RU2463610C1 - METHOD FOR MAKING PANEL OF HBsAg SUBTYPES AD AND AY SERUMS FOR QUALITY CONTROL OF DIAGNOSING HEPATITIS B - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a method for making a panel of serums for the purpose of quality control of diagnosing hepatitis B with the certified low concentration of HBsAg subtypes AD and AY which involves the analysis of donor serums for the presence of anti-HIV, anti-HCV and anti-HBs antibodies and the nonspecific HBsAg binding; the selection of donor serums with said parameters not found; the use of the selected serums to prepare diluting solutions with a stabilising additive introduced; the certification of the monopreparations of HBsAg subtypes AD and AY by titration dilutions of international standards and the preparation of a reference panel from the certified monopreparations of HBsAg subtypes AD and AY within the range of 0.01 to 0.5 IU/ml; the lyophilisation of the serums followed by thermal degradation panel testing to determine a shelf life. The use of a new formulation of the stabilising additive containing proline 200-250 mM and benzoic acid 0.05-0.08 wt %, and the selection of an optimum temperature storage conditions promote the prolonged activity preservation at annual loss (at +4°C) making less than 0.5%.

EFFECT: higher shelf life.

3 cl, 5 tbl, 1 dwg, 1 ex

Description

The invention relates to a technology for the manufacture of reference panels of sera 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 diagnosed 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 the quality of immunological preparations improved, 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 the staff of control and diagnostic laboratories (CDL), the quality of test systems and vaccines against hepatitis B.

Commercial test systems with a declared sensitivity of 0.01 IU / ml have already appeared (Diagnostic Systems CJSC, Vector Best CJSC, etc.). However, to confirm this sensitivity and implement 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.

Methods analogous to the manufacture of panels are known from the literature, for example, a method for preparing serum panels developed in the laboratory of Red Cross standards (Holland) for the preparation of PELICHECK and PELISPY RUN controls reference panels [l]. These panels are samples of reference AY and AD 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 includes the preparation of standard serums 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 (Product catalog 2003/2004 Boston Biomedica, Inc., 2004, p. 28) [2]. Panel samples represent HBV DNA sera 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 that make up these panels are presented in liquid form and therefore should be stored frozen. Distribution and storage of BBI panels require strict adherence to low temperature conditions (Damen, M., Cuypers, HTM, Zaaijer, HL, Reesink, HW, Schaasberg, WP, Gerlich, WH, Niesters, HGM, Leiie PN (1996) International collaborative 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 serum panel for monitoring the quality of diagnosis of hepatitis B, which provides samples of the indicated serum panel with a certified extremely low concentration of AD and AY subtypes of HBsAg and preserves 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 of HBsAg AD and AY subtypes for monitoring the quality of diagnosis of hepatitis B includes the verification 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 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 re ultat in enzyme immunoassay, production of dilution solutions based on selected serums, certification of mono preparations of HBsAg AD and AY subtypes by enzyme immunoassay methods, titration in a dilution solution of samples of the indicated mono preparations of HBsAg AD and AY subtypes from 0.01 to 0.5 IU / ml, construction of calibration calibrations 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 is determined one that can be injected into the dilution solution to obtain their final concentration in accordance with the calibration curves, and the manufacture of a panel of serums with extremely low concentrations of HBsAg AD and AY subtypes by diluting the HBsAg AD and AY subtypes with dilution 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.

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

As a stabilizing additive use 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 wt.%.

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 HBsAg concentration is carried out using dilution factors of the initial AY & AD subtypes calculated using the equations of the calibration curves of AY & AD HBsAg preparations in a diluting stabilized solution to the required concentration of HBsAg (0.1 ME / 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 sera with a minimum level of suppression for the manufacture of samples of a panel of serums with extremely low concentrations (PNA) 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 HBsAg standard (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.

The proposed model of the serum panel of extremely low concentrations of HBsAg AD and AY subtypes includes 8 samples: 2 samples of donor serums, 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 ME / 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, IP-27 [four]. 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 OH in ELISA) for each panel sample. At the same time, a prerequisite for the use of HBsAg reference preparations is the unchanged 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 0.01% and gentamicin 0.2%) is known from RF patent No. 2265028, IPC G01N 33/96, 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 No. 163646 1206) and 0.05-0.08 wt.% Benzoic acid to 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 add foreign ingredients to the solution (Bolli R., Woodtii K., Bartschi M. et al. L-proline reduces IgG dimmer content and enhances the stability of intravenous immunoglobulin solutions // Biologicals 38 (2010), 150-157) [8]. 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) [6].

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 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 concentration main subtypes of HBsAg, which allows us to evaluate the sensitivity of test systems at the level of 0.01 IU / ml and specificity to various subtypes of HBsAg, to control the level of professional skills of clinical diagnostic laboratories and quantify hepatitis B vaccines.

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

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

LS - laboratory standard of SSC VB Vector;

R.Er. - standard of institute P. Ehrlich

Ycp - the average value of Ln OP

SY - dov. interval

When processing titration curves, the values of OP * = OPec-Optf are used.

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 No. 1, the intra-group errors associated with titration errors in various laboratories are 4.25% (VB) and 9.22% (DS).

The intergroup spread between the 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 at the selected 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

An example of a method of manufacturing a panel of sera with extremely low concentrations of HBsAg AY and AD subtypes

Preparation of dilution solution. As RR for the preparation of positive panel samples with a certain concentration of HBsAg, donor serums 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, amber-colored sera samples are taken that are transparent in the light. A total of 9 high-quality sera were selected.

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

After preparation, all sera are analyzed for anti-HBs 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 was 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 in donor blood serum with 0.2 IU / ml HBsAg Controls Sample No. OH, p. Sample No. OP, p.u. K-0.011 7831 1,741 10384 1,656 K-0.011 10345 1,345 10379 1,326 0.019 10373 0.814 10390 1,483 K + 3.666 10364 1,520 8947 2,947 K + low 0.457 10376 1,569 Note. Samples 10373 and 8947 are not included in the PP pool

Despite the fact that the properties of the individual components of the stabilizing additive are known from the prior art, said stabilizer is a composition that has a synergistic stabilizing effect, ensuring the preservation of the properties of extremely low concentrations of antigens containing the AD or AY subtype of HBsAg during the preparation of the serum panel, drying and its storage.

Experimental studies indicate that maintaining the properties of extremely low concentrations of antigens containing the AD or AY subtype of HBsAg in the serum panel is a complex task that cannot be solved by the usual trial and error method. The serum panel requires special temperature conditions to maintain stability.

An analysis of the research results shows that separately proline at a concentration of 200-250 mM or more, as well as separately benzoic acid at a concentration of 0.05-0.08% and more, do not ensure the preservation of the properties of antigens in the serum panel for a long time (see table).

In addition, the introduction of proline is an important methodological technique. Proline has a powerful disintegrating potential that prevents the process of association of HBsAg monoparticles. Proline, as a primary amino acid, does not introduce foreign ingredients into solutions (Bolli 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). Benzoic acid is a classic antioxidant used to stabilize medications and food products, exhibits antimicrobial and antifungal effects, and has a depressing effect on mold, yeast and some types of bacteria.

The low viscosity of the stabilizing 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.

Table 1a shows comparative activity data of HBsAg PNA samples for the proposed method based on accelerated tests at elevated storage temperatures using the inventive stabilizer composition and its individual components.

Table 1a Comparative activity data of HBsAg PNA panel samples based on their accelerated tests obtained by the claimed method at elevated storage temperatures Storage temperature, deg. FROM Loss of activity for the year,% Monthly activity loss,% It is declared. structure Proline Benz acid It is declared. structure Proline Benz acid four 0.48 49 57 0.04 1.9 2.5 37 53 one hundred one hundred 6.1 17 21 fifty 76 one hundred one hundred 11,2 26 29th

Preparation of positive samples of the standard panel. To prepare samples of a standard panel of HBsAg PNA subtypes, the HBsAg subtype AY preparation (NPK Preparation, NN) and the HBsAg AD preparation, heat-inactivated (GeneLabs Diagnostis, Singapore, # 118259) are used.

Table 2 shows the weighted average titration curves of the AD sample of HBsAg (figure 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 H ₂ SO ₄ .

table 2 Weighted Average Titration Curves of the AD HBsAg Sample CCA HBsAg, ad subtype The concentration of HBsAg, IU / ml OP ₄₅₀ Breeding OP ₄₅₀ 0.5 3,436 3,253 ± 0.33 0.1 ME AU / 10 ⁵ 1.051 0.985 ± 0.15 0.05 0.482 0.455 ± 0,092 0.05 IU AC / 2 × 10 ⁵ 0.584 0.565 ± 0,092 0.05 0.472 0.459 ± 0,092 0.017 IU AC / 6 × 10 ⁵ 0.308 0.279 ± 0,087 0.02 0.208 0.206 ± 0,061 0.2 ME HELL / 5 × 10 ⁶ 0.821 0.837 ± 0.123 0.02 0.209 0.196 ± 0,061 0.1 ME HELL / 10 ⁷ 0.532 0.521 ± 0.15 0.01 0.123 0.115 ± 0,057 0.03 ME HELL / 3 × 10 ⁷ 0.263 0.253 ± 0.118 0.01 0,126 0.119 ± 0,057 Pool Neg. 0.134 0.126 ± 0,063 Control n.h.s. 0,022 0.024 ± 0.013 Pulotr. 0.148 0.128 ± 0,063 Tilt angle, b -0.637 ± 0.073 Tilt angle, b - 0.66 ± 0.072

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:

The HBsAg standard of Institute P. Ehrlich, Ad subtype is 100 IU / ml and the WHO 2 ^nd International standard HBsAg, adw2 subtype, code 00/588 (NIBSC, UK).

Semilogarithmic direct titrations with a correlation coefficient - R ² > 0.99 - were statistically processed to determine the slope angle - bi, cutoff on the OY axis - a 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 HBsAg of each subtype must be introduced into the PP so that the final antigen concentration corresponds to 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 HBsAg PNA Sample Certification Results OP controls t / s DS-IFA - HbsAg Samples of the panel received. way OP, [th] (Vector-Best) OP, [th] (Diagn. Syst.) CV,% CCA 0.4 ME-1.933 1-ay 1.132 ± 0.056 1.27 ± 0.064 7.75 CCA 0.2 ME-1.015 2-ay 0.643 ± 0.078 0.590 ± 0.086 14.0 CCA 0.1 ME-0.538 3-ay 0.171 ± 0.14 0.171 ± Y-0.15 60.0 OCO 0.05 ME-0.311 4-ad 0.946 ± 0.048 1.03 ± 0.069 13,4 OCO 0.02 ME-0.173 5-ad 0.528 ± 0.075 0.648 ± 0.086 14.0 P.Ehr-1: 200-2.822 6-ad 0.174 ± 0.13 0.175 ± 0.15 56.7 2nd Internat Standard (0.33ME) - 1,560 7 (donor) 0.042 ± 0.01 0.035 ± 0.01 10,4 Cc - 0.003 8 (donor) 0.028 ± 0.01 0.046 ± 0.01 9.1 Cut off 0.121 0.118 Note. Statistical processing performed for OP * = OPex-OPfon OSO - industry standard sample of the GISK named after L.A. Tarasevich Drugs - laboratory standard of SSC WB Vector P.Ehr. - P. Erlich Institute Standard

To prepare 3 samples of a standard AD subtype 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 the test systems "HBsAg - ELISA - Best - 0.01" and "DS - ELISA - HBsAg - 0.01" by adding either HBsAg or PP.

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

The 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 sample No. 1, 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 of OP (AU) = 0.1 ME is

Таким образом, CV, % = 9,3/1,2=7,75%.

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 preparation in bottles is frozen at a counter temperature of (50-60) ° С 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 ° С. The temperature of the drug at the stage of sublimation dehydration is maintained 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 throughout the drying is maintained 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 should not exceed minus 30 ° C. The average heating rate of the material at the stage of drying is 6 ° C / min, thermostating time at 27 ° C is 10 hours. The bottles with the drug are corked 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 of 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. Samples were thermostated under controlled temperature conditions at –20 ° C, + 4 ° C + 37 ° C and + 50 ° C, removed at regular intervals, dissolved in double-distilled water and analyzed in duplicates in the HBsAg - IFA - Best test system. The activity of thermostated samples was determined relative to the activity of samples stored at -20 ° C.

Estimates of the activity of preparations stored at elevated temperatures were further used to predict their stability during prolonged storage at various temperatures using the formal Arrhenius kinetics (Kirkwood, TBL Principles for the preparation and approval of international and other standards and reference materials for biological preparations. Ser. Tech. Reports . N 37. WHO, Geneva, 1990. 14a // J. biol. Standardization. - 1984. - Vol. 12. - P.207-224) [7]. 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 are certain reasons to assume that both candidates of 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 samples of the panel of the PNS HBsAg based on accelerated testing of samples of the panel of serum obtained by the claimed method at elevated storage temperatures Storage temperature, ° С Loss of activity for the year,% Monthly activity loss,% four 0.48 0.04 37 53 6.1 fifty 76 11,2

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. 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 (3)

1. A method of manufacturing a panel of serums with HBsAg AD- and AY-subtypes for monitoring the quality of diagnosis of hepatitis B, comprising checking donor sera for anti-HIV, anti-HCV and anti-HBs antibodies, as well as non-specific binding of HBsAg; sampling with a negative (according to ELISA and spike / recovery test) results for the indicated indicators; preparation based on selected sera of dilution solutions (PP), involving the introduction of a stabilizing additive; certification of ELISA of mono-preparations of AD- and AY-subtypes of HBsAg by titration of their dilutions in PP relative to international standards with the determination of the exact quantities (dilutions) of certified single preparations of HBsAg in PP, providing the desired final concentrations; freeze drying of sera to a residual moisture content of about 1-1.5%; conducting a panel test for thermal degradation to determine the expiration date, characterized in that the titration in a dilution solution of samples of the indicated HBsAg AD and AY subtypes is performed in the range from 0.01 to 0.5 IU / ml to obtain their final concentration of 0.1; 0.05 and 0.01 IU / ml, and proline in the amount of 200-250 mM and benzoic acid in the amount of 0.05-0.08 wt.% Are used as a stabilizing additive in the manufacture of diluting solutions. 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. 3. The method according to claim 1, characterized in that for the formation of a panel of subtypes produce 6 samples with a concentration of HBsAg of 0.1; 0.05 and 0.01 IU / ml, including 3 samples of the AY subtype and 3 samples of the AD subtype, and 2 negative samples to control the specificity of the test systems.

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