B carriers with a very low concentration of HBsAg can be detected, in particular among individuals who are carriers of anti-HBc antibodies.

The use of different HBsAg subtypes in reference drugs in everyday work will expand the range of diagnosable markers and use these materials to detect HBsAg in blood products intended for transfusion or processing.

According to experts in the field of molecular biology, the number and types of mutations in biomolecules are largely determined by regional characteristics: climate, food mix, and the representativeness of the chemical elements Fe, Co, Cr, Ni, and Cu.

Improving the quality of diagnosis of HBV is in parallel with increasing the sensitivity of diagnostic test systems. As 5 of improving the quality of immunological preparations, the sensitivity of diagnostic systems for the detection of HBsAg increased from 0.1 IU / ml to 0.05 IU / ml. Now, practical health care has been given the task of diagnosing HBsAg in blood products at a level of 0.01 IU / ml to monitor the qualifications of employees of the control diagnostic laboratories (CDL), the quality of test systems and hepatitis B vaccines

Currently, commercial test systems with a declared sensitivity of 0.01 IU / ml have appeared (Diagnostic Systems CJSC, Vector Best CJSC (Russia), etc.). However, to confirm this sensitivity and the 15 introduction of these tests in clinics and blood transfusion stations, certified standard drugs are needed. The task is extremely difficult because It is necessary to validate samples with a concentration of 0.01 IU / ml using test systems with a sensitivity of 0.01 IU / ml.

20 Analogous methods for producing panels are known from the literature, for example, a method for preparing serum panels for preparing reference panels (M. Ferguson, Holmes N. and Sands D. National Institute for Biological Standards and Control / UK Blood Transfusion Service working standards for HBsAg, anti-HCV and anti-HIV-1 ('go / no-go' controls) // Blackwell

25 Science Ltd. Vox Sanguinis (2001) 80, 205-210). [one]. These panels are samples of reference drugs AY and AD of the HBs Ag subtypes, diluted in a pool of donor sera that do not contain markers of dangerous viral infections, at a concentration of up to 0.1 ng / ml. The method involves the preparation of standard sera containing HBs Ag and having different concentrations of this protein.

The closest technical solution (prototype) is a method for preparing serum panels HBV DNA Genotype Performance Panel, developed in the laboratory of Boston Biomedical Inc. (USA) - BBI. This panel includes 10 samples with A-D genotypes (BBI Product catalog

35 2003/2004 Boston Biomedica, Inc., 2004, p. 14) - www.bbii.com [2]. Samples 5 panels represent HBV DNA serum diluted in a pool of donor sera that do not contain markers of dangerous viral infections.

However, these panels do not meet the needs of practical healthcare, as Qualitatively certified by the concentration of DNA of the hepatitis B virus, and not by the concentration of HBsAg. In addition, the serums contained in these panels are presented in liquid form and therefore must be stored in a frozen state. Distribution and storage of BBI panels require strict adherence to low temperature conditions (Damen, M., Cuypers, N.T.M., Zaaijer, HL, Reesink, HW, Schaasberg, WP, Gerlich, WH, Niesters, HGM, Lelie PN (1996) International collaborative

15 study on the second EUROHEP HCV-RNA reference panel. J. Virol. Methods 58, 175-185) [3].

The technical result of the claimed invention is the creation of such a method of manufacturing a panel of sera to control the quality of diagnosis of hepatitis B, which provides samples

20 of the indicated serum panel with a certified extremely low concentration of AD and AY subtypes of HBsAg and preservation of the specific characteristics of these samples for a long time.

The specified technical result is achieved by the fact that a method of manufacturing a panel of serums with extremely low concentrations

25 HBsAg AD and AY subtypes for quality control of the diagnosis of hepatitis B includes testing of donor sera by polymerase chain reaction for the presence of hepatitis B virus DNA, anti-HIV, anti-HCV and anti-HBs antibodies of non-specific binding of HBsAg, selection of donor serums that do not contain specific antibodies to the main markers of viral infections by the values of the effect of suppressing HBsAg (spike / recovery test) and having a negative result in an enzyme-linked immunosorbent assay, the manufacture of dilution solutions based on selected sera, certification monoprep subtypes of HBsAg AD and AY subtypes by enzyme-linked immunosorbent assay, titration in a diluting solution of samples

35 of these single drugs HBsAg AD and AY subtypes from 0.01 to 0.5 IU / ml, construction of calibration curves for titration in a diluting solution from 0.01 to 0.5 IU / ml AD and AY of HBsAg subtypes relative to international standards and their statistical analysis, according to which the number of HBsAg monopreparations of each subtype required for introduction into the diluting solution is determined to obtain their final concentrations in accordance with the data of the calibration curves and the manufacture of a panel of serums with extremely low concentrations of HBsAg AD and AY subtypes by dilution of single drugs HBsAg AD and AY subtypes with a dilution solution d To obtain their final concentrations of 0.1 IU / ml; 0.05 IU / ml and 0.01 IU / ml, respectively, in accordance with the data of the calibration curves.

As monopreparations of HBsAg, plasma 100% antigens containing AD and AY subtypes of HBsAg are used.

As a stabilizing additive used proline in an amount of 200 - 250 mm and benzoic acid in an amount of 0.05 - 0.08 wt.%.

The prepared samples of the serum panel with extremely low concentrations of HBsAg AD and AY subtypes are lyophilized to a residual moisture content of less than 2.0 mass. %

Samples of the serum panel with extremely low concentrations of HBsAg AD and AY subtypes are examined using a thermal degradation test to determine their shelf life.

Alignment of standard samples to the required concentration of HBsAg is carried out using dilution factors of the initial AY & AD subtypes calculated according to the equations of the calibration curves of AY & AD of HBsAg preparations in a diluting stabilized solution to the required concentration of HBsAg (0.1 IU / ml, 0.05 IU / ml and 0.01 IU / ml). As starting preparations of HBs Ag, preparations with a content of HBsAg of up to 100% for the desired protein are used.

The following is a manufacturing chart for a serum panel with extremely low concentrations of HBsAg AY & AD subtypes. 1. Testing of samples of donor sera using the laboratory standard HBsAg to study the amount of suppression of the analytical signal by the components of the tested plasma. A dilution solution (PP) is prepared from donor serums with a minimum level of suppression for the manufacture of samples of a panel of serums with extremely low concentrations (PN) of HBsAg.

2. Titration of the HBsAg AY subtype preparation (manufactured by NPK Drug, Nizhny Novgorod) and the AD subtype preparation (co-generation GeneLab Diagnostics, Singapore) in the prepared PP on one tablet together with international standards - 2 ^nd International Standard ADW2 code 00 / 588 (NIBSC, England) and P. Erlich Institute Standard (Germany) - AD subtype in various test systems.

3. Statistical processing of the results of calibration titrations at a confidence level of 95% to calculate dilution factors of 100% of HBsAg preparations in stabilized PP to the required concentration.

4. Preparation of candidate PNA AY & AD subtypes of HBsAg.

5. Validation of candidates for PNA AY & AD subtypes in ELISA test systems with a declared sensitivity of 0.01 ME (in different laboratories) in parallel with the 2nd International Standard HBsAg (NIBSC, 33 IU / ml) and P. Ehrlich standard (100 IU / ml).

6. Preparation of a dry, stable form of PNA candidates by lyophilization.

7. Testing the accelerated inactivation of PNA candidates at elevated temperatures to determine the shelf life of the standard.

To determine the relationship between the value of the analytical signal of ELISA and the amount of HBs Ag in the dilution solution, it is necessary to use ELISA test systems, which in the range from 0.3 to 0.02 IU / ml maintain a linear relationship between the optical density and the concentration of HBs Ag in the solution. To solve the problem of certification of serum panel samples by the concentration of HBs Ag, the following actions should be carried out:

- to use for creating panel samples plasma samples containing various concentrated preparations of plasma or recombinant antigens with an HBs Ag content of at least 95% of the total protein that do not have an infectious hazard;

- use a stabilized pool of normal human blood serum as a diluting solution (PP), which does not contain markers of major infections and does not suppress the analytical signal from HBsAg.

Proposed Extreme Low Serum Panel Model

The HBsAg AD and AY subtypes include 8 samples: 2 samples of donor sera, 3 samples of AY subtype of HBsAg and 3 samples of AD subtype of HBsAg prepared by diluting samples in a stabilized pool of donor serums (PP) to a concentration of 0.1 IU / ml, 0.05 IU / ml and 0.01 IU / ml antigen.

Concentrated preparations of AD and AY subtypes of HBsAg, but not native serums, are used to make samples of the serum panel. In the process of concentration of HBsAg preparations, the sera are treated with trypsin, filtered through 0.45 μm and 0.22 μm filters, high-speed centrifugation and concentration. The resulting product, called "purified plasma HBs antigen," is certified for protein content and activity with anti-HBs antibodies. The content of the desired HBsAg protein in such preparations exceeds 95%.

The importance of using serum panels in clinical diagnostic laboratories is the correctness of the analysis results. Rezapkin G., Dragunsky E. and Chumakov K. '(2005) Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) // Biologicals 33, 17- 27 [4]. The main criterion for the correctness of the results of serological analysis is the certified value of the analytical signal (for example, the optical density of the OD in the ELISA) for each panel sample. Wherein A prerequisite for the use of reference HBsAg preparations is the constant level of this analytical signal during long-term storage.

Preservation of the specific properties of panel sera containing IgG antibodies by introducing a preservative - a bactericidal additive (merthiolate and 0.01% gentamicin and 0.2% gentamicin) is known from RF patent 2265028, IPC G01N33 / 96, publ. 11/27/2005 [5]. In the present invention, to stabilize the level of HBs Ag antigen, it is proposed to use a stabilizer comprising 200 - 250 mM proline (L-proline, 99%, for synthesis, catalog N ° 163646 1206) and 0.05 -0.08 wt.% Benzoic k- you. The introduction of proline is an important technique. Proline has a powerful disintegrating potential that prevents the process of association of HBsAg monoparticles. In addition, proline, as a primary amino acid, does not introduce foreign ingredients into solutions (Voshn R., Woodtli K., Bartschi M. Et al. L-proline reduces IgG dimmer content and enhances the stability of intravenous immunoglobulin solutions // Biologicals 38 (2010), 150-157) [6]. Benzoic acid is a classic antioxidant used to stabilize medications and foods, exhibits antimicrobial and antifungal effects, and inhibits mold, yeast and certain types of bacteria.

The low viscosity of the stabilizer solution for a standard preparation is a critical parameter for the detection of HBsAg in practice, because many manufacturers of test systems are trying to reduce analysis time. Under these conditions, a decrease in diffusion inhibition of the MCA + HBsAg interaction process is of great importance, due primarily to a decrease in the solution viscosity (Kanev AN, Bakirov TS, Yudina IV and Zaitsev BN Simulation of interaction of particles HBsAg with antibodies immobilized on a surface of the microplate well at ELISA // Biotechnology, 2005, 6, 74-82) [7]. Thus, the panel serum certification procedure includes three stages, the results of which are independent and allow reliable identification of the concentration of various subtypes of HBs Ag. Comparison of HBsAg PNA serum panels prepared in accordance with the claimed method with analog panels of the same purpose showed that a combination of essential features that is different from the prototype provides a new previously unknown property: the possibility of manufacturing HBsAg panels in a dry (lyophilic) form with an extremely low certified the concentration of the main subtypes of HBsAg, which allows us to evaluate the sensitivity of the test systems at the level of 0.01 IU / ml and specificity to various subtypes of HBsAg, to control the level of professional asterstva employees clinical diagnostic laboratories and control the quantity of hepatitis B vaccine

The invention is illustrated by the drawing in FIG. 1, which shows the weighted average AY & AD titration curves of HBsAg samples and the standard deviation curves Υ ± δΥ, where:

CCA - industry standard sample GISK them. L.A. Tarasevich

LS - laboratory standard of SSC VB Vector;

R.E. - standard of institute P. Erlich

Yep is the average value of Ln OD

SY - dov. interval

When processing titration curves, the values of OP * = OPEC -

Opp.

The total mean square error at the selected level is calculated (table 2) as the square root of the sum of the squares of the intra-group and inter-group errors. For example, for sample Ν ° 1, the in-group errors associated with titration errors in various laboratories are 4.25% (VB) and 9.22% (DS).

The intergroup spread between test systems for AD samples is (1.03-0.946) / 2 * 100% = 4.42% plus the maximum additional error spread between the average values and the values for the selected 5

level 7.2-4.2 = 4.33%. In total, the intergroup scatter at this level of OP is 8.75%. The total error (Δ) of determination at a fixed level of HBsAg concentration (0, 1 IU / ml) is

Δ = V4.25 ² + 9.22 ² + 8.75 ² = 13.4%

Example of a method for manufacturing a panel of serums with extremely low concentrations of HBsAg AY and AD subtypes Preparation of a dilution solution. As RR for the preparation of positive panel samples with a certain concentration of HBsAg, donor sera that do not contain markers of viral infections are used. Serum is prepared immediately after blood sampling. Whole blood is centrifuged at a speed of 2800-3200 * rpm at a temperature of 10 - 12 ° C for 15 minutes and the supernatant is decanted. Store until use at a temperature of + (2 - 4) ° C. To prepare the panel, samples of 20 amber-colored sera are taken, which are transparent in the light. A total of 9 high-quality sera were selected.

Control of donor sera intended for dilution of HBsAg preparations for total protein content (norm 6.5 - 8.5%).

After preparation, all sera are assayed for anti-HBs 25 antibodies and viral contamination.

At the next stage, they put a test to suppress the analytical signal by the components of the tested plasma. For this purpose, 1 ml aliquots of each serum number are taken and the same amount of HBsAg is calculated, calculated on the final concentration of 0.2 IU / ml for each serum (Table 1). Taking into account the results of the suppression test allows you to reject 2 sera by the value of OD in ELISA.

Next, prepare the stabilizer (stabilizing additive) according to the following recipe. To 1 l of the pool of donor serums add 250 mm L- proline and 0.07% benzoic acid to you as an antioxidant. The pool is adjusted to a pH of 6.8-7.1 with 0.1 M NaCl. As a bactericidal additive, merthiolate is introduced in an amount of 0.01% and gentamicin - 0.2% by weight of the solution. PP is poured into containers of 250 cm ³ and used immediately or stored at a temperature of (2-8) ° C for no more than 1 month.

Table 1. Spike / recovery test results for HBsAg donor blood serum with 0.2 IU / ml HBsAg

Note. Samples 10 373 and 8947 are not included in the PP pool

Preparation of positive samples of the standard panel. For the preparation of samples of a standard panel of HBsAg PNA PNA subtypes, the HBsAg subtype AY preparation (NPK Preparation, NN) and the HBsAg AD thermally inactivated preparation (GeneLabs Diagnostic, Singapore, # 118259) are used.

Table 2 shows the weighted average titration curves of the AD sample of HBsAg (Fig. 1)

III. test system "DS-IFA-HBsAg" (DS)

HBsAg, Ad (GeneLab Diagnostic)

The drug was diluted in PP with 0.05 M FSB during titration.

Analysis Mode:

1.2 hours at 37 ° C on a shaker, 400 rpm 2. Flushing 5 times with FSBT

3. Incubation with the conjugate monoclonal At: HRP, 1 hour, 37 C, shaker;

4. Flushing 5 times with FSBT;

5. Incubation with a peroxide solution of the substrate - TMB, 25 min;

6. Stop reagent 2M H2S04

Table 2. Weighted average titration curves of the AD sample of HBsAg.

These preparations are titrated in increments of 2 in PP to dilutions corresponding to concentrations below 0.01 IU / ml on one microplate with standard preparations:

Standard HBsAg Institute of P. Ehrlich, Ad subtype - 100 IU / ml and

WHO 2 ^nd International standard HBsAg, adw2 subtype, code 00/588 (NIBSC, UK) Semilogarithmic direct titrations with a correlation coefficient - R> 0.99 - were statistically processed to determine the angle of inclination -, cutoff on the OY - axis, and the coefficient of variation CV,%, which should not exceed 10%. Recalculate direct titrations to one b = b (standard P. Ehrlich). The average calibration curve determines how much 17

12 HBsAg preparations of each subtype must be introduced into PP so that the final antigen concentration is 0.1 IU / ml, 0.05 IU / ml and 0.01 IU / ml. Findings. Based on the measurements, the concentration of the ad-subtype of HBsAg in a 100% preparation in ME is approximately ⁶ ME.

Table 3. The results of the certification of samples of HBsAg PN

Note. Statistical processing was performed for OP * = OPex-OPfon OSO - industry standard sample of GISK named after L.A. Tarasevich

LS - laboratory standard of SSC VB Vector;

P.Ehr. - P. Erlich Institute Standard

To prepare 3 samples of a standard subtype AD panel, the HBsAg subtype AD preparation, (Genelab Diagnostics, Singapore), is used. Similarly, AY HBsAg samples are prepared from the HBsAg AY subtype preparation (NPK Preparation, NN) with a concentration of 0.1 IU / ml 0.05 IU / ml and 0.01 IU / ml. Adjustment of standards is performed using test systems.

"HBsAg - ELISA - Best - 0.01" and "DS - ELISA - HBsAg - 0.01" by the addition of either HBsAg or PP.

At the last stage, all panel samples are filtered through 0.45 μm filters.

Determination of the OD of sera of all numbers is carried out in ELISA test systems of various formats of Russian and foreign production.

According to the results of testing samples of HBsAg PNA, the following results are shown in table 3.

The total standard error of the AY preparations at the selected level is calculated in the same way as the square root of the sum of the intragroup and intergroup variances. For example, for the Nel sample, the intragroup dispersions associated with the titration error in various laboratories are 4.95% and 5.03%. The intergroup dispersion between laboratories is (1.27-1.132) / (1.27 + 1.132) * 100% = 5.99%. The standard error of the selected level ΟΠ (ΑΥ) = 0.1 ME is

V4.95 ² + 5.03 ² + 6 ² = 9.3%. Thus, CV,% = 9.3 / 1, 2 = 7.75%

Filtered HBsAg PNA samples are poured in a 0.5 ml laminar cabinet into 2 ml vials, closed with rubber stoppers and placed in metal cassettes for freezing. The drug in bottles is frozen at a counter temperature of 50-60 ° C for 10-12 hours. Frozen samples in bottles are transferred to the sublimation chamber of the TG-50 lyophilic unit, the shelves of which are cooled to minus 32 ° C. The temperature of the drug at the stage of sublimation dehydration maintain below its glass transition temperature (-30 ° C). The compressor of the installation for cooling the desublimator and the shelves of the drying chamber is turned on. On each of the five shelves placed on a cassette with bottles, one of which is frozen in a sensor for measuring the temperature of the material. The set temperature on everything 5 during the drying is supported automatically with an accuracy of 1-3 degrees. The temperature values of each shelf, the temperature of the material on each shelf, as well as the temperature of the sublimator are constantly recorded by the KP-35 recorder. The temperature of the material at the time of switching on the vacuum pump must not exceed minus 30 ° C. The average heating rate of the material at the second stage of drying is 6 ° С / min, thermostating time at 27 ° С is 10 hours. The bottles with the preparation are capped under vacuum. Monitoring the end of the drying process is carried out according to the readings of the residual pressure sensor. Freeze-dried preparation has a residual water content of about 1.0 - 1.5 wt.%. The determination is carried out according to the method

15 thermogravimetry. The total average drying time is 32 hours

A pooled sample of lyophilized candidates for AY & AD subtypes was investigated in an accelerated thermal degradation test. The samples were thermostatically controlled under conditions of controlled temperature at -20 ° C, + 4 ° C + 37 ° C and + 50 ° C; they were removed at regular intervals,

20 was dissolved in double-distilled water and analyzed in duplicates in the HBsAg-IFA-Best test system. The activity of thermostatically controlled samples was determined relative to the activity of samples stored at -20 ° C. The activity estimates of preparations stored at elevated temperatures were then used to predict their stability

25 during prolonged storage at different temperatures using the formal Arrhenius kinetics (Egan W., Schofield T. Basic principles of stability // Biologicals 37 (2009) 379 - 386) [8]. The calculated% loss of activity for the month and year are shown in Table 4. Samples showed high stability. The predicted loss of activity for the year at + 4 ° C is about 0.3% for candidates.

The predicted loss of activity per month is approximately 4.4% at 37 ° C.

Therefore, there is some reason to believe that both candidates of the AY and AD subtypes require compliance with the temperature regime to maintain stability. Table 4 shows the forecast activity of samples of HBsAg PNA for the proposed method based on accelerated tests at elevated storage temperatures.

Table 4. The forecast activity of the samples of the panel of the PNS HBsAg based on accelerated testing of samples of the panel of sera obtained by the claimed method at elevated storage temperatures

Dissolved samples of the panel of sera obtained by the claimed method, it is recommended to store at + 4 ° C for 1 week. The stability of liquid HBsAg PNA samples was tested in the freeze-thaw test. The test results showed that the specific properties of the candidates do not change during 3 cycles of freezing and thawing.

Economic efficiency. Subtype HBsAg AY & AD is used as a prognostic marker of hepatitis B. It circulates in the blood of infected people 2-3 weeks earlier than antibodies appear. The earlier detection of HBsAg with increased sensitivity of test systems helps to reduce the time needed to diagnose infected patients. Earlier medical care and controlled by the number and subtypes of HBsAg are socially significant cost-effectiveness associated with reducing the time of treatment of people. The introduction of the Russian panel of serum PNA HBsAg, obtained by the claimed method, will significantly reduce the costs of the control organizations of health care for the purchase of expensive foreign analogues.

Claims

5 claims

1. A method of manufacturing a panel of sera with extremely low concentrations of HBsAg AD and AY subtypes for monitoring the quality of diagnosis of hepatitis B, comprising checking donor sera by polymerase chain reaction for the presence of hepatitis B virus DNA, anti-HIV, anti-HCV and anti-HBs antibodies , non-specific binding of HBsAg, selection of donor sera that do not contain specific antibodies to the main markers of viral infections according to the values of the effect of suppressing HBsAg (spike / recovery test) and have a negative result in

15 enzyme-linked immunosorbent assay, production of dilution solutions based on selected serums, including the introduction of stabilizing additives, certification of single drugs HBsAg AD and AY subtypes by enzyme-linked immunosorbent assay, titration in a dilution solution from 0.01 to 0.5 IU / ml of samples of the indicated single preparations HBsAg AD, and AY subtypes

20 construction of calibration titration curves in a dilution solution from 0.01 to 0.5 IU / ml AD and AY of HBsAg subtypes relative to international standards and their statistical analysis, according to which the number of HBsAg monopreparations of each subtype required for introduction into the dilution solution to obtain them is determined end

25 concentrations in accordance with the data of the calibration curves and the manufacture of a panel of serums with extremely low concentrations of HBsAg AD and AY subtypes by diluting single drugs HBsAg AD and AY subtypes with a diluting solution to obtain their final concentrations of 0.1 IU / ml; 0.05 IU / ml and 0.01 IU / ml, respectively. In accordance with the data of the calibration curves.

2. The method according to claim 1, characterized in that the plasma antigens containing the AD or AY subtype of HBsAg are used as HBsAg preparations.

5 3. The method according to claim 1, characterized in that proline in an amount of 200-250 mM and benzoic acid in an amount of 0.05-0.08 wt.% Are used as a stabilizing additive.

4. The method according to p. 1, characterized in that for the formation of a panel of subtypes produce 6 samples with a concentration of HBsAg of 0.1 IU / ml, 0.05 IU / ml and 0.01 IU / ml, including 3 samples AY subtype, 3 samples of AD subtype, prepared on the basis of plasma antigens, and 2 negative samples to control the specificity of test systems.

5. The method according to p. 1, characterized in that the prepared samples of the serum panel with extremely low concentrations of HBsAg AD and AY

15 subtypes freeze-dried to a residual moisture content of less than 2.0 May. %

6. The method according to p. 1, characterized in that the samples of the panel of sera with extremely low concentrations of HBsAg AD and AY subtypes are examined using a thermal degradation test to determine their shelf life. .

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