RU2179726C2 - Method for producing blood serum control panels for controlling hepatitis b diagnosis quality - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: panel has five native blood sera diluted to HBs Ag concentration being from 1 MU/ml to 0.1 MU/ml, the sera belonging to different HBs Ag subtypes, and three native blood sera as negative samples. The prepared sera are dried with lyophilization with residual moisture content being not greater than 1.5 % by mass. EFFECT: wide range of action; prolonged storage life of serum panels containing dangerous virus antigens. 5 cl, 1 dwg, 2 tbl

Description

 The invention relates to the field of immunology and standardization and can be used in the production technology of serum panels containing antigens of the most dangerous viral infections, and in the organization of external control of laboratory studies.

 External quality control of laboratory tests is an integral part of ensuring the information content and reliability of laboratory diagnostics [l]. Serological diagnosis of HBsAg is the main tool for monitoring and preventing hepatitis B.

 For this purpose, the enzyme-linked immunosorbent assay, ELISA, is most commonly used for screening. To perform ELISA, test systems are used, which are multicomponent sets of ingredients. The effectiveness of the enzyme-linked immunosorbent assay is influenced not only by the quality of these ingredients, but also by the professional preparedness of the laboratory personnel providing the test [2]. To ensure an objective external assessment of the quality of laboratory research, specially prepared and certified control materials are extremely necessary.

 The WHO Expert Committee on Standardization of Biological Products has been preparing control materials and an external quality assessment of laboratory tests for HIV antibodies since 1988. At the WHO Expert Meeting in 1996, particular attention was paid to the certification of national standards used to control test systems and quality control tests for the identification of markers of HIV, hepatitis C and B. The expert council recommended the certification of national standards for immunobiological preparations and preparations based on human serum relative units (ME), but not in absolute (ng) as the procedures of concentration and isolation of the desired proteins can uncontrolledly change their specific properties [3].

 When implementing an external quality control program (PACC), it should be borne in mind that laboratory studies of serum samples for the presence of HBsAg are performed using test systems from different companies. The spectrum of detected subtypes and the limiting sensitivity of the analysis are the main criteria for evaluating the quality of diagnosis of hepatitis B in serological laboratories of clinics and blood transfusion stations.

 The test systems used in laboratories have a different structure and different modes of analysis. Some test systems are based on 2-3 monoclonal antibodies (MAT), while others include additional polyclonal antibodies (PAT). The introduction of PAT is known to eliminate the “hook-effect” and expand the spectrum of detectable HBsAg subtypes [4,5]. The main advantage of MAT-based kits is their high specificity. Unfortunately, monoclonal test systems are often not able to detect all subtypes of HBsAg. They have a significantly higher sensitivity to “their subtype” than test systems with the participation of PAT, but can absolutely not identify the “alien subtype”.

 In addition, in the general case, the optical density for solutions of control materials is different for different diluting solutions. In connection with these and similar problems, the WHO Expert Council recommends that instead of describing the concentration of HBsAg in ng / ml, use the classical method for biochemical components in international units (ME).

To establish the relationship between ME and ng / ml use:
- HBsAg preparation - plasma purified, the content of the desired protein>95%;
- control samples ser. 2 and 4 tests of "Labsystems", certified relative to the standard sample (CO) of the Institute of P. Ehrlich, ay - subtype, approximately 1 IU / ml.

 Titration of certified preparations is carried out in the test systems "Recomatgen B" and "Labsyatems". As a result of the tests, it was found that for the Rekomatgen B test system the equivalent is 1 U / ml = 4.1 ± 0.3 ng / ml and for the Labsystems test system the equivalent is 1 U / ml = 5.2 ± 0.2 ng / ml.

The solution to the problem of creating a control panel of human serum for the content of HBsAg is proposed to be carried out in the following way:
- use for the creation of a control panel the native sera of patients diagnosed with hepatitis B, belonging to different subtypes, with a concentration of HBsAg certified in international units;
- use a pool of sera of healthy donors that do not contain markers of major infections as a diluting solution;
- use known material as a stabilizer [6].

 Boston Biomedical Inc. serum panels are known. (USA): Hepatitis B Surface Antigen Sensitivity Channel (PHA 806) [7]. The panel includes samples of sera that are simultaneously certified for HBsAg concentration in absolute - from 2.5 ng / ml to 0.1 ng / ml and in relative units from 0.31 to 0.01 IU / ml and one negative serum.

 The closest technical solution (prototype) is the method developed in the Central laboratory (Dutch Red Cross standards for the preparation of Pelichek control panels [8]. These panels are 8-10 diluted samples of the standard HBsAg preparation in a pool of donor serums that do not contain hazardous markers viral infections.

The prototype method involves the preparation of a standard serum from a pool of reactive native sera containing HBsAg and having different concentrations of this protein. The main steps in the production of standard serum are:
- collection of reactive sera for the preparation of the standard HBsAg;
- collection of donor sera that do not contain antiHBs antitol and markers of major viral infections;
- the manufacture of diluting serum, not containing fibrinogen and lipids, from a pool of selected donor serums;
- dilution of the standard HBsAg in diluting serum 4, 8, 16, 32, 64, 128, 256, 1024, 2048 and 8192 times.

 The advantages of the prototype method include the development of a general scheme for the preparation of the HBsAg standard and the preparation of diluting serum, which can be implemented in the manufacture of serums for control panels with HBsAg and other markers (anti-HIV and anti-HCV).

The main disadvantages of the prototype:
- breeding serum is an idealized model (i.e., fibrin, fibrinogen and lipids have been removed from the serum) as a model of native human blood serum;
- the Pelicheck panel was prepared on one serum pool, the quantitative concentration of HBsAg was not certified, and only the multiple dilution method was used. For another pool of sera, other HBsAg concentrations will respond to the same dilutions;
- the panel does not show negative sera;
- serum panel prototype is not stabilized and not lyophilized.

 The objective of the present invention is to provide such a method that provides the manufacture of a control panel from native sera containing HBsAg of different subtypes with normalized concentrations of HBsAg in international units, to assess the quality of the HBsAg analysis and allows you to save the specific characteristics of the panel sera for a long time (about 2- x years).

 The problem is solved in that in the method of obtaining serum panels for the quality control of laboratory tests for hepatitis B, including the selection of donor sera that do not contain specific antibodies to the main markers of viral infections, the preparation of a dilution solution from a pool of selected sera, the selection of reactive native HBsAg sera and dilution them in a diluting solution, conducting a thermal degradation test to establish the shelf life of the control panel, according to the invention, the selection of diluting serums for the panel The first is carried out according to the scheme, including their certification by ELISA and PCR for the presence of HBsAg and DNA of hepatitis B virus (HBV) and titration of samples of HBsAg, and negative serum panels are formed from native sera with zero titer, which have a negative result in PCR DIC of HBV, and in ELISA - the value of OD is less than critical (in the range of 0.6-0.9 OD of crit.). Positive panel sera are formed from the native sera of patients diagnosed with hepatitis B, containing various subtypes of HBsAg and diluted to a concentration of HBsAg from 1.0 to 0.1 IU / ml in a diluting solution containing a known stabilizer. The prepared whey is freeze-dried to a residual moisture content of not more than 1.5 wt.%.

The selection of donor sera is carried out in 2 stages:
1) selection of sera that do not contain antibodies to HIV, hepatitis B and C viruses, syphilis, having OD ≤0.5 OP crit;
2) selection of donor sera having an optical density of from 0.6 to 0.9 OD crit.

 Donor sera selected after 2 stages are monitored by PCR for the absence of HBV DNA. From donor sera having an OD value in HBsAg ELISA of less than 0.5 OD crit., Prepare a pool. The stabilizer is added to the pool in dry form, the components of which do not have an inhibitory effect on the analytical signal of HBsAg in ELISA. To prevent bacterial growth, bactericidal components are added to the pool. A pool of donor sera prepared in this way is used as a reconstitution solution for diluting native reactive samples to the desired concentration of HBsAg in the range from 1 IU / ml to 0.1 IU / ml.

 The main stages of preparing positive samples of the control panel are as follows.

 HBsAg reactive sera are selected according to the results of blood serum screening of patients diagnosed with hepatitis B using test systems certified in the order of the Ministry of Health of the Russian Federation on the first list.

 To establish a relationship between the analytical signal of ELISA and the amount of HBsAg, it is necessary to use ELISA test systems, which in the range from 1.5 to 0.1 IU / ml show a direct relationship between the optical density and the concentration of HBsAg in the dilution solution.

 Donor negative sera having an OD in ELISA from 0.6 to 0.9 OD crit., Are used in their native form as negative panel samples. The sera are stabilized and 0.015% merthiolate is administered.

 Samples of the prepared stabilized sera of the control panel with normalized HBsAg content were analyzed in highly sensitive test systems of various formats (one- and two-stage, accelerated and with night incubation) of domestic and European production. If the obtained concentrations of HBsAg differ from the set values, their content is adjusted upward by introducing an aliquot of reactive serum or downward using a dilution solution. Liquid samples are lyophilized according to a regimen specially developed for this type of preparation and stabilizer composition according to the method described in [9].

 The applicability of the serum preparations created according to the present invention with a normalized level of HBsAg is checked by sending panels to various laboratories and control services of manufacturers of test systems in accordance with the program for certification of diagnostic laboratories developed by the external quality control department of the Ministry of Health of the Russian Federation [1].

 Thus, the certification procedure for the control panel sera includes three stages, the results of which are independent and allow reliable identification of the amount of HBsAg.

The concentration of HBsAg established during this certification is entered into the passport on the control panel of this series. The service life of the control preparations created according to the present invention is established according to the results of the thermal degradation test at temperatures of 4 ^° C and 37 ^° C in accordance with [9].

 To conduct an external assessment of the quality of laboratory tests on HBsAg, control panels with encrypted samples are transferred to the diagnostic laboratories of clinics and blood transfusion stations. Thus, all laboratories receive identical and certified control materials.

 To exclude the effect on the results of each serum formulation of possible differences in antigen reactivity in different wells of the immunosorbent, each encrypted panel sample is analyzed during the study in less than 4-6 replicates.

New in comparison with the prototype is:
- the use as samples of the control panel of native human blood serum;
- the use as a positive sample of a panel of reactive sera containing various subtypes of HBsAg;
- preparation of serum with a concentration of HBsAg in the range from 1 IU / ml to 0.1 IU / ml.

 - manufacture of the control panel in a dry, stabilized form.

 The proposed method for producing a serum panel for monitoring the quality of analysis of human serum (plasma) and hepatitis B vaccines is not described in the literature, therefore, it can be concluded that the technical solution meets the criteria of the invention of "novelty" and "inventive step".

 In FIG. Figure 1 shows the titration curves of HBsAg reactive sera in reconstitution solution.

 Examples of the method.

 Example 1

 Preparation of HBsAg control panel.

 Donor K-serums are obtained with SEC certified for the absence of anti-HIV, anti-HCV, HBsAg and anti-syphilis antibodies. In the laboratory, sera are additionally monitored for the presence of anti-HBs and anti-PVA antibodies in negative sera. Selected at the 1st stage of K-serum (plasma) is filtered and centrifuged. To separate serum (plasma) from flakes and clots, a filtration unit from a flask and funnel is collected. As a filter element, 3 layers of gauze are used, between which a layer of cotton wool is laid. Then the donor K-serum (plasma) is centrifuged at a speed of 1,200 rpm./min for 10 minutes The precipitate is discarded. The clarified donor sera are analyzed in the test systems "Rekomatgen V" (CJSC Maistar) and "Vectogen B 1,2" (CJSC "VectorBest"). Serum with OD <0.5 OD crit. used to prepare the working solution, and donor serum with an OD> 0.6 OD crit. used to prepare negative samples of the control panel.

 Serum reactive to HBsAg in the amount of 8 samples is obtained from patients with a diagnosis of hepatitis B and with an OD in ELISA of more than 2.0 p.u. These reactive sera are analyzed on a monoclonal antibody panel to determine the HBsAg subtype. All sera from this control panel series were found to belong to the ayw l-2, ayw 3, var A and ayw 3, var B subtypes.

 At the 2nd stage, a reconstitution solution is prepared from donor serums and a stabilizer.

 Donor sera with OD <0.5 OD crit. mixed in one container on a magnetic stirrer. At the same time, a stabilizer is prepared per 1 liter of mixed serum, sequentially dissolving peptone, sucrose, KC1 and EDTA in a pool of donor serums in the following amounts: peptone (Serva) 25 g, sucrose 60 g, KCl 10, casein 7 g.

 The dilution solution was adjusted to pH 6.8-7.1 with 0.1 M NaCl. As a bactericidal additive, merthiolate is introduced in an amount of 0.01% by weight of the solution. The serum solution with the stabilizer components added is filtered sequentially through 0.8 μm and 0.45 μm filters for clarification.

 Reactive sera of various HBsAg subtypes are titrated in a dilution solution simultaneously with the Labsystems standard, certified with respect to the primary standard of the P. Ehrlich Institute of the subtype in the amount of 1 IU / ml, on one microplate in the Recomatgen B test system in steps of 3 (cm .Drawing). Joint processing of titration curves of reactive candidate serums and a standard sample allows one to select dilution factors for a given dilution solution to obtain HBsAg concentrations in the range from 1 IU / ml to 0.1 Me / ml.

Positive serum of the control panel of each number is obtained by mixing the candidate serum with a dilution solution in the ratio specified by the dilution coefficient. Dilution for each of the 6 numbers is performed in a container with a volume of 500 cm ³ mounted on a magnetic stirrer for 10-15 minutes. The determination of the OD of samples of the control panel of all numbers is carried out in ELISA test systems of the same type that were used in the titration of primary solutions.

 Diluted samples suitable for testing to prepare a control panel are transferred to the bottling operation. For sera whose optical density does not match the required concentration of HBsAg, the concentration is adjusted using the original candidate serum or dilution solution. In this way, 6 panel samples are prepared having different contents of HBsAg: 2 samples of each subtype in the range from 1 to 0.1 IU / ml.

 Negative reference panel sera are prepared from native donor sera that do not contain anti-HIV, anti-HCV and anti-syphilis antibodies, as well as HBsAg. To this end, they add a stabilizer of the same composition and in the same amount as in the preparation of the dilution solution. In total, 4 samples are prepared that do not contain HBsAg, but have increased OD values in ELISA from 0.6 to 0.9 OD crit.

 All 10 samples of panel sera are monitored for the absence of HBV DNA by PCR in the HBV PCR DNA system (Vector Best CJSC). From each sample number, control samples for ELISA of 1.5-2.0 ml are taken into penflon bottles.

Serum panels are poured in a 0.8 ml laminar cabinet into 5 ml vials, closed with rubber stoppers and placed in metal cassettes for freezing. Serum freezing is carried out at a temperature of minus (60 ± 5) ^o С, keeping them at this temperature for at least 20 hours. The temperature during the freezing process is maintained automatically and controlled by the machine operator.

The frozen preparation in vials is transferred to the sublimation chamber of the TG-50 freeze dryer, the shelves of which are cooled to -35 ^o C. The material temperature at the freeze-drying stage is maintained below its glass transition temperature (-35 ^o С). The average heating rate of the material at the stage of drying is 4 ^o C / min, the drying time at 27 ^o C is 10 hours

 Lyophilized dried samples of the control panel have a residual water content of about 1.0-1.5 wt.%. The determination is carried out according to the method. Vials with the drug are sealed under vacuum. The vacuum in the chamber is controlled by a vacuum gauge. The total average drying time is 36 hours.

 The control of the specific activity of lyophilized and initial liquid serum with a prepared concentration of HBsAg is carried out in the test systems "Recomatgen B" and "Vectogen B2". According to the results of preliminary certification, one reactive serum and one negative serum were discarded. Thus, 5 positive samples and 3 negative samples remained in the control panel. The results of the analysis are given in table. 1.

The thermal stability of the samples of the control panel is estimated by the results of the thermal degradation test conducted at 37 ^o C for 3 weeks. The results of ELISA are presented in table. 2 in comparison with the results for panel sera stored in a refrigerator at 4 ^° C.

Based on the results of the thermal degradation test, it is possible to evaluate the shelf life of drugs with a certified concentration of HBsAg by a standard method [10]. For storage of drugs at a temperature of 4 ^o With an expiration date t (4 ^o ) is estimated:
t (4 ^o С) = 3 weeks. • 24 = 72 weeks.

 Thus, the estimated shelf life of the serum HBsAg panels meets the requirements of WHO experts on standard biological products.

 The economic efficiency of the proposed method for the manufacture of samples of HBsAg sera from the control panel is that the control panel is formed from native serums with HBsAg of various subtypes. Such a panel significantly increases the effectiveness of external quality control of laboratory diagnoses of hepatitis B.

 Using this approach for markers of other dangerous infections will significantly reduce the cost of their development and manufacture. This applies primarily to the diagnosis of HIV and hepatitis C.

 In addition, the introduction into serology of serum calibrators with a known concentration of HBsAg and various subtypes provides the basis for checking the correctness of the analyzes for the quantitative content of this marker in the blood and thereby the legal basis for resolving conflicts between manufacturers and users of HBsAg test systems.

 Improving the quality of analysis contributes to a more accurate diagnosis and a reduction in social tension in society.

Industrial applicability
The invention can be used in biotechnology, medicine and veterinary medicine for an external assessment of the quality of detection of markers of various viral or bacterial infections in the diagnostic laboratories of the Ministry of Health of the Russian Federation.

Sources of scientific, technical and patent information
1. Quality control of clinical diagnostic laboratory tests. Principles and methods. Moscow - 1994, 154 pp.

 2. Carrett A.J., Seagroatt V., Supran E.M. at all. WHO Bulletin 1988, vol. 66, N1, p. 44-60.

 3. WHO Symposium. Report BS / 96 1848. Geneva 1996.

 4. Janot S., Courouse A.-M., Maniez-Montreuil M. et al. Reevaluation of the sensivity of 19 HBs antigen screening kits. La Revue francaise des Laboratories, fevrier / mars 1996, N282 (translation).

 5. Palomaki P. Simultaneous use of poly-and monoclonal antibodies as enzyme tracers in one-step enzyme immunoassay for the detection og hepatitits B surface antigen. J. Immmunol. Methods, 1991, 145, 55-63.

 6. E.A. Gutova, A.N. Kanev, E.Yu. Shalaev Stabilizing composition for control sera positive for antibodies to viral and microbial antigens. RF patent N2011202, 1993

 7. Newsletter of Boston Biomedica, Inc., 1996, Hepatitis B surface antigen sensitivity panel (PHA806).

 8. Pelichesk sensitivity HBsAg panel. Central Laboratory of the Netherlands Red Cross. Amsterdam, 1996.

 9. A.N. Kanev, A.P. Sadovsky, E.Yu. Shalaev, M.Yu. Rukavishnikov. A method for determining the technological parameters of lyophilization of biological products. RF patent N 2010220.

Claims (5)

 1. A method of manufacturing control panels of sera for monitoring the quality of diagnosis of hepatitis B, including the selection of reactive sera of individuals diagnosed with hepatitis B for the formation of positive samples of the panel and donor sera that do not contain specific antibodies to the main markers of viral infections, the manufacture of a dilution solution based on selected donor sera diluting reactive sera in a diluting solution, conducting a thermal degradation test to determine the shelf life of the panel, characterized in that negative panel samples are formed from sera having a negative result in the polymerase chain reaction to hepatitis B DNA, and in an enzyme immunoassay, the optical density is less than critical in the range of 0.6-0.9 OD crit. and the selection of reactive sera for the panels is carried out according to a scheme that includes their certification by enzyme-linked immunosorbent assay and polymerase chain reaction for the presence of hepatitis B DNA, determination of the HbsAg subtype and titration of positive panel samples containing various HbsAg subtypes in a diluted stabilized solution to the required concentration HbsAg from 1 IU / ml to 0.1 IU / ml.  2. The method according to p. 1, characterized in that the negative serum of the reference panel is formed from native sera.  3. The method according to p. 1, characterized in that as a positive panel samples using native serum of patients diagnosed with hepatitis B, containing various subtypes of HbsAg.  4. The method according to p. 1, characterized in that 5 sera with an HbsAg concentration of from 1 IU / ml to 0.1 IU / ml and 3 native donor serums are taken as negative samples to form a control panel.  5. The method according to p. 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. %

Images