RU2271011C1 - METHOD FOR PREPARING WORKING STANDARD SERUM COMPRISING HBsAg AY AND AD SUBTYPES - Google Patents

Abstract

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

Description

The invention relates to the field of biotechnology and immunology and can be used in the technology of manufacturing control samples for test systems for the detection of HBsAg.

Working Standard (PC) is a reference drug that is designed to:

1. For the preparation of control samples for test systems for the detection of HBsAg.

2. For use as semi-finished products of standard preparations with a low concentration of HBsAg.

Known reference drug CCA (GISK them. L.A. Tarasevich) for monitoring the diagnosis of hepatitis B [1].

Its disadvantage is that it is made on the basis of an unknown subtype of HBsAg and does not allow effective control of blood samples, because its activity cannot be attributed to a specific subtype or genotype of HBV. In addition, it is impossible to solve the problems of quality control of analyzes in clinical diagnostic laboratories with one drug.

The closest (prototype) is the working standard (working standard) of the AD subtype (genotype A), which is manufactured in NIBSC and used to control the quality of HBsAg tests in diagnostic laboratories of infectious diseases clinics and in blood transfusion centers in the UK [2].

British PC prepared as follows.

The serum of an asymptomatic patient who has been a carrier of HBV (genotype A) for at least 7 years is used. Serum is diluted in a ratio of 1: 4 in water for injection and sequential heating at a temperature of 60 ° C for 10 hours to minimize infectivity. Pasteurized serum is diluted in phosphate-buffered saline (PBS) containing 5% BSA and 0.1% sodium azide and poured into 1 ml ampoules and stored at -20 ° C or lower.

This material at the NIBSC Standards Institute (Potter Bar, UK) was given the status of the British subtype AD standard with a specific activity of 100 British U / ampoule.

The disadvantages of this drug are:

1. For the manufacture of PC using a model solution (5% BSA), and not true human serum.

2. Only one subtype of HBsAg is used - (AD).

The objective of the invention is to create such a method that would ensure the manufacture of 2 working standards of sera containing AY and AD subtypes of HbsAg, and would allow to preserve the specific characteristics of these samples for a long time (at least 1 year) when stored from 2 to 6 ° C. .

The problem is solved by the production of working standards of sera (PC) containing AY and AD subtypes of HBsAg at a concentration of 5 to 10 IU / ml and having a viscosity equal to the viscosity of normal human serum (NP).

Production Scheme of HBsAg Operating Standards for AY and AD Subtypes

1. Testing samples of donor sera using the laboratory standard HBsAg to study the magnitude of the suppression of the analytical signal from the entered amount of HBsAg. Laboratory standard (SL) - stabilized plasma HBsAg, titrated in Russian and foreign test systems. From donor sera with a minimum level of suppression, a reconstitution solution (PP) is prepared.

2. Titration of HBsAg AY and AD subtypes in the prepared PP together with the CCA of the Ministry of Health (production of Diagnostic Systems, NN) and international standards - NIBSC (England) - AD subtype or P. Erlich Institute (Germany) - AY subtype in various tests -systems.

3. Determination of the dilution coefficient for each subtype to obtain candidate PC with a HBsAg concentration of 5-10 ME / ml.

4. Preparation of candidates PC AY and AD subtypes (in the amount of 30-50 copies each).

5. Determination of the concentration of HBsAg in PC according to the results of ELISA in different test systems in different laboratories.

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

7. Conducting a thermal degradation test of PC candidates at elevated temperatures to determine shelf life.

8. Certification of PC candidates in GISK them. L.A. Tarasevich.

To establish the relationship between the analytical signal of ELISA and the mass concentration of HBsAg in solution, it is necessary:

- to study and bring into compliance the fractionally dispersed composition (FDS) of the international standard and the test drug;

- use ELISA test systems, which in the range from 1.5 IU / ml to 0.1 IU / ml demonstrate a direct relationship between the optical density and the amount of HBsAg in solution.

To certify the mass concentration of the HBsAg preparation in PC, it is necessary to select an international standard with an identical FDS. The difference in the FDS of reference drugs can be explained by the fact that the proportionality coefficients between the standards used in the production of test systems are not consistent with each other. Tests of standards conducted under the auspices of the WHO showed that 1 IU of WHO is equivalent to 0.584 units of the standard Institute P. Ehrlich or equivalent to 5.587 ng Abbott [3, 4]. Electron microscopy and atomic force microscopy methods are used to evaluate the PDF of HBsAg preparations used in the manufacture of national standards.

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 series of the working standard. PC candidates were titrated in various matrices: NSF, 5% BSA, and fetal serum relative to saline supplemented with 0.2% casein.

PC Materials

1. Reconstitution solutions

The following solutions are used as PP:

- Normal human serum (NPP);

- A solution of 5.5% BSA in 0.05 M phosphate-buffered saline (0.15 M NaCl) - FSB;

- A solution of 5.5% tryptone in the FSB;

- A solution of 0.2% casein in the FSB.

2. Test systems for detecting HBsAg

3. Standard HBsAg preparations

The following are examples of specific implementation of the method.

Example 1

Collection and preliminary analysis of blood serum. Freshly prepared blood from a vein is poured into hemacons containing 0.12 ml of 0.34 M EDTA, so that the final concentration of EDTA is 2 mM (20 ml). Direct separation of whole blood is carried out by centrifugation at 1000g for 15 min at room temperature. The supernatant - the serum is decanted into sterile dishes, visually discarded and stored until use in a frozen state. For the preparation of standards, transparent amber-colored sera are used. Turbid and chylous samples are rejected.

HBV DNA detection. HBV DNA is detected in donor serum pool samples and in plasma preparations using polymerase chain reaction (PCR) as described [5].

Analysis of donor sera includes:

1. Determination of total protein content.

2. Transparency and color.

3. The concentration of hydrogen ions.

4. Donor sera are analyzed for anti-HIV, anti-HCV, anti-HBs and antibodies to syphilis and HBsAg using certified commercial test systems.

For certification of PC samples, test systems are additionally used: "HBsAg EIA Plus" (hereinafter "HBsAg EIA") (Labsystems, Finland); "HBsAg / M" (Diagnostic systems, N. Novgorod) and "Monolisa Ag HBs 2eme generation" (hereinafter "Monolisa Ag HBs") (Sanofi Pasteur, France) [6].

HBsAg preparations. To prepare a working standard for the AY subtype, purified plasma HBsAg produced by NPK "Drug" (Nizhny Novgorod, Russian Federation) (0.4 mg / ml, AYw subtype) is used. In accordance with the passport data, the content of the main protein in the preparations exceeds 95%, and the impurities of other proteins in total do not exceed 1%.

Highly purified plasma or recombinant proteins used in the manufacture of hepatitis B vaccines are used as ADs of the HBsAg subtype:

- Plasma purified and concentrated HBsAg from a pool of sera of infected AD subtype patients (GLD's Ltd., Singapore).

- Recombinant (yeast) vaccine subtype ADW "H-B VAX II" manufactured by "Merck Sharp & Dohme Haarlem" (Holland), 10 μg / ml. As impurities, the vaccine may contain less than 1% yeast protein. Isolation of HBsAg from vaccine preparations is carried out under conditions of gentle desorption of HBsAg particles while maintaining their structure in 0.4 M phosphate buffer. Desorbed preparations are certified by fractional dispersed composition (FDS) and HBsAg concentration.

The purity analysis of HBsAg preparations is performed by gel chromatography, electrophoresis, and atomic force microscopy.

Reference drugs HBsAg. To perform certification determinations in the work, standard preparations are used, certified in absolute or relative units:

- Industry standard sample "OCO-HBsAg" (GISK named after L.A. Tarasevich, Moscow).

- WHO International Standard, code 80/549 ADW of the subtype HBsAg, 100 IU / ml (NIBSC, UK).

Preparation of a pool of donor sera. From native blood serums that do not contain markers of viral infections, a pool of at least 10 donors is prepared.

A stabilizer, including sucrose, KCl, and EDTA Na-salt, is added in dry form with constant stirring in each pool with constant control of the viscosity of the solution.

A stabilized serum pool is used as dilution solutions for the preparation of PC subtypes of AD and AY. The pH of the solution was adjusted to 6.8-7.2 at room temperature with 0.1 N NaOH or 0.1 N HCl. As a bactericidal additive, merthiolate is introduced in an amount of 0.012% by weight of the solution and gentamicin in an amount of 0.02% w / v. Solutions are poured sterilely in 250-500 ml containers and stored at 2-8 ° C for no more than 1 month. In frozen form, at temperatures below minus 30 ° C, solutions can be stored for 1 year.

The stabilized serum pool is normalized by total protein and viscosity relative to human donor serum and a dilution solution is obtained - PP. The viscosity control of the PP is carried out using a glass viscometer according to the classical method using a calibrated capillary.

PC sample preparation. The stabilized serum pool (PP) is divided into 2 equal parts: use one part to prepare the PC AY subtype. The second part is used to prepare the PC subtype AD. The estimated amount of HBsAg preparations that must be added to the PP to obtain a concentration of 5-10 IU / ml is determined by the ELISA curves of the titration of AY and AD drugs in the PP simultaneously with the titration of standard preparations of the same subtypes.

In total, 30-50 PC candidate samples are prepared in this way.

Filling and lyophilization of PC samples. The prepared PC samples are filtered sequentially through 1.2 μm filters to remove debris and insoluble impurities of the stabilizer, and then through 0.45 μm to remove microorganisms from solutions. Pour 800 μl of each PC into FI-5 vials with constant stirring to create homogeneous conditions in each vial. After bottling, PCs are immediately frozen at temperatures below -30 ° C.

The lyophilization regime for the selected stabilizer composition is selected according to a specially developed technique [7].

Stability tests. Vials with lyophilized PC samples are kept under thermostated conditions at 37 ° C and 50 ° C, because at temperatures above 56 ° C the solubility of the tablets deteriorates sharply. At fixed intervals, part of the vials is removed from the thermostats and stored at minus 20 ° C until the entire series of stability tests is completed.

PC samples, held at different temperatures for different times, are analyzed simultaneously with the samples, all the while standing at minus 20 ° C (reference point). Additionally, it is necessary to test the preservation of the properties of PC in a liquid state when stored at 4 ° C for at least 3 months.

The results of measuring PC activity are shown in table 1.

Table 1
Decrease in PC activity after storage at different temperatures. Storage time, day PC activity as a percentage PC activity logarithm 50 ° C 37 ° C 50 ° C 37 ° C one 2 3 four 5 0 one hundred one hundred 4.60 4.6 7 94.0 97.5 4,54 4,58 fourteen 88.4 94.9 4.48 4,55 thirty 76.8 89.5 4.34 4.49 162 24.1 55.0 3.18 4.01

In this table 1 in column 1 indicates the storage time of the PC in days. The initial activity value at time 0 days is taken as 100%. Columns 4-5 show the natural logarithms of these values. The deactivation rate constant is defined as the slope of the straight line to the abscissa. Using this, the rate constants are calculated, they turn out to be equal to 0.0037 1 / day and 0.0088 1 / day at temperatures of 37 and 50 ° C, respectively.

The stability condition will be the limiting change in PC activity by no more than 10% of the initial value. In this approximation, the storage time of the drug will be

T = 2.303 log (100/90) / K = 369.7 days or 1.03 years

The stability of PC preparations in the liquid and lyophilic states is determined by thermostating at elevated temperatures and in a 3-fold freeze-thaw cycle.

According to the results of the accelerated inactivation test, the shelf life of the PC at 4-6 ° C is 1 year.

Example 2. Expert selection of test systems

Using the HBsAg dilution method for PC manufacturing, three main factors must be considered:

- the effectiveness of the diagnosticum in relation to AD and AY subtypes of HBsAg,

- the ability to suppress the analytical signal of HBsAg in the native sera of absolutely healthy donors,

- the initial purity of the HBsAg preparations used to prepare the PC.

To carry out an examination to evaluate the effectiveness of the Vector Best and BiAlgam test systems, two working standards of AD and AY subtypes are prepared by diluting HBsAg preparations in the same PP, certified by the value of the inhibitory effect. PP is prepared from a pool of 9-10 donor sera, each of which shows the suppression of the analytical signal in ELISA by no more than 10%. After homogenizing the pool and introducing stabilizer components

The working standard AY subtype is prepared from plasma AYW1-2 HBsAg (N. Novgorod) and

the working standard of the AD subtype of HBsAg (sample No. 10) was prepared from the plasma ADW HBsAg of GLD's (Singapore).

The certification of working standards for HBsAg concentration is carried out by joint titration of PC and CCA samples (20 IU / ampoule) and with the international standard ADW of the HBsAg subtype (100 IU / ampoule, NIBSC).

To determine the specific characteristics of PC, they are titrated in various buffer solutions (matrices), because different test systems use different PPs. Table 2 shows the results of the titration of PC candidates of both subtypes in the solutions of the NES with the addition of tryptone and BSA as stabilizers.

Tab. 2.
Concentration of candidate PC AY subtype according to ELISA results in two test systems Buffer "Recomatgep V" Bialgam ME / ml "Rekomatgep V" Vector Best ME / ml 0.2% casein in 0.05 M FSB 7.1 6.53 Fetal Serum 11.69 8.22 5% BSA solution in 0.05 M FSB 13.31 7.71 Average value 10.7 +/- 3.2 7.49 +/- 0.88 Standard sample SL, 16 IU / ml CCA, 20 IU / ampoule

In this table, each manufacturer uses its own standard for assessing the concentration of HBsAg. As follows from the table, this approach corresponds to the concentration of HBsAg, which differs by almost 1.5 times.

The economic feasibility of a set of working standards is that they can be used in the simultaneous certification of test systems for the detection of HBsAg in low concentrations and at the same time different subtypes. In diagnostic laboratories, PCs can be used in the form of an encrypted sample to control the quality of analysis by laboratory staff.

Information sources

1. CCA HBsAg Instructions for use, 1999

2. Ferguson M., Pipkin PA, Heath AB, Minor PD: Working standards for hepatitis B surface antigen for use in the UK Blood Transfusion Service: Results of a collaborative study.//Vox Sang 1993, 65: 303-309.

3. J.A. J. Barbara. Questions of quality: how much HBsAg is there in this sample and is our assay sensitive enough to detect it? Vox sang 1993: 65: 249-250

4. M. Ferguson, A. Heath. WHO Working Group on Hepatitis and HIV Diagnostic Kits. WHO, Geneva, October 3-5,2003.

5. Larzul D., Guige P., Sninsky J.J. et al -Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J. Virol. Methods 1988, 20, 227-237.

6. MONOLISA Ag HBs 2eme generation. Instructions for use. Kit for detection of the surface antigen of hepatitis B by the enzyme immunoassay technique. Sanofi Pasteur, 1990.

7. Kanev A.N., Gutova E.A., Shalaev E.Yu. and others. A method of obtaining a standard positive panel of sera for quality control of test systems and immunoblots. - Pat. No. 048529, 1996. RF.

Claims (4)

1. A method of obtaining working standards of serum AY and AD subtypes of HBsAg, including the selection of donor sera that do not contain specific antibodies to the main markers of viral infections, diluting the HBsAg preparation in a diluting solution, conducting a thermal degradation test to establish the shelf life of working standards, characterized in that the selection diluting sera are carried out by analysis of donor sera by enzyme immunoassay and polymerase chain reaction for the presence of hepatitis B virus DNA, anti HBs antibodies, non-specific factors specific binding of HBsAg, and serums with a negative result in these methods are selected as diluting serums, titration of highly concentrated HBsAg preparations in a stabilized diluting solution to the required concentration of HBsAg (5-10 IU / ml) AY and AD subtypes. 2. The method according to claim 1, characterized in that to obtain a diluting solution, a pool of 9-10 donor sera is used, each of which shows the suppression of the analytical signal of HBsAg in ELISA by no more than 10%. 3. The method according to claim 1, characterized in that as the preparation of HBsAg use plasma purified and / or recombinant antigens belonging to different subtypes of HBsAg and containing at least 95% of the main protein (antigen), and each positive panel sample in a liquid state has a viscosity equal to the average viscosity of a pool of healthy donor sera. 4. The method according to claim 1, characterized in that the prepared working standards of sera are freeze-dried to a residual moisture content of not more than 1.5 wt.%.