A method of obtaining a reference panel for quality control of test systems and vaccines against hepatitis b

 

(57) Abstract:

The invention relates to biotechnology and immunology, and can be used for fabrication of panels of sera containing antigens of hepatitis C. In accordance with the present method are selected donor serum by their analysis methods of ELISA and PCR for the presence of DNA of hepatitis b virus and anti S antibodies, prepared diluting the solution on the basis of selected sera, diluted standard HBsAg in razvodami solution, conduct test thermodegradation and stabilization of the solution. The invention allows to obtain a reference panel with a constant concentration of viral protein for a long time, which, in turn, can increase the level of quality control of the vaccine against hepatitis C. 4 C.p. f-crystals, 1 Il., table 2.

The invention relates to the field of biotechnology and immunology, and can be used for fabrication of panels of sera containing antigens of the most dangerous viral infections, and in production test systems for the determination of antigens as a positive control sera. Certified content of HBsAg in samples of reference of the panel allow you to use these standartinis HBsAg.

Known methods of preparation and certification of sera containing specific amounts of specific proteins must be used in conjunction with the manufacture of sera with the certified concentrations of HBsAg and require the development of:

- reliable method of preparation of non-communicable drug HBV;

- physico-chemical method of determining the concentration of HBsAg in the serum of human blood;

- selection of the stabilizer composition and mode of freeze drying of samples of the reference panel for receiving drugs with a normalized concentration of HBsAg, remaining unchanged for a long period of time in accordance with the requirements of the who expert Committee for reference materials.

To maintain a constant concentration of a specific protein is a complex problem. An additional problem faced during the evaluation of the amount of HBsAg in the serum, is that the optical density (OD) of the same solution changes when using different test systems and even for a single test system when using different modes of analysis.

For example, the standard sample (CO) Institute R. Erlich subtype ay, zyme HBsAg" company "Murex" has a threshold sensitivity of 0.08 ng/ml during the incubation of the samples during the night and the sensitivity of 0.17 ng/ml by incubation for 30 min at 50o[3]. In addition, in the General case, the size of the OD of the solution WITH different for different planting solutions. Solution certification sera panel on the content of HBsAg proposed to be done in the following way:

- be used to create a reference panel plasma purified and recombinant antigen belonging to different subtypes of known mass content of HBsAg and no infectious risk;

as the diluent solution is to use a pool of sera of healthy donors, which do not contain markers of major infections and does not significantly inhibit the OP HBsAg ELISA relatively OP buffered saline containing inert protein to prevent "hook"effect (for example, SPR + 0.2% casein);

as components of the stabilizer to use substances that do not affect the level of analytical signal.

The main criterion for the sensitivity-based assays for serological assays is the value of the analytical signal of the positive control serum. When this prerequisite health test systems is the level of the analytical signal, which should not be lower than the certified values.

systematic analyses [RF patent 2094807 - a panel of anti-HIV]. However, unknown to the methods of manufacture of sera, in which standardized quantitative content viral markers.

Known panels of sera Boston Biomedical Inc. (USA): Hepatitis Surface Antigen In Sensitivity Panel (PHA 806) [1]. The panel includes: serum concentration of HBsAg from 2.5 ng/ml to 0.1 ng/ml and negative serum. However, these panels are not used as standard reference materials of the United States and are intended for use only for research purposes. In addition, serum included in these panels, supplied in a liquid form and therefore must be stored at low temperatures. As a consequence, very strict (temperature) requirements for their storage and distribution create serious problems for use in other countries, not having a reliable cold chain [instructions on the use of PHA 806. W. Bridgewater, MA 02379, 1996].

These panels of sera can be used as an analogue for the proposed reference panels.

The closest technical solution (prototype) is a method for preparing serum panels, developed in the Central laboratory standards of the Dutch red cross to prepare control panels brand Pelichek [2].

Prototype method includes preparation of a standard serum containing HBsAg and with different concentrations of this protein. The main stages of production of standard sera are:

- collection of reactive sera for preparation of standard HBsAg;

- collecting donor sera not containing anti-HBs antibodies and markers of viral infections;

- production of planting serum that does not contain fibrinogen and lipids, selected from the pool of donor sera;

- breeding standard HBsAg in planting serum 4, 8, 16, 32, 64, 128, 256, 1024, 2048 and 8192 times;

- test thermodegradation to establish the shelf life of the panel.

The advantages of the prototype method should include the development of the General scheme of preparation of standard HBsAg and preparation of planting serum, which is implemented in the manufacture of control sera for serum panels with other markers.

The main disadvantages of the prototype:

- planting serum is an idealized (i.e., the serum is removed fibrin, fibrinogen and lipids) model of native human serum;

- in the "Pelicheck" not defined and no attestator the same dilutions will meet other concentrations of HBsAg;

serum panel of the prototype is not stable and not dried.

As follows from the above description, shipping and storage panels-prototypes require strict adherence to a low-temperature regimes.

The purpose of this invention is to provide such a method which enables the production of a reference panel of sera with normalized concentrations of HBsAg in the range of 6 ng/ml to 0.25 ng/ml, for assessing the quality of test systems and allows you to save specific characteristics of these sera for a long time (not less than 2 years).

The problem is solved in that in the method of producing panels of sera for quality control of test systems and vaccines against hepatitis b, including the selection of donor sera not containing specific antibodies to the major markers of viral infections, preparation of diluent solution on the basis of selected breeding standard HBsAg in razvodami solution, test thermodegradation to establish the shelf life of the reference panel, according to the invention the selection of planting sera panels is carried out according to the scheme, including their certification methods ELISA and PCR for the presence of HBV DNA (hepatitis b virus) from, who have negative results in PCR HBV DNA and ELISA value OP less critical (in the range of 0.5-0.8 OP Crete). As HBsAg is used plasma purified and recombinant antigen belonging to different subtypes of HBsAg containing not less than 95% the main protein, separation of plasma and recombinant HBsAg produce pooled donor sera containing the following stabilizing composition: 15-20 g BSA, 45-55 g sucrose, 7-15 g KCl, 5-10 g of casein per 1 liter of the mixed serum; the panel is made up of 10 sera from HBsAg concentration from 6 to 0.25 ng/ml and 6 native sera of donors; prepared serum dried to a residual moisture content of not more than 1.5 wt.% lyophilisation.

The selection of donor sera carried out in 2 stages:

a) selection of serum samples containing antibodies to HIV, hepatitis b and C, syphilis;

b) selection of sera with suppression ratio less than 1.3.

Donor serum taken after the two phases are pooled. Pooled contribute stabilizer in the dry form, the components of which also do not have an overwhelming effect on the analytical signal of HBsAg. To prevent bacterial gains in pool make bactericidal components.

Ovline sample reference panel, containing HBsAg in the required concentration of 6 ng/ml to 0.25 ng/ml (further increasing the value of HBsAg does not affect the value of the OP, as seen in the drawing).

Sera reactive for HBsAg, selected based on the results of the screening of native serum samples in serological laboratories using test systems, certified in the Ministry of health of the Russian Federation on the 1st list. Reactive serum processed for cleaning particles from deyna for inactivation of HBsAg solution. In the processes of separation and concentration of proteins allocate fractions with HBsAg content above 95% in accordance with the data of electrophoresis. In the process of concentration of HBsAg 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. To establish the relationship between the analytical signal ELISA and the amount of HBsAg in the solution of the distribution of serum must be used ELISA test systems, which range from 6 to 0.25 ng/ml show a linear relationship between optical density and the amount of HBsAg in the solution.

Samples prepared stabilised serum reference the applications, expedited and overnight incubation) of domestic production. When the difference in the concentrations of HBsAg from the specified content to correct them upward through the introduction of plasma and recombinant antigen or downward with diluent solution. Liquid samples lyophilizer regime specifically designed for this type of drugs and composition of the stabilizer according to the method described in [5].

Known method of stabilizing the specific properties of positive sera [4] and in the present invention is to stabilize the level of HBsAg is encouraged to use a stabilizer, containing 5-10 g of casein.

-Casein is M m, close to the mass of HBsAg, and therefore the introduction of inert molecules of similar size will not create steric for the occurrence of specific interaction: AG+At. Introduction casein is equivalent to creating around the desired molecules additional protective membrane to hydrophobic regions of the molecule. At the same time the introduction of a new component in the composition of the protective environment causes a change in the temperatures of phase transformations diluent solution that entails a change in the mode lyophilization.

Thus, the procedure of attestation of serum reference panel consists of three phases, the results of which are independent and reliably identify the number of HBsAg.

Established in this certification, the concentration of HBsAg entered in the passport on the reference panel. The life created by the present invention reference preparations are established according to the test results of thermodegradation at temperatures of 4oWith the 37oIn accordance with [6].

New compared to the prototype is:

- use as a plasma HBsAg purified and recombinant antigen belonging to different subtypes of HBsAg containing not less than 95% of the basic protein;

- use as a diluent solution pooled donor sera not containing main markers of viral infections and do not reduce the analytical signal from HBsAg relatively buffered saline;

- manufacture of sera with normalized concentric is of casein in the composition a stabilizing environment.

The proposed method of obtaining a reference panel of sera to control the quality test kits and vaccines against hepatitis B are not described in literature, therefore, we can conclude that the technical solutions according to the criteria of the invention of "novelty" and "inventive step".

The drawing shows a titration curve of the working standard in razvodami solution.

Examples of the way

Example 1. Preparation of reference of the panel.

Donor K-serum get with the SEC, certified for the absence of anti-HIV, anti-HCV, HBsAg and antibodies to syphilis. In the laboratory serum additionally control for the presence of anti-HBs and anti-HBC antibodies. Selected at the 1st stage K-serum (plasma), filtered and centrifuged. For separating serum or plasma from flakes and clots gather filtration installation from a flask and funnel. As the filter element used 3 layers of gauze, between which laid a layer of cotton wool. Then the donor K-serum (plasma), centrifuged at a speed of 1200 rpm for 10 minutes the Precipitate discarded. At the 2nd stage, prepare working solutions of plasma purified HBsAg subtype ayw2 (production of CJSC ImBio", Nizhny Novgorod) and recombinant HBsAg ayw4, subs estimated concentration of 10 ng/ml each.

Prepared solutions titrated simultaneously on one microplate in the test system Vectora 2 with step 3. In the direction of the titration curves and the ratio of OP (SFR)/OP (test serum) make a conclusion about the suitability of the test serum as a component of the diluent solution. In particular, OP(SFR)/OP (so R-RA) < 1,3 when parallelism titration curves of both antigens with their solutions in SFR-casein whey include pooled donor sera for the preparation of diluent solution.

At the same time preparing the stabilizer on 1 liter of the mixed serum, consistently dissolving BSA, sucrose, KCl and EDTA pooled donor sera in the following amounts:

BSA - 15-20

Sucrose - 45-55

KCl - 7-15

Casein - 5-10

Diluting the solution brought to a pH of 6.8 to 7.1 using 0.1 M NaCl. As bactericidal additive injected thimerosal in an amount of 0.01% by weight of the solution. A pool of sera made with components of the stabilizer is filtered sequentially through the filter of 0.8 μm and 0.45 μm.

Cooked razvodami solution prepare solutions plasma AG and recombinant AG to obtain reference solutions with a concentration of 10 ng/ml Obtained working standards Ista "HBsAg ELISA" (JSC "Vector master" of Alexey Verbitskiy). At the same time on tablets titrated solution WITH HBsAg (GISC or other) with a concentration of 10 ng/ml in SFR-casein.

Titration curves for treated and determine the average titration curve for each working standard and the exact initial concentration of HBsAg in the working standards (see drawing). On average titration curve to determine the coefficients of the breeding of working standards to sample reference panel HBsAg concentrations: 6 ng/ml, 2.0 ng/ml, 1.0 ng/ml, 0.5 ng/ml and 0.25 ng/ml

Serum reference panel, each room comes with in the process of mixing the reference solution with HBsAg razvodami solution in the ratio specified by the dilution factor. Mixing for each of the rooms is carried out in a volume of 500 cm3set on a magnetic stirrer for 5-7 minutes Determining the OD of the samples of the reference panel of all rooms is carried out in ELISA test-systems of the same type that was used in the titration of the reference solutions.

When assessing the correctness of the cultivation compare the magnitude of the OD obtained in the analysis of prepared samples with OD of sera, shown in the drawing. Diluted samples suitable for testing for cooking referencename of titration curve, spend an adjustment of concentration using the original reference HBsAg solution or diluent solution. Thus prepared 10 samples of panels having different contents of HBsAg: 5 samples on the basis of plasma cleared of AG and 5 samples on the basis of recombinant AG with equal concentrations of 6 ng/ml, 2.5 ng/ml, 1 ng/ml 0.5 ng/ml and 0.25 ng/ml

Negative serum reference panel prepared from donor sera not containing anti-HIV, anti-HCV and antibodies to syphilis and HBsAg. Just prepare 6 samples not containing HBsAg, but having a high OD values in the ELISA from 0.5 to 0.8 OD Crete.

All serum samples of the control panel in the absence of HBV DNA by PCR system (JSC "Vector-best").

Serum panel is poured in a laminar flow Cabinet in 0.7 ml vials with a volume of 5 ml, closed with rubber stoppers and placed in a metal cassette for freezing. Freeze serum is carried out at a temperature of minus 60+/-5)oWith, maintaining at this temperature for at least 20 hours. The temperature in the freezing process is automatically maintained and controlled by the operator.

Frozen drug in vials are transferred into the freeze chamber leonovo dehydration support below the glass transition temperature (-35oC). The average heating rate of the material at the final stage is 4oC/min, time-keeping at the 27oWith is 10 hours.

Lyophilized samples of the reference panel has a residual water content of about 1.0 to 1.5 wt.%. The determination carried out by the method of [7]. The vials with the product is sealed under vacuum. The vacuum in the chamber is monitored by the vacuum gauge. Total average drying time is 36 hours. Control specific activity liofilizovannyh sera with a normalized level of HBsAg carried out in test systems for domestic production. The results of the analysis are shown in table 1.

Example 2. Preparation of the reference panel HBsAg

The selection of donor sera, filtering and polerowanie implement the same methods as in example 1. When stabilization pool in diluting solution make the same amount of substances, except that casein contribute 3 g per 1 l of solution. Other operations were carried out as in example 1.

Example 3.Preparation of the reference panel HBsAg

The selection of donor sera, filtering and polerowanie implement the same methods as in example 1. When stabilization pool in diluting solution make the same number of the example 1.

Example 4. Preparation of the reference panel HBsAg

The selection of donor sera, filtering and polerowanie implement the same methods as in example 1. When stabilization pool in diluting solution make the same amount of the substance, except that casein contribute 15 g per 1 l of solution. Other operations were carried out as in example 1.

Prepared in examples 1-4 diluting the solutions were used to prepare positive samples panel HBsAg. thermal stability of the samples prepared with different amounts of casein, is estimated by the test results of thermodegradation conducted at 37oC for 2 weeks. The results are presented in table 2 in comparison with serum panels were stored in a refrigerator at 4oC.

Serum panel after soaking in thermostat were set in a test-system "Vectored-2" and in the "HBsAg ELISA". As follows from the results table. 2, the optimum amount of casein is from 5 g to 10 g per 1 liter of diluent solution. When lower concentrations of casein OD of positive samples panel in ELISA reduced after 2 weeks. For samples prepared with the addition of casein more than 10 g/l, not marked improvements in srestore, what hampers the IFA.

From the test results it follows that the concentration of HBsAg in the tested sera decreased significantly when the casein content of less than 5 g/l and virtually unchanged at a content of 5 to 15 g/l after storage for 4 weeks at a temperature of 37oC. However, increasing the concentration of casein in the sample is accompanied by a decrease in OD in ELISA, which is undesirable. Therefore, the recommended content of casein per liter stabilizing solution is from 5 to 10 g/L.

On the basis of the obtained results is possible to estimate the shelf life of drugs with a normalized level of HBsAg according to standard methods [7]. For storage of drugs at a temperature of 4oWith a shelf life:

t=4 weeks. * 24 = 96 weeks.

Thus, estimation of the shelf-life of serum HBsAg panel meets the requirements of the who expert standard biologics.

Economic efficiency of application of the proposed method of manufacture of sera with the certified concentration of HBsAg is that the corresponding protein is diagnosed in blood of patients with hepatitis b at all stages of infection. He appears in the blood of infected 2-3 weeks after INFI the medical care and treatment are of social importance and economic efficiency, associated with the reduction of treatment time and dates of employment to the inability of the people.

In addition, the introduction of serology serum calibrator with known concentration of HBsAg provide the basis for monitoring the validity of tests on the contents of this marker in the blood and thus a legal basis for resolving conflicts between producers and users of HBsAg test systems and between producers and users of vaccines for hepatitis b, which must contain a certain amount of HBsAg.

Industrial applicability. The invention can be used in biotechnology, medicine and veterinary medicine in assessing the quality of test systems for the detection of markers of various viral or bacterial infections.

Sources of information

1. Newsletter company Boston Biomedica, Inc., 1996, Hepatitis B surface antigen sensitivity panel (RNA).

2. Pelicheck sensitivity HBsAg panel. Central Laboratory of the Netherlands Red Cross. Amsterdam, 1996.

3. Janot C., Courouce, 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, 282 (translation).

4. E. A. Gutov, A. N. Kahn, E. Y. Shalaev. Stabilizing composition for control sera positive for antibodies to viral and microbial antigens. Patent R. the parameters of the freeze-drying of biological products. RF patent 2010220, 1993.

6. OST 42-2-72 "Drugs. The procedure for establishing shelf life". The Ministry of health USSR, Moscow, 1972.

7. "Determination of the residual moisture of small amounts of biological products", MI 123.39.002.89, SRC VB "Vector", 1990.

1. The method of obtaining the reference panels of sera containing sAg for quality control of test systems and vaccines against hepatitis b, including the selection of donor sera not containing specific antibodies to the major markers of viral infections, manufacturing diluent solution based on selected serum dilution standard HBsAg in razvodami solution, test thermodegradation to establish the shelf life of the panel, characterized in that conduct stabilizing diluent solution, and the selection of planting sera carried out by analysis of donor sera methods enzyme-linked immunosorbent assay and polymerase chain reaction for the presence of DNA of hepatitis b virus and anti s antibodies, nonspecific suppression of HBsAg, titrated samples of HBsAg in razvodami stable solution to the concentration of HBsAg from 6 to 0.25 ng/ml, and as diluting sera choose sera with negative results in these methods and having the value of OPM, as HBsAg using plasma purified and recombinant antigen belonging to different subtypes of HBsAg containing not less than 95% the main protein.

3. The method according to p. 1, wherein the stabilizer contains 15-20 g SA, 45-55 g sucrose, 7-15 g KCL, 5-10 g of casein per 1 liter of the mixed serum.

4. The method according to p. 1, characterized in that for forming the standard Referans panel select the 10 sera from HBsAg concentration from 6 to 0.25 ng/ml, including 5 samples prepared on the basis of plasma purified antigen, and 5 samples prepared on the basis of recombinant antigen belonging to different subtypes of HBsAg, and 6 native donor sera.

5. The method according to p. 1, characterized in that the prepared serum in Referans-panel freeze-dried to a residual moisture content of not more than 1.5 wt. %.

 

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