RU2346282C2 - Method of making panels of sera of different sub-types and genotypes of hepatitis b virus for controlling quality of hepatitis b diagnosis - Google Patents

Abstract

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

Description

The invention relates to the field of biotechnology and immunology and can be used in the technology of manufacturing reference panels of sera containing antigens of the most dangerous viral infections.

Blood samples from donors in Russia must be screened for the presence of HIV 1 + 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 confirming the results depends not only on the quality of the test systems, but also on the availability of standard drugs for the analyzed infectious markers in the control laboratories [1, 2].

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

The use of reference drugs of different genotypes and subtypes of HBsAg in everyday work will expand the range of diagnosed markers and use these materials to control the quality of blood tests for the detection of hepatitis B.

According to experts in 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.

This hypothesis was confirmed by Dr. H.Fields, who, when forming the HBV Master Panel, assembled a collection of 1000 patient plasma samples from all 6 HBV DNA genotypes. When sequencing these samples, H. Fields found that in almost 100% of cases, genotype D is found in sera from Tunisia and Jordan, genotype A is common in Egypt and the United States. In Russia, the distribution of genotypes: 90% genotype D and 5% genotype A.

Thus, in solving the problem of assessing the quality of the diagnosis of viral infections in Russia, a collection of plasma of patients with an established diagnosis should be collected, mainly from local infectious disease clinics. And on the basis of this material form national serum panels. This conclusion has a statistical justification, as More than 90% of samples from the indigenous population pass through diagnostic laboratories; the number of samples from visitors and tourists does not exceed 5-7%.

Consequently, national standard panels should include plasma samples with genotypes and subtypes characteristic of Russia.

In order to solve such problems, a method for the manufacture and certification of human blood serums containing various HBV genotypes and HBs Ag subtypes at a concentration above 0.06 IU / ml was developed. Such panels are designed to control HBV DNA analysis and to analyze the concentration of HBsAg in blood samples and its components.

Methods for 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 [1]. 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 in titers of about 0.1 IU / 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). This panel includes 10 samples with A-D genotypes [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 are intended for use in laboratory tests only. In addition, the serums that make up these panels are presented in liquid form and therefore should be stored frozen. Serum panel prototype is not stabilized and not lyophilized. Distribution and storage of BBI panels require strict adherence to low temperature conditions [3].

It should be understood that the diagnostic task is to identify carriers of hepatitis B infection, and not just HBV DNA or HBsAg. Thus, researchers armed with one PCR or ELISA method can detect carriers of hepatitis B in the general population only partially. For completeness, each blood sample should be tested for both HBV DNA and HBsAg. Therefore, you need a standard that allows you to simultaneously record the quality of PCR diagnostic kits and ELISA test systems. Such a role can be played by the national panel of subtypes, the sera of which are certified for HBV DNA content and HBsAg concentration.

This panel is based on a sample of 80 primarily HBsAg positive plasma samples genotyped and subtyped for HBV DNA.

The specificity of PCR and ELISA diagnostic kits for the detection of HBV markers can be certified on a sample of 6 pools of negative serum donors and persons of the blood group.

Thus, a panel of subtypes was developed and manufactured for state control and for conducting national tests of ELISA test systems to identify various subtypes of HBsAg. With additional certification, a standard panel can be used to certify PCR diagnostic kits for the quantitative identification of HBV DNA.

The objective of this invention is to create such a method that would ensure the manufacture of panel samples with a certified concentration of various subtypes of HBsAg and allow you to save the specific characteristics of these samples for a long time (at least 2 years) when stored at 2-6 ° C.

The problem is solved in that they use a method of manufacturing panels of serums containing HBV DNA and HBsAg to control the quality of test systems and vaccines against hepatitis B, including the selection of donor serums that do not contain specific antibodies to the main markers of viral infections, the manufacture of dilution solutions based on selected sera, performing molecular analyzes to determine the HBsAg subtypes, diluting them in a diluting solution, conducting a thermal degradation test to determine the shelf life of the panel, according to Retenu selection is carried out by distributing the analysis sera donor sera by enzyme immunoassay and polymerase chain reaction for the presence of HBV DNA, and antiHIV HBs antibodies, nonspecific binding of HBsAg, wherein as distributing sera selected whey negative result in these methods. The HBsAg samples are titrated in a diluted stabilized solution to the required concentration of HBsAg in the range 0.06-1.0 IU / ml. As HBsAg preparations, the sera of patients with a diagnosed viral hepatitis and positive in PCR studies for HBV DNA are used.

To form the panel, 30 candidate samples were selected containing HBsAg subtypes adw2 and ayw1, awy2, ayw3 and ayw4 (ayw1-4). These samples are diluted in a stabilized pool of donor sera to HBsAg concentrations from 1.0 IU / ml to 0.06 IU / ml. To control specificity, the panel included 6 samples of native donor sera negative for PCR and not containing anti-HBc antibodies.

The prepared samples of the subtype panel are freeze-dried to a residual moisture content of not more than 1.5 wt.%.

Such a model is selected on the basis of the following premises. From the literature it is known that the main subtypes of HBsAg in Russia are ayw2 and ayw3, ayw1 and ayw4 are less common [5]. However, in Europe and Japan, the main subtype is adw2 [6]. Therefore, all of these subtypes are included in the Russian panel of subtypes.

Unfortunately, the best Russian test systems have a sensitivity of 0.1-0.2 IU / ml, but only for the AY subtype of HBsAg. Often, the sensitivity of tests to the AD subtype usually does not exceed 0.3 IU / ml. This is what determines the relevance of creating a panel of HBsAg subtypes.

To determine the HBsAg subtype, PCR sequencing of positive plasma samples should be used.

In this work, the isolation of HBV DNA is carried out using kits for the isolation of viral DNA from blood serum of the company "QIAGEN".

Sequencing / genotyping of a 404 bp nucleotide sequence including determinant A is carried out according to the method and using the primers developed by Dr H. Fields (CDC, USA). Four primers were calculated using the computer program "Oligo v.5.0" and synthesized (see table 1). The length of the first round PCR product using the primers RMB1 and RMB2 is 467 bp. The length of the second round PCR product using primers RMB3 and RMB4 is 454 bp. Sequencing is performed using the CEQ DTCS Kit on a Beckman CEQ2000XL automatic sequencer ..

Knowledge of the nucleotide sequence in the study region provides an opportunity for real determination of the HBsAg subtype in the sample. In this work, the determination of the antigen subtype is carried out according to the results of the nucleotide sequence using the computer program "subtyper", presented by Dr. X. Fields

As an example, we give several sequences.

In the course of work, genotypes and subtypes are determined for 80 samples selected for the manufacture of the panel (distribution by genotypes and regions is presented in Table 2).

table 2 Generalized genotyping data City \ Genotype BUT AT C D E F G Total Krasnodar (I) 0 0 0 10 [100%) 0 0 0 10 [13%) Nizhny Novgorod (II) 1 (16%) 0 0 5 (84%) 0 0 0 6 (8%) Mountain Altai (III) 0 0 0 5 (100%) 0 0 0 5 (7%) Saint Petersburg (IV) 2 (12%) 0 0 19 (88%) 0 0 0 21 (24%) Novosibirsk (V) 0 0 0 16 (100%) 0 0 0 16 (20%) Khabarovsk (VI) 1 (7%) 0 0 14 (93%) 0 0 0 15 (19%) Irkutsk (VII) 0 0 0 7 (100%) 0 0 0 7 (9%) Total 4 (5%) 0 0 76 (95%) 0 0 0 80 (100%)

In the course of the work, genotypes A and D and subtypes adw2, ayw2, ayw3 and ayw4 were identified.

To establish a relationship between the value of the analytical ELISA signal and the amount of HBsAg in the diluting solution, it is necessary to use ELISA test systems that maintain a straightforward relationship between the optical density and the titer of the HBsAg solution in the range from 1.0 to 0.1 IU / ml.

Our model of the HBsAg subtype panel is designed to monitor the effectiveness of test systems to detect various subtypes and antigen concentrations and to monitor the professional level of diagnostic laboratory workers.

Thus, a complete set of subtype panel should include 36 samples: 6 samples of donor serum pools and 30 samples of different HBsAg subtypes prepared by diluting native plasma samples in a stabilized pool of donor sera to clinically significant antigen concentrations.

The use of 6 pools with different levels of nonspecific binding as negative samples allows us to differentiate commercial test systems by specificity at a higher level than in the case of BBI panels, which include only 1 or 2 negative samples.

An important problem when using HBsAg test systems in clinical diagnostic laboratories is the correctness of the analysis results. The main criterion for the correctness of the results of serological analysis is the certified value of the analytical signal (for example, OD in ELISA) for each panel sample. In this case, a prerequisite for the validity of test systems is a constant level of this analytical signal during long-term storage.

The question of preserving the specific properties of panel sera containing IgG antibodies was resolved in [7]. In the present invention, to stabilize the level of HBsAg, it is proposed to use a stabilizer comprising not more than 1.0 wt.% Hydrolyzed (enzymatic) peptone. The introduction of peptone is an important technique. The fact is that enzyme peptone has 2 positive qualities that distinguish it among other protein supplements: it has a low specific viscosity (0.029) [5] and contains about 10% of free amino acids. The low viscosity of the stabilizer solution for the standard panel is a critical parameter for detecting HBsAg in practice, as 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.

The applicability of the reference preparations created according to the present invention — serums of various subtypes with a normalized level of HBs Ag — was confirmed by analyzing the panels in scientific laboratories and control laboratories of the service of manufacturers of test systems in accordance with their certification program developed by the national control authority of the Russian Federation - GISK them. L.A. Tarasevich. Based on the results presented by participating analytical laboratories, the effectiveness of detecting various subtypes in the test systems used for analysis will be finally established and on this basis certified values of HBsAg concentrations in panel samples will be established.

Thus, the certification procedure for panel sera includes three steps, the results of which are independent and allow reliable identification of various subtypes of HBsAg. Comparison of the reference panels of subtypes, prepared in accordance with the claimed method, with the prototype panels of the same purpose, showed that a combination of essential features different from the prototype provides a new previously unknown property: the ability to manufacture subtype panels in lyophilic form with a certified concentration of different HBsAg subtypes, which allows to evaluate the sensitivity of test systems and specificity to various subtypes of HBsAg and to monitor the level of professional excellence of clinical workers their diagnostic laboratories.

Figure 1 - The results of the test suppression of HBsAg in serum donor blood.

Figure 2 - Average weighted AY & AD titration curves of HBsAg samples.

An example of the method

Preparation of serum pools. The selection of donor sera is carried out in several stages:

a) Selection of sera that do not contain antibodies to HIV, hepatitis B and C viruses, syphilis; each serum is analyzed for the presence of HBV DNA. The results of HBV DNA analysis for all donor sera are negative.

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

c) Determination of the level of non-specific binding of HBsAg in test sera.

d) Formation of serum pools with equal levels of non-specific binding.

Donor sera selected after 3 steps are pooled. A stabilizer is added to the pool in dry form, the components of which do not have an effect on the analytical signal of HBsAg and on the viscosity of the serum. A stabilized serum pool is a dilution solution (PP) for preparing positive subtype panel samples.

Preparation of dilution solution

Stabilized donor serums that do not contain markers of viral infections are used as PP to obtain positive panel samples with a certain concentration of HBsAg. Serum is prepared immediately after blood sampling. Whole blood is centrifuged at a speed of 1800-2000 * g at a temperature of 10-12 ° C for 15 minutes and the supernatant is decanted. Store until use at 2-4 ° C. To prepare the panel, amber-colored sera samples are taken that are transparent in the light. A total of 65 high-quality sera were selected.

After preparation, all sera are analyzed for anti-HBs antibodies and viral contamination. 9 sera containing antibodies were found, they were rejected.

In the next step, they put down a suppression test. For this purpose, 1 ml aliquots of each serum number are taken and the same amount of HBsAg calculated for a final concentration of 2 IU / ml and 1 mg / ml for each serum is introduced into them (Fig. 1). As follows from figure 1, almost every serum non-specifically interacts with HBsAg. Taking into account the results of the suppression test allows you to divide the serum into 3 pools, similar in magnitude to the OD in the ELISA.

A stabilizer is prepared according to the recipe described in previous patents [7].

Each 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 - 0.02% by weight of the solution. PP is poured into containers of 250-500 cm ³ and used immediately or stored at a temperature of 2-8 ° C for no more than 1 month.

Preparation of positive samples of the standard panel. To prepare samples of a standard subtype panel, 30 plasma samples were taken (6 samples of each ayw subtype (1-4) and adw2 subtype were prepared)

To select candidates for the joint venture, 80 plasma samples positive for HBV DNA were genotyped and subtyped. Based on the results of subtyping and ELISA, 6 samples of the indicated subtypes with the highest concentration of HBsAg were selected.

The method of statistics from individual titration curves build a weighted average curve (figure 2). The average curve determines how much HBsAg should be added to each PP so that the final antigen concentration corresponds to 0.5-1.0 IU / ml. 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.

The properties of the samples of the HBsAg subtype panel are presented in Table 3.

At the last stage, all panel samples are filtered sequentially through 0.8 μm and 0.45 μm filters.

Filtered samples of the subtype panel are poured in a 0.8 ml laminar cabinet into 2 ml bottles, closed with rubber stoppers and placed in metal cassettes for freezing. The freeze drying mode is similar [7]. Freezing of the drug in the bottles is carried out at a counter temperature of 50-60 ° C for 10-12 hours. Frozen samples in the bottles are transferred to the sublimation chamber of the TG-50 lyophilic unit, the shelves of which are cooled to minus 30 ° С. The temperature of the drug at the stage of freeze-drying 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 process is maintained automatically with an accuracy of 3-5 degrees. The temperature values of each shelf, the temperature of the material on each shelf, as well as the sublimator temperature are recorded continuously 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. 24 hours after the temperature of 10 ° C was established on the shelves, the RP-32 setter raised the temperature of the material to dry to 27 ° C. The material is kept at this temperature for 6-8 hours. The average heating rate of the material at the 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 1.0-2.0 wt.%. The determination is carried out according to the method [7]. 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, removed at regular intervals, dissolved in bidistilled water and analyzed in duplicates in the Recomatgep B (Vector) 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 [8]. The calculated% loss of activity for the month and for the year are given in Table 4. Samples showed high stability. The predicted loss of their activity for the year at 4 ° C is about 0.3%. The predicted loss of activity per month is approximately 4% at 37 ° C, giving the possibility of short-term storage at elevated temperatures, for example when transporting samples.

Table 3
Predict the activity of MP samples based on accelerated tests Storage temperature, ° С Loss of activity for the year,% Monthly activity loss,% four 0.28 0.0215 37 44 4.4 45 86 fourteen 60 99 63

It is recommended that dissolved MP samples be used when stored at 4 ° C for a month. The stability of liquid MP samples was tested in the freeze-thaw test. The test results showed that the specific properties of the candidates do not change during 4 cycles of freezing and thawing.

The economic efficiency of the proposed method for the manufacture of HbsAg panel samples of various subtypes is that these antigens are used as prognostic markers of hepatitis B. They circulate in the blood of infected people 2-3 weeks earlier than antibodies appear. It is the reduction of the time needed to establish diagnoses for infected patients and the earlier medical care and the treatment controlled by the number and subtypes of HbsAg that make up socially significant and economic efficiency associated with a reduction in the time of treatment and the duration of labor incapacity for people.

The introduction of the Russian panel of subtypes will significantly reduce the cost of funds of controlling healthcare organizations for the acquisition of expensive foreign analogues.

Table 4 Characteristics of HBsA9 Subtype Panel Samples No. Hepastrip B Ecolab AgHBs Monolysis Plus Subtypes Genotypes Op oe OP OP one Ayw1 DAyw1 0.338 0.197 0.666 2 DAyw1 0.186 0.170 0,307 3 DAyw1 0.145 0.139 0.172 four DAyw1 0.250 0.245 0.646 5 DAyw1 0.121 0.138 0.287 6 DAyw1 0,078 0,112 0.165 7 Ayw2 DAyw2 0.319 0.331 0.774 8 DAyw2 0.165 0.171 0.442 9 DAyw2 0.124 0.118 0.277 10 DAyw2 0.283 0.469 0.942 eleven DAyw2 0.198 0.250 0.46 12 DAyw2 0,083 0,078 0.156 13 Ayw3 DAyw3 0.462 0,200 0.652 fourteen DAyw3 - 0,096 0.535 fifteen DAyw3 0.174 0,100 0.424 16 DAyw3 0.216 0.153 0.32 17 DAyw3 0.159 0,095 0.27 eighteen DAyw3 0.144 0,078 0.239 19 Ayw4 DAyw4 0.656 0.737 twenty DAyw4 0.377 0.434 21 DAyw4 0.201 0.244 22 DAyw4 0.238 0,203 0.548 23 DAyw4 0.150 0.136 0.317 24 DAyw4 0.160 0.154 0.555 25 ADW2 Dadw2 0.175 0.498 0.49 26 Dadw2 0,090 0.259 0.191 27 Aadw2 0.485 0.490 1,542 28 Aadw2 0,200 0.192 0.588 29th Aadw2 0.130 0.119 0.33 thirty Aadw2 0.106 0,076 0.186 31 Gen a 0,054 0,046 0,046 32 Gen a 0,065 0,072 0,054 OP Crete 0.114 0,076 0,099 33 Negative 0,071 0.058 0,068 34 Negative - 0,064 0,047 0,041 35 Negative - 0,068 0,078 0,085 36 Negative - 0,062 0,054 0.48 37 Negative - 0,07 0,067 0,049 38 Negative - 0,066 0,054 0,048 OP Crete 0,100 0,084 0,095

Literature

1. VQC Pelispy Multi-Marker, run control. Central Laboratory of Red Cross, Netherlands, 1998.

2. Product catalog 2003/2004 Boston Biomedica, Inc., 2004, p. 28.

3. Damen, M., Cuypers, H.T.M., Zaaijer, H.L., Reesink, H.W., Schaasberg, W.P., Gerlich, W.H., Niesters, H.G.M., Lelie P.N. (1996). International collaborative study on the second EUROHEP HCV-RNA reference panel. J. Virol. Methods 58, 175-185.

4. Thon E.V., van Loon A., Schrim J. et al. (2001). European Proficiency Testing Program for molecular detection and quantitation of hepatitis B virus DNA. 39, 12, 4407-4412.

5. Netesova I.G., Swenson P.D., Osipova L.P., Gubina M.A., Posukh O.L., Netesov S.V. (2003). Determination of HBsAg subtypes in Western Siberian part of Russia. // J Med Virol 71, 183-187.

6. H.A. Fields. Diagnosis of Viral Hepatitis in the Blood Transfusion Services: NAT or NOT. CDC, Atlanta 2004.

7. Kanev A.N., Fields H.A., Bektimirov T.A., Shalunova N.V., Maksyutov A.Z., Yudina I.V., Cherepanova N.S., Musina E.E., Bocharova N.G. A method of manufacturing a control panel of serum AY & AD subtypes of HBsAg for monitoring the quality of diagnosis of hepatitis B, RF patent No. 2265028, published 2003.

8. Kirkwood, T.B.L. The principles of preparation and approval of international and other standards and reference materials of biological preparations. Ser.techn. doc. N 37. WHO, Geneva, 1990. 14a // J.biol. Standardization. - 1984. - Vol. 12. - P.207-224.

Claims (2)

1. A method of manufacturing a panel of sera of various HBsAg subtypes and genotypes for monitoring the diagnosis of hepatitis B, including the selection of donor sera that do not contain specific antibodies to the main markers of viral infections, the manufacture of dilution solutions based on selected sera, the dilution of HBsAg preparations in a dilution solution, the formation of a panel of sera of various subtypes and thermal degradation test to determine the shelf life of the panel, characterized in that the selection of panel sera is carried out according to the results of the gene otyping and subtyping of reactive sera by enzyme-linked immunosorbent assay and sequencing of hepatitis B DNA samples; moreover, serums with a negative result in these methods are selected as diluting sera; plasma antigens belonging to the ADW2 & AYW1-4 subtypes are used as HBsAg drugs; titration of selected drugs HBsAg in a diluting stabilized solution is carried out to the desired concentration of HBsAg 0.06-1.0 IU / ml ADW2 & AYW 1-4 subtypes, and 6 negative ones are made to form a panel of subtypes samples and 30 samples with HBsAg concentration from 0.06 IU / ml to 1.0 IU / ml, including 6 samples of AYW1 subtype, 6 samples of AYW2 subtype, 6 samples of AYW3 subtype, 6 samples of AYW4 subtype and 6 samples of ADW2 subtype, prepared on the basis of plasma antigens. 2. The method according to claim 1, characterized in that the prepared samples of the control panel are freeze-dried to a residual moisture content of not more than 1.5 wt.%.

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