Method for making panel of hbsag subtypes ad and ay serums for quality control of diagnosing hepatitis b

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

 

The invention relates to manufacturing techniques reference panels of sera containing antigens of the most dangerous viral infections, particularly hepatitis b virus (HBV), and can be used in medicine and biotechnology.

Samples of blood donors in Russia, you must examine for the presence of markers of HIV 1 and 2, hepatitis b and C and syphilis. Approximately 1% of all samples sent for re-confirmed in a reference laboratory. However, the effectiveness of the confirmation of the results depends on the qualifications of the staff of the clinical diagnostic laboratory quality test systems and the availability in the control laboratories standard drugs during infectious markers (VQC Pelispy Multi - Marker, run control. Central Laboratory of the Red Cross, Netherlands, 1998) [1].

When using highly sensitive HBsAg screening tests are carriers of hepatitis b with a very low concentration of HBsAg can be detected, particularly among individuals who are carriers of anti-HBC antibodies.

Use in everyday work reference preparations of various subtypes of HBsAg will allow you to extend the range of diagnosed markers and use these materials for the detection of HBsAg in blood products intended for transfusion or for processing.

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

Improving the quality of diagnosis of HBV runs parallel with the increased sensitivity of diagnostic test systems. With the improvement of the quality of immunological preparations sensitivity of diagnostic systems for the detection of HBsAg was increased from 0.1 IU/ml to 0.05 IU/ml Now before practical health tasked to diagnose HBsAg in the blood at the level of 0.01 IU/ml for the control of employees ' qualifications control and diagnostic laboratories (CDL), quality test kits and vaccines against hepatitis C.

Already have a commercial test system with a claimed sensitivity of 0.01 IU/ml (CJSC Diagnostic systems, CJSC Vector best and others). However, to confirm this sensitivity and the implementation of these tests in clinics and blood transfusion stations required certified standard drugs. The task is extremely difficult because validate samples with a concentration of 0.01 IU/ml is necessary with the use of test systems with a sensitivity of 0.01 IU/ml

From the literature known methods analogous to the construction of the panels, such as the method of preparation of serum panels, developed in the laboratory of the standards of the red cross (Netherlands) for the preparation of reference panels PELICHECK and PELISPY RUN controls [l. These panels represent the samples of the reference drugs AY and AD subtypes HBs Ag, diluted in pooled donor sera not containing markers dangerous viral infections, in concentrations up to 0.1 ng/ml. the Method includes preparation of a standard serum containing HBs Ag and with different concentrations of this protein.

The closest technical solution (prototype) is a method for preparing panels of serum HBV DNA Genotype Performance Panel, developed in the laboratory of the Boston Biomedical Inc. (USA) - BBI. This panel includes 10 samples with genotypes A-D (the Product catalog 2003/2004 Boston Biomedica, Inc., 2004, p.28) [2]. Sample panels are HBV DNA serum, diluted in pooled donor sera not containing markers dangerous viral infections.

However, these panels do not meet the practical needs of health, as certified quantitatively concentration of DNA of hepatitis b virus, and not on the concentration of HBsAg. In addition, serum included in these panels, supplied in a liquid form and therefore must be stored in a frozen state. Distribution and storage BBI panels require strict adherence to a low-temperature regimes (Damen, M., Cuypers, H.T.M., Zaaijer, H.L., Reesink, H.W., Schaasberg, W.P., Gerlich, W.H., Niesters, H.G.M., Leiie P.N. (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 so the first method of manufacturing a panel of sera for quality control of diagnosis of hepatitis b, which provides obtaining samples of the specified panel, serum with certified very low concentration of AD and AY subtypes of HBsAg and preserving the specific characteristics of these samples for a long time.

This technical result is achieved by a method of manufacturing a panel of sera with extremely low concentrations of HBsAg AD and AY subtypes for quality control of diagnosis of hepatitis b includes examining donor serum by polymerase chain reaction for the presence of DNA of hepatitis b virus, anti-HIV, anti-HCV and anti-HBs antibodies, nonspecific binding of HBsAg, the selection of donor sera not containing specific antibodies to the major markers of viral infections on the values of the effect of suppression of HBsAg (spike/recovery test and have negative results in ELISA analysis, manufacturing, distributing solutions on the basis of selected sera, certification of plain products HBsAg AD and AY subtypes methods of ELISA, titration in razvodami solution samples from the specified plain products HBsAg AD and AY subtypes from 0.01 to 0.5 IU/ml, construction of calibration curves titration in razvodami the solution is from 0.01 to 0.5 IU/ml AD and AY subtypes of HBsAg in relation to international standards and their statistical analysis, under which determine the number of plain products HBsAg each subt the PA, it is necessary to incorporate in diluting solution to obtain a final concentration in accordance with the data of the calibration curves and the production of panels of sera with extremely low concentrations of HBsAg AD and AY subtypes by cultivation of plain products HBsAg AD and AY subtypes razvodami solution to obtain final concentrations of 0.1 IU/ml to 0.05 IU/ml and 0.01 IU/ml, respectively, in accordance with the data of the calibration curves.

As plain products HBsAg using plasma 100%green antigens containing AD and AY subtypes of HBsAg.

As stabilizing additives used Proline in the amount of 200-250 mm and benzoic acid in a quantity of 0.05 to 0.08 wt.%.

Prepared samples panel of sera with extremely low concentrations of HBsAg AD and AY subtypes freeze-dried to a residual moisture content of less than 2.0 wt.%.

The samples panel of sera with extremely low koncentracije HBsAg AD and AY subtypes examined using test thermodegradation to establish the expiry date.

Sterowanie standard samples to the desired concentration of HBsAg is done using the coefficients dilution of the initial preparations AY & AD subtypes, calculated according to the equations of the calibration curves AY & AD drugs HBsAg in razvodami stable solution to the desired concentration of HBs Ag (0.1 ME/ml of 0.05 IU/ml and 0.01 IU/ml). As the original p is aparatow HBs Ag use preparations containing the HBsAg to 100% for the desired protein.

Below is a diagram of the manufacturing panel of sera with extremely low concentrations of HBsAg subtypes AY & AD.

1. Testing of samples of donor sera using laboratory standard HBsAg to study the magnitude of the suppression of the analytical signal components of the tested plasma. From the donor sera with a minimum level of suppression is prepared diluting solution (RR) for prototype panel of sera with extremely low concentrations (NCP) HBsAg.

2. Titration of the drug HBsAg AY subtype (PR-in JSC "Drug", Nizhny Novgorod), and drug AD subtype (production of the company GeneLab Diagnostics, Singapore) in the prepared PP on one tablet in conjunction with international standards - 2ndInternational Standard ADW2 code 00/588 (NIBSC, UK) and standard Institute P. Ehrlich (Germany) - AD subtype in different test systems.

3. Statistical processing of the results of the calibration tetravac at confidence level 95% for the calculation of the coefficients of cultivation 100% preparations of HBsAg in stabilized PP to the required concentration.

4. Preparation of candidates NCP AY & AD subtypes of HBsAg.

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

6. PR is cooking, dry stable form candidates NCP method lyophilization.

7. The test accelerated inactivation of candidates NCP at elevated temperatures to determine the expiration date of the standard.

To determine the ratio between the value of the analytical signal ELISA and the number of HBs Ag in razvodami solution must be used ELISA test system, which is in the range from 0.3 to 0.02 IU/ml retain the linear relationship between absorbance and concentration of HBs Ag in solution.

To solve certification sample panel of serum concentration of HBs Ag should perform the following steps:

- use to create a sample panel of plasma samples containing different concentrated preparations of plasma or recombinant antigens with the content of HBs Ag is not less than 95% of the total protein, no infectious risk;

as diluent solution (PP) to use stable pool of sera of normal human blood that does not contain markers of major infections and does not inhibit the analytical signal from HBsAg.

The proposed model panel of sera extremely low concentrations of HBsAg AD and AY subtypes includes 8 samples: 2 sample donor sera, 3 sample AY subtype of HBsAg and 3 sample AD subtype of HBsAg prepared by dilution of the samples in stable pooled donor sera (PP) to a concentration of 0.1 ME/ml of 0.05 IU/ml and 0.01 M is/ml of antigen.

For prototype panel of sera using concentrated products AD and AY subtypes of HBsAg, but not native serum. In the process of concentration of HBsAg preparations spend processing of serum trypsin, filtration through 0.45 μm and 0.22 μm filters, high-speed centrifugation and concentration. The obtained product called "purified plasma HBs-antigen" certificate on the protein content and the activity of interaction with anti-HBs antibodies. The content of the desired protein HBsAg in such preparations is greater than 95%.

An important value when using panels of sera in clinical diagnostic laboratories is the correctness of the results of the analysis Rezapkin G., E. Dragunsky and Chumakov, K. (2005) Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) // Biologicals 33, IP-27 [4]. The main criterion for the correctness of the results of serological analysis is the certified value of the analytical signal (e.g., optical density IT in ELISA) for each sample panel. The obligatory condition for application of the reference preparations of HBsAg is a constant level of this analytical signal during long-term storage.

Preserve specific properties of the sera panel containing IgG antibodies by injection of preservative - bactericidal additive (thimerosal 0.01% gentamicin and 0.2%), it is known from the patent of the Russian Federation No. 2265028, IPC G1N 33/96, publ. 27.11.2005 [5]. In the present invention to stabilize the level of HBs antigen Ag is invited to use the stabilizer comprising 200-250 mm Proline (L - Proline, 99%, for synthesis, catalogue No. 163646 1206) and 0.05 to 0.08 wt.% benzoic to you. The introduction of Proline is an important activity. Proline has a powerful dezintegriraat potential, preventing the Association process monoparticles HBsAg. In addition, Proline, as a primary amino acid does not contribute to the solutions of extraneous ingredients (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 pharmaceuticals and food products, exhibits antimicrobial and antifungal action, has a dampening effect on mold, yeast and certain bacteria.

The low viscosity solution of the stabilizer for the standard preparation is a critical parameter for the detection of HBsAg in practice, because many manufacturers of test systems try to reduce the analysis time. Under these conditions, a greater importance is the reduction of the diffusion inhibition of the interaction process µa+HBsAg, driven primarily by a decrease in solution viscosity (Kanev A.N., Bakirov T.S., Yudina I.V. and Zaitsev B.N. Simulation of interaction of particles with HBsAg antibodies immobilized on a surface of the micropate well at ELISA // Biotechnology, 2005, 6, 74-82) [6].

Thus, the certification procedure sera panel consists of three phases, the results of which are independent and reliably identify the concentration of various subtypes HBs Ag. Comparison of serum panels NCP HBsAg, prepared in accordance with the inventive method, with panels of peers of the same purposes showed that different from the prototype set of essential features provides a new previously unknown property: the ability to manufacture panels of HBsAg in the dry (lyophilic) form with extremely low certified concentration of major subtypes of HBsAg, which allows to estimate the sensitivity of the test systems at the level of 0.01 IU/ml and specificity to different subtypes of HBsAg, to control the level of professional skill of workers of clinical diagnostic laboratories and to control quantitatively the vaccine against hepatitis C.

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

CCA - industry standard sample gisk named after. Lautareasca

Drug - laboratory standard SRC VB Vector;

Reg. standard in-TA Parlia

Ycp is the mean value of Ln OD

SY - Dov. interval

When processing titration curves using values OP*=OPEC-BPA.

About the General mean-square error at the selected level is calculated (table 2) as the square root of the sum of squared intra-group and intergroup bias. For example, for sample No. 1 within-group errors errors titration in different laboratories be 4.25% (VB) and which 9.22% (DS).

Intergroup variation between test systems for AD samples is(1,03-0,946)/2*100%=4,42% plus the maximum additional variance of the error between the average values and the values at the selected level of 7.2-4,2=4,33%. In the amount of inter-group variation at this level the OP is of 8.75%. Total deviation (Δ) of the definition at a fixed concentration of HBsAg (0.1 IU/ml) is

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

Preparation of diluent solution. As PP for the preparation of positive samples panel with a certain concentration of HBsAg in the use of donor serum that does not contain markers of viral infections. Serum is prepared immediately after blood sampling. Centrifuged speed 2800-3200*rpm whole blood at a temperature of 10-12°C for 15 min and decanted the supernatant solution. Stored until use at a temperature of +(2-4)°C. To prepare a panel of selected serum samples amber, translucent in the light. Only selected 9 quality serums.

Control donor sera intended for cultivation HBsAg PR the preparations the content of total protein (normal range 6.5-8.5%).

After cooking all serum analyzed for anti-HBs antibodies and viral contamination.

In the next step, put the test on the suppression of the analytical signal components of the tested plasma. With this purpose, selected aliquots of 1 ml from each of the rooms serum and give them the same amount of HBsAg, calculated on the final concentration of 0.2 IU/ml for each serum (table 1). Results suppression test allows to reject 2 serum largest OD in ELISA.

Next, prepare the stabilizer (stabilizing additive) according to the following recipe. To 1 l of pooled donor sera add 250 mm L-Proline and 0.07% benzoic to you as an antioxidant. The pool was adjusted to pH 6.8 to 7.1 using 0.1 M NaCl. As bactericidal additive injected thimerosal in an amount of 0.01% and gentamicin - 0.2% by weight of the solution. PP is bottled in containers of 250 cm3and used immediately or stored at a temperature (2-8°C for up to 1 month.

Table 1
Results suppression test (spike/recovery) HBsAg in the sera of blood donors with the introduction of 0.2 IU/ml HBsAg
ControlsSample # HE PU Sample # OP, O.E.
K-0,01178311,741103841,656
K-0,011103451,345103791,326
0,019103730,814103901,483
To+3,696103641,52089472,947
K+ low 0,457103761,569
Note. Samples 10373 and 8947 not included in the pooled RR

Despite the fact that the properties of the individual components of the stabilizing additives known from the prior art, this stabilizer is a compound which has a synergistic stabilizing effect, ensuring the preservation of the properties of the extremely low concentrations of antigens containing AD or AY subtype of HBsAg preparation of a panel of sera, drying and storage.

xperimentally research suggests, the preservation of the properties of the extremely low concentrations of antigens containing AD or AY subtype of HBsAg in a panel of sera is a complex task, which is not solved in the usual trial and error. Sera panel requires compliance with special temperature conditions to maintain stability.

The analysis of the results shows that separately Proline at a concentration of 200-250 mm and more, as well as separately benzoic acid at a concentration of 0.05-0.08% or more does not ensure the preservation of the properties of the antigens in the panel of sera for a long time (see table).

In addition, the introduction of Proline is an important activity. Proline has a powerful dezintegriraat potential, preventing the Association process monoparticles HBsAg. Proline, as a primary amino acid does not contribute to the solutions of extraneous ingredients (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 pharmaceuticals and food products, exhibits antimicrobial and antifungal action, has a dampening effect on mold, yeast and certain bacteria.

The low viscosity solution of the stabilizer for the standard preparation is a critical parameter for the detection of HBsAg in practice, K many manufacturers of test systems try to reduce the analysis time. Under these conditions, a greater importance is the reduction of the diffusion inhibition of the interaction process µa+HBsAg, driven primarily by a decrease in solution viscosity (Kanev A.N., Bakirov T.S., Yudina I.V. and Zaitsev B.N. Simulation of interaction of particles with HBsAg antibodies immobilized on a surface of the microplate well at ELISA//Biotechnology, 2005, 6, 74-82.

In table 1A below provides comparative data activity NCP samples for HBsAg proposed method based on accelerated testing at elevated temperatures storage using the claimed composition of the stabilizer and its individual components.

Table 1A
Comparative activity data samples panel NCP HBsAg based on accelerated testing, obtained by the claimed method at elevated storage temperature
Storage temperature, deg. The loss of activity for the year %The loss of activity for the month, %
Stated. compositionProlineBenzo. acidStated. composition ProlineBenzo. acid
40,4849570,041,92,5
37531001006,11721
507610010011,22629

Preparation of positive samples the standard toolbar. For sample preparation the standard panel NCP HBsAg subtypes drug use HBsAg subtype AY (CDD Drug, NN) and the preparation of HBsAg AD, thermoinactivation (GeneLabs Diagnostis, Singapore, # 118259).

Table 2 shows the average titration curves for AD sample HBsAg (Fig 1)

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

Preparation of HBsAg, Ad (GeneLab diagnostics)

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

Analysis mode:

1. 2 hours at C on the shaker, 400 rpm;

2. Washing 5 times FSBT;

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

4. Washing 5 times FSBT;

5. Inka is the situation with the peroxide substrate solution - TMB, 25 min;

6. Stop reagent 2M H2SO4.

td align="center"> ±0,087
Table 2
Weighted average titration curves for AD sample HBsAg
CCAPreparation of HBsAg, ad subtype
The HBsAg concentration, IU/mlOP450BreedingOP450
0,53,4363,253±0,330.1 IU AU/1051.0510.985±0,15
0,050,4820,455±0,092of 0.05 IU AU/2×1050.5840.565±0,092
0,050,4720,459±0,0920.017 IU AU/6×1050.3080.279
0,020,2080,206±0,0610,2 ME HELL/5×1060.8210.837±0,123
0,020,2090,196±0,0610,1 ME HELL/1070.5320.521±0,15
0,010,1230,115±0,0570,03 ME HELL/3×1070.2630.253±amount of 0.118
0,010,1260,119±0,057Pool OTP.0.1340.126±0,063
Control NCS0,0220,024±0,013Pultr.0.1480.128±0,063
The angle b-0,637±0,073The angle b- 0,66±0,072

Titrated in increments of 2 these drugs in PP before dilution, corresponding to concentrations below 0.01 IU/ml on the same microplate with standard drugs:

Standard HBsAg in-TA R. Ehrlich, Ad subtype - 100 IU/ml and WHO 2ndInternational standard HBsAg, adw2 subtype, code 00/588 (NIBSC, UK).

Semi-log straight titration with correlation coefficient - R2>0,99 - were statistically processed to determine the angle - bi cutoff on the axis OY and the coefficient of variation CV%, which should not exceed 10%. Recalculate direct titration to one b=b (standard R. Ehrlich). On average calibration curve to determine how much of the drug HBsAg each subtype must be entered in PP, so that the final concentration of the antigen corresponded to 0.1 IU/ml to 0.05 IU/ml and 0.01 IU/ml

The conclusions. On the basis of measurements of the concentration of ad-subtype of HBsAg 100%drug in ME is approximate6ME.

Table 3
The evaluation results of samples NCP HBsAg
OP controls the t/s DS-EIA - HbsAg The swatches panel, obtained Appl. wayOP,[th] (Vector-best)OP,[th] (automotive technician. Syst.)CV %
CCA 0,4 ME-1,9331-AY1,132±0,0561,27±0,064of 7.75
CCA 0,2 ME-1,0152-AY0,643±0,0780,590±0,08614,0
CCA 0,1 ME-0,5383-AY0,171±0,140,171±-0,1560,0
OCO 0,05 ME-0,3114-AD0,946±0,0481,03±0,069the 13.4
OCO 0,02 ME-0,1735-AD0,528±0,0750,648±0,08614,0
P.Ehr-1:200-2,8226-AD0,174±0,130,175±0,1556,7
2nd Internat Standard (0,33ME) - 1,5607 (donor)0,042±0,010,035±0,01 10,4
Cc - 0,0038 (donor)0,028±0,010.046±0,019,1
Cut off0,121amount of 0.118
Note. Statistical processing is performed to the OP*=OPEX-Opton
CCA - industry standard sample risk imlotence
Drug - laboratory standard SRC VB Vector
P.Ehr. standard Institute P. Ehrlich

For the preparation of 3 samples, the standard panel AD subtype drug use HBsAg subtype AD (Genelab Diagnostics, Singapore). Similarly, samples of HBsAg AY is prepared from preparation of HBsAg AY subtype (CDD Drug, NN) with a concentration of 0.1 IU/ml to 0.05 IU/ml and 0.01 IU/ml

Alignment of standards is carried out using the test systems HBsAg EIA - bis 0,01" and "DS - EIA - HBsAg - 0.01 to" add or HBsAg, or PP.

In the last step, all the swatches panel is filtered through the filters of 0.45 µm.

The definition of OP sera of all rooms is carried out in ELISA test systems of different formats of Russian and foreign production.

According to the results of testing the second sample NCP HBsAg obtained the following results, given in table 3.

The total mean-square error of drugs AY at the selected level is calculated as the square root of the sum of intra-group and inter-group dispersions. For example, for sample No. 1 within-group variance associated with the error titration in different laboratories, amount of 4.95% to 5.03%. Intergroup variance between laboratories is(1,27-1,132)/(1,27+1,132)*100%=5,99%. The RMSE at the selected level, OP(AC)=0.1 TO ME is

Thus, CV, % = 9,3/1,2=7,75%.

Filtered samples NCP HBsAg poured in hoods 0.5 ml vials with a volume of 2 ml, closed with rubber stoppers and placed in a metal cassette for freezing. Freezing of the drug in vials carried out at a temperature of counter -(50-60)°C for 10-12 hours Frozen samples in vials are transferred into the freeze chamber lyophilic install TG-50, shelves which chilled to minus 32°C. the Temperature of the preparation at the stage of freeze dehydration maintain temperature below its glass transition

(-30°C). Include a compressor installation for cooling desublimator and shelves of the drying chamber. For each of the five shelves are placed on the cassette with the bottles, one of which is frozen sensor measuring the temperature of the material. The preset temperature n the entire drying is maintained automatically with an accuracy of 1-3 degrees. The temperature of each shelf, the temperature of the material on each shelf, and the temperature of sublimator write constantly chart CP-35. The material temperature at the time of activation of the vacuum pump must not exceed minus 30°C. the Average rate of heating of the material at the final stage is 6°C/min, time of incubation at 27°C is 10 o'clock Bottles with the product is sealed under vacuum. The control end of the drying process is carried out based on the sensor readings residual pressure. Lyophilized drug has a residual water content of about 1.0 to 1.5 wt.%. The determination is carried out by the method of thermogravimetry. Total average drying time is 32 hours

The pooled sample liofilizovannyh candidates AY & AD subtypes was investigated in the test accelerated thermodegradation. Samples thermostatically under conditions of controlled temperature - 20C, +4C +S and +50C, was removed after a certain period of time, was dissolved in bidistilled water were analyzed in duplicate in a test system HBsAg ELISA - the best". The activity of the temperature-controlled sample was determined relative to the activity of samples stored at -20°C.

Evaluation of activity of preparations stored at elevated temperatures were then used to predict their stability during prolonged storage at different is cnyh temperatures using formal kinetics Arrhenius equation (Kirkwood, T.B.L. Principles of preparation and approval of international and other standards and reference materials for biological products. Ser. technology. Dokl. N 37. Who, Geneva, 1990. 14a // J. biol. Standardization. - 1984. - Vol.12. - P.207-224) [7]. Calculated % loss of activity for the month and the year are given in table 4. The samples showed high stability. The predicted loss of activity per year at 4°C is about 0.3% for candidates. The predicted loss of activity for the month is about 4.4% at 37°C.

Therefore, there is some evidence to suggest that both candidates AY and AD subtypes require observance of a temperature mode to maintain stability. Table 4 shows the forecast activity NCP samples for HBsAg proposed method based on accelerated testing at elevated temperature storage.

Table 4
Forecast activity of the samples panel NCP HBsAg based on accelerated testing of samples panel of sera obtained by the claimed method at elevated storage temperature
Storage temperature, °CThe loss of activity for the year %The loss of activity for the month, %
40,480,04
37536,1
507611,2

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

Economic efficiency. HBsAg subtypes AY & AD is used as a prognostic marker of hepatitis C. It circulates in the blood of infected 2-3 weeks before antibodies appear. Earlier detection of HBsAg with increasing sensitivity test systems reduces the time of diagnosis to infected patients. Earlier medical care and controlled by the number and subtypes of HBsAg are socially meaningful economic efficiency associated with the reduction of treatment time people. The introduction of the Russian panel of sera NCP HBsAg obtained by the claimed method, will significantly reduce the costs of control and health organizations to purchase expensive is the present foreign drugs in its class.

1. A method of manufacturing a panel of sera from HBsAg AD and AY subtypes for quality control of diagnosis of hepatitis b, including verification of donor sera for the presence of anti-HIV, anti-HCV and anti-HBs antibodies and nonspecific binding of HBsAg; selection of negative samples (according to ELISA and spike/recovery test results on these indicators; preparation based on selected serum diluting solutions (PP), introducing a stabilizing additive; certification IFA plain products AD and AY subtypes of HBsAg by titration their dilutions in PP relative to international standards definition exact quantities (dilutions) certified plain products HBsAg in PP that provide the specified final concentrations; lyophilic drying sera to a residual moisture content of about 1-1 .5%; the test panel on thermodegradation to determine shelf life, wherein the titration in razvodami solution samples from the specified plain products HBsAg AD and AY subtypes produce in the range from 0.01 to 0.5 IU/ml with obtaining final concentrations of 0.1; 0.05 and 0.01 IU/ml, and as a stabilizing additive in the manufacture of distributing solutions used Proline in the amount of 200-250 mm and benzoic acid in the amount of 0.05-0.08 wt.%.

2. The method according to claim 1, characterized in that as HBsAg preparations used in isout plasma 100%green antigens, containing AD or AY subtype of HBsAg.

3. The method according to claim 1, characterized in that for forming the panel subtypes produce 6 samples with HBsAg concentration of 0.1; 0.05 and 0.01 IU/ml, including 3 sample AY subtype and 3 sample AD-subtype, and 2 negative sample to control the specificity of the test systems.



 

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8 dwg

FIELD: medicine.

SUBSTANCE: there is involved revealing differences between measurement of the analysed substance level in blood samples and measurement of the analysed substance level in reference solutions used for testing of optical devices used to perform these measurements. The reference solution contains: a marking substance revealed by the optical device to distinguish measurements, e.g., of glucose level received for the reference solutions from measurements of glucose level received for biological fluid sample, e.g., blood.

EFFECT: use of the invention allows revealing differences between measurements of the analysed substance level in blood and reference solutions.

2 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology, namely to immunological diagnostics. There is disclosed a manufacturing technique of serum panels containing antibodies of danger viral infections. For making the serum panel containing various antibodies, within primary screening (by enzyme immunoassay (EIA) and polymerase chain reaction (PCR)), 60 immune-specific bloods serums of HBV-infected persons with positive PCR and 25 donor serums are sampled. The analysis of the EIA and PCR data ensures to establish that the immune-specific serums used in making said panels contain antibodies to HBs, HBc and to HBe antigens. Simultaneously, candidates are sampled as HBV DNA negative and not containing antibodies to HBV antigens. This sample is used to prepare 6 pools of negative samples for the panel.

EFFECT: development of the serum panel which contains all range of antibodies to hepatitis B virus and enables to control clinical effectiveness in treating hepatitis B.

3 tbl, 1 dwg, 2 cl

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biotechnology and immunology and can be used in making standard sera panels, containing DNA and antigens of viruses of dangerous infections, and in production of a test-system for detecting HBV DNA and antigens as control positive sera. The sub-type panel contains 30 samples with HBsAg concentration from 1.0 IU/ml to 0.06 IU/ml, including 24 samples of AY sub-type, prepared from a plasma antigen. Introduction into serology of standard panels with certified concentration of HBsAg of different sub-types forms the basis for controlling accuracy of analysing content of these markers in blood.

EFFECT: formation of the basis for controlling accuracy of analysing content of hepatitis virus in blood.

4 tbl, 2 dwg, 2 cl

FIELD: biotechnology, immunology, vaccines.

SUBSTANCE: invention relates to Master-panel used for assay of hepatitis B. Invention proposes Master-panel of lyophilized sera comprising and not comprising HBsAg wherein the positive part of Master-panel comprises plasma samples obtained from patients suffering from hepatitis B and consists of three blocks: block 1 comprises sera of A & D genotypes of different subtypes HBsAg (ayw2, ayw3, adw4 and adw2), block 2 comprises positively attested sera diluted to mean and low concentrations (in the range from 1.5 IU/ml to 0.05 IU/ml), block 3 comprises sera showing OD values near or below ODcr and positive by PCR method or having high level of aminotransferases activity; negative part of panel comprises plasma samples obtained from donors, persons from risk groups and vaccinates persons. Master-panel must store at temperature 2-6°C and diluted sera for panel are stored at temperature 2-6°C for one month and at temperature -24°C for 6 months. Master-panel provides standardizing sera used in carrying out analysis for hepatitis B.

EFFECT: valuable medicinal and analytical properties of sera.

4 cl, 1 tbl, 2 ex

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the present innovation deals with evaluating autoimmunity to ganglioside of human hepatocytes. In blood serum due to immunoenzymatic assay one should detect the content of autoantibodies IgM and IgG to ganglioside of hepatic tissue, calculate the ratio of the content of autoantibodies in tested serum against their content in negative control, and at its value being above 1 it is possible to detect the presence of autoimmune process in human liver. The innovation enables to fulfill diagnostics of autoimmune process in hepatic tissue at higher accuracy and specificity.

EFFECT: increased accuracy and efficiency of evaluation.

FIELD: medicine.

SUBSTANCE: method involves taking synovial fluid sample for testing it with chondroitin sulfate as antigen. Critical optical density value and optical density of the sample at 450 nm is determined. The optical density of the sample being found greater than the critical optical density value, autoimmune process in osteoarthrosis patient articulations is to be diagnosed.

EFFECT: high accuracy in determining contraindications to intra-articular chondroprotectors introduction.

FIELD: immunology.

SUBSTANCE: method involves selection of diluting sera by analysis of donor sera by immunoenzyme analysis and polymerase chain reaction for the presence of hepatitis B virus DNA, anti-HBs antibodies and factors of nonspecific binding HBsAg. Method involves selection of sera with negative result in these methods as diluting sera, titration of highly concentrated HBsAg preparations in diluting stabilized solution to the required concentration HBsAg (5-10 IU/ml) of AY and AD subtypes. Invention provides preparing two working serum standards comprising AY and AD subtypes of HBsAg retaining specific indices of these samples for a long time in storage at from 2°C to 6°C. Invention can be used in technology for preparing control samples for test-systems for detection of HBsAg.

EFFECT: improved preparing method.

4 cl, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: by means of bioluminescent method determined is activity of NADPH- and NADP-dependent reactions of glutamatdehydrogenases of peripheral blood lymphocytes of patients with chronic hepatitis C, and value of coefficient NADPH/NADP+ in enzymatic reactions of glutamatdehydrogenase is used to make conclusion about possibility to develop chronic hepatitis C exarcerbation. If coefficient value is ≥25, development of exarcerbation is predicted, if coefficient value is <25 - absence of exarcerbation.

EFFECT: application of method allows to make quick prediction of CHC exacerbation in children with clinical-biochemical remission at early stages.

1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: in order to realise invention in lymphocytes, which are separated from venal blood, glutationreductase enzyme (GR) activity is determined. If index value equals and more than 2.06 mcU/10000 cell first-second stages of chronisation are diagnosed, and if value is less than 2.06 mcU/10000 cell - third stage of chronisation of infection process. Method requires single sampling of 4-6 ml of blood from vein.

EFFECT: possibility to predict disease course and carry out proper therapy correction.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: serum content of easily detected biological markers - apolypoprotein A1, alpha.2-macroglobulin, alanine-aminotransferase, gamma-glutamine transpeptidase and triglycerides- is determined. Invention also relates to diagnostic sets for method realisation.

EFFECT: increase of accuracy and self-descriptiveness of liver steatosis diagnostics.

14 cl, 7 dwg, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: blood serum is analysed for ceruloplasmin activity. If ceruloplasmin is 37.9 relative units and lower, high degree of activity of chronic hepatitis is diagnosed, the value of ceruloplasmin within 38.0 relative units to 46.4 relative units show moderate degree of activity, and mild degree is indicated by ceruloplasmin within 46.5 to 57.9 relative units.

EFFECT: use of the method allows for the most precise determination of degree of activity of chronic hepatitis in out-patient conditions.

3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to laboratory diagnostics of internal diseases. In order to realise the method of differential diagnostics of chronic viral hepatitis and liver cirrhosis in patients' blood plasma activity of von Willebrand factor (vWF) is determined, which is evaluated on aggregometre by induced by examined plasma agglutination of standard platelet preparation. If vWF value is less or equal 102% chronic viral hepatitis is diagnosed, if vWF is higher than 102% - liver cirrhosis.

EFFECT: application if invention allows to realise available, non-traumatic, sensitive, specific and efficient differential diagnostics of chronic viral hepatitis and liver cirrhosis of viral genesis by degree of endothelium injury.

1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method includes certification of serum samples for observed antibodies to hepatitis C virus to assess availability thereof as an operational standard. The serum samples are certified by immunobiological markers of HBs antigen, hepatitis C virus antibodies, HIV types 1, 2 and anti-HBs, anti-HBc, anti-HBe antibodies, and by the biochemical properties: enzyme level, total protein, pH. The immune-specific samples are serums containing RNA of hepatitis C virus and positive results of the immune-enzyme assay ("ОП<ОПкр."), negative plasma samples of "ОТ" - PCR with "ОП<ОПкр." The operational standard represents a complete set of negative and a number of positive serums containing various types of antibodies to hepatitis C virus.

EFFECT: according to the invention, the methods allows making the operational standard containing serums close to the human native serums, and also assessing the quality of each antigen: core, NS3, NS4 and NS5, used in development of the immune-enzyme test systems for detecting antibodies to various proteins of hepatitis C virus, storing and handling the operational standard at higher temperatures.

4 cl, 5 tbl

FIELD: medicine.

SUBSTANCE: in order to optimise antigen composition, polypeptides containing additionally epitopes characteristic of most hepatitis C viruses are included in the set as antigens.

EFFECT: high specificity properties.

2 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, namely to diagnostics of infectious diseases and concerns a method of revealing antibodies to antigens of hepatitis viruses. The essence of the way consists in use of an immunochromatographic strip which consists from fiber glass non-sorbing matrix (6) on which the strips containing conjugates of painted particles with monoclonal antibodies to human immunoglobulins G and M (7,8) plastic substrates are placed and immobilised by means of drying, covered with an adhesin layer with low penetrating ability (1) on which a microporous nitrate cellulose membrane with evaporated on a side close to the substrate with a protective polymeric basis (2) is mounted, and admixtures of antigens of viruses of a hepatitis A, B, C (3, 4, 5) are placed perpendicularly to the long side of the strip on the membrane from the non-protected side. The strip is moistened with investigated blood serum and at detection of the painted strip around the strip of antigens of hepatitis A, antibodies to an antigen of a virus of hepatitis A are detected. At detection of the painted strip around the strip of antigens of hepatitis B, antibodies to an antigen of a virus of hepatitis B are detected. At detection of the painted strip around the strip of antigens of hepatitis C, antibodies to an antigen of a virus of hepatitis C are detected, which are formed at linkage of antibodies of blood serum with conjugates of antibodies to immunoglobulins G and M with the painted particles.

EFFECT: method allows to tap simultaneously antibodies to antigens of viruses of a hepatitis A, B, C and to define to what type they are specific to.

2 ex, 1 tbl, 1 dwg

FIELD: medicine.

SUBSTANCE: detection or confirmation of hepatitis B virus surface antigen (HBsAg). Immune-enzyme test system for HBsAg identification in blood serum (plasma) and blood preparations (albumin, fibrinolysin, immunoglobulins and leukocytic interferon) containing: solid phase containing immunosorbent that is mice HBsAg monoclonal antibodies fixed on tray strips; conjugate -1 - biotin with HBsAg monoclonal antibodies and conjugate-2 - streptavidine marked with horseradish peroxidase as liquid concentrates; positive control sample; negative control sample; washing solution; solution for conjugates dilution; substrate buffer solution; tetramethyl benzidine as chromogen; sulfuric acid as stop-reagent; neutralizing reagent, liquid or lyophilized that is goat HBsAg antibodies immunoglobulins; control reagent liquid or lyophilized that is goat immunoglobulins without HBsAg antibodies; weakly-positive control sample that is HBsAg lyophilized blood serum in concentrations 0.02-0.06 ME/ml. Method of hepatitis B virus surface antigen (HBsAg) detection is performed through immune-enzyme test of biological sample by means of the given test system.

EFFECT: invention allows to detect required antigen of low sensibility at high specificity level.

4 cl, 1 ex, 8 tbl

FIELD: medicine, immunology.

SUBSTANCE: invention relates to methods for in vitro diagnosis of infection caused by microorganism, and using reagents and sets for detection of antigens and antibodies raised against infectious microorganism. Method involves carrying out simultaneous detection of microorganism antigen and antibodies raised against this antigen. For detection of infection method involves carrying out contacting biological sample with a binding antibody raised against microorganism and a binding antigen from this microorganism. Mixture is incubated and formed complexes "antigen-antibody" are revealed using labeled and, if necessary, detecting antibody and/or detecting antigen. Binding and/or labeled detecting antigen comprises fragment of microorganism antigen wherein at least one epitope of that is inactivated, and a binding and/or detecting antibody recognizes native epitope of bound antigen. Also, invention proposes a set for detection of infection and peptides or polypeptides of hepatitis C and HIV proteins using of that allows carrying out a method for diagnosis of these diseases. Using the proposed method allows enhancing precision of diagnosis of infection caused by microorganism.

EFFECT: improved detecting method.

42 cl, 13 tbl, 2 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: what is offered is an expression construct for expression of single- or multipass transmembrane polypeptides in a bacterial host cell. Said construct contains a protein-coding polynucleotide, a strictly sensitive promoter of lower basal activity in the host-cell, and a leader sequence comprising a translation initiation enhancer. The strictly sensitive promoter comprises at least one positive control element and at least one negative control element. One or more positive and negative control elements represent a heterologous control element. Besides, what is offered is a method for producing the expressed transmembrane polypeptide and a method of recovering it form the host cell.

EFFECT: methods provide producing the high-yield transmembrane polypeptides either of native conformation, or in a soluble form.

53 cl, 28 dwg, 5 tbl, 10 ex

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