RU2342158C2 - Method for branch standard sample production of hepatitis b viral surface antigen, used in quality control of test systems and antigen quantitative immune-enzyme determination in human blood serum (plasma) and preparations - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for branch standard sample (BSS) production of hepatitis B viral surface antigen (HBsAg), used in quality control of test systems, as well as in antigen quantitative immune-enzyme determination (IEA) in human blood serum (plasma) and preparations; and quality control kit for test systems, applied in HBsAg detection, and in antigen quantitative IEA in human blood serum (plasma) and preparations; are claimed.

EFFECT: shelf life prolongation and providing possibility of repeated use of preparation.

2 cl, 3 ex, 3 dwg

Description

The industry standard sample (CCA) is a substance of synthetic origin used to control medical immunobiological preparations used to diagnose human diseases.

The invention relates to the field of immunology and standardization and can be used to determine the sensitivity of diagnostic test systems used to detect the surface antigen of hepatitis B virus (HBsAg), as well as to determine the quantitative content of HBsAg in serum (plasma) and human blood products by enzyme immunoassay (IFA).

The invention:

1. A set of OCO-HBsAg, including the preparation of OCO-HBsAg, which is made from plasma or recombinant HBsAg of different subtypes and lyophilized in 1% solution of bovine serum albumin (BSA); 0.75% glycine solution; 0.01% thimerosal solution and has a concentration of 20 IU / ml.

Each component that is part of the solution for lyophilization of antigen is added based on the required volume of this solution.

For example, per 100 ml of solution you will need: 1 g of bovine serum albumin (BSA); 0.75 g of glycine; 0.1 ml of 10% thimerosal solution; 100 ml distilled water.

The CCA-HBsAg kit also includes 9 vials of lyophilized negative serum (Reagent 1) used to dilute CCA.

2. A method of manufacturing a preparation of CCA-HBsAg, in which the drug can be used repeatedly and stored for a long time.

Advantages of the invention:

1. The ability to prepare the required number of dilutions of OCO-HBsAg in various concentrations, for example 0.01; 0.05; 0.1; 0.25; 0.5; 1.0 IU / ml. For this purpose, 9 vials with Reagent 1 are included in the kit, which can be used gradually, preparing two to three concentrations for one ELISA.

2. Repeated use of the OCO-HBsAg preparation due to the possibility of long-term storage of the re-concentrated concentrate, previously dispensed in aliquots, at a temperature of minus 20 ° C for a month or at minus 70 ° C until the end of the shelf life of the drug (3 years).

3. The invention provides the ability to compare the results of evaluating the sensitivity of test systems to detect HBsAg of various manufacturers, as well as the results of quantitative determination of HBsAg in laboratories.

Description of the invention

The invention relates to the field of immunology and standardization and can be used in biotechnology and medicine to determine the sensitivity of diagnostic test systems designed to detect hepatitis B virus surface antigen (HBsAg), as well as to determine the quantitative content of HBsAg in serum (plasma) and blood products human ELISA.

Hepatitis B virus is one of the most dangerous infections in humans. Modern detection methods allow the detection of HBsAg in low concentrations. Currently, work is underway to create and implement test systems with higher sensitivity in practical healthcare. In this regard, the need for an affordable quantitative standard is increasing.

The closest technical solution (prototype) to the claimed invention is a method of preparing "Second International Standard for HBsAg, subtype adw2, genotype A," NIBSC ", United Kingdom, code number 00/588" containing 33IU / vial, which consists in the following: plasma antigen isolated from a human plasma pool containing HBsAg (anti-HCV negative, anti-HBs, anti-HIVI + 2) is dissolved in a certain concentration in non-immune human plasma and lyophilized (Instructions for Use (Version 1, February 24, 2004)) .

The disadvantages of this method:

1. There is no solution for the preparation of dilutions. Therefore, each laboratory is forced to use the available plasma for the preparation of dilutions, which may affect the results and effectiveness of the analysis. To ensure a reliable and objective assessment of the sensitivity of test systems, it is necessary to use a unified solution that has passed reliable control to prepare dilutions.

2. The bottle of standard before use must be stored at minus 20 ° C, and after rehydration storage of the drug is not provided.

The objective of the present invention is a method of manufacturing OCO-HBsAg for determining the sensitivity of diagnostic test systems and for determining the quantitative content of hepatitis B virus surface antigen in serum (plasma) and human blood products by enzyme-linked immunosorbent assay, providing for a long shelf life and the possibility of repeated use.

The problem is solved by the developed method for manufacturing an industry standard sample (CCA) of hepatitis B virus surface antigen (HBsAg) to control the quality of test systems, as well as to determine the quantitative content of antigen in serum (plasma) and human blood products by ELISA, which consists in the fact that in 1% aqueous solution of bovine serum albumin containing 0.75% glycine, 0.01% thimerosal, add plasma or recombinant HBsAg hepatitis B virus surface antigen to a concentration of 20 IU / ml, pipet, mix t, preventing foaming, within 30-60 minutes and lyophilized. This obtained OCO-HBsAg preparation is included in the kit for quality control of test systems used to detect hepatitis B virus surface antigen, as well as to determine the quantitative content of HBsAg antigen in serum (plasma) and human blood products by ELISA, characterized in that it includes an industry standard sample (CCA) of hepatitis B virus surface antigen (HBsAg) obtained according to claim 1, and reagent 1, which is negative, freeze-dried human serum.

Reagent 1 is prepared by centrifuging the selected negative human blood serums, pooling, adding thimerosal to 0.01%, with 20-30% of its total dilution mixture added to the treated pool, including: 23.36 g (0.4 M) NaCl; 0.16 g (0.002 M) KCl; 0.16 g (0.001 M) KH ₂ PO ₄ ; 17.28 g (0.12 M) of Na ₂ HPO ₄ · 12H ₂ O; 10 g (1%) of bovine serum albumin; 0.5 ml (0.05%) of EDTA - ethylenediaminetetraacetic acid; 0.1 ml of a 10% thimerosal solution, followed by lyophilization of the finished solution to a residual moisture content of not more than 3%.

The technical result consists in increasing the duration of storage and providing the possibility of repeated use of the drug.

The invention can be illustrated by the following examples.

Example 1

For the manufacture of OCO-HBsAg, a plasma or recombinant hepatitis B virus surface antigen (HBsAg) can be used. This type of raw material can be isolated and purified by the method of Ananiev V.A. (purification of HBsAg from blood serum), or obtained according to the original methodology of the NGO Diagnostic Systems.

Recombinant HBsAg Production Plan:

1. Obtaining a yeast lysate of HBsAg producing cells.

2. PEG-pH precipitation of clarified lysate.

3. Obtaining an extract of HBsAg - enriched fraction.

4. Dialysis of the obtained HBs-Ag - enriched fraction into the buffer for HAP.

5. Adsorption chromatography.

6. Gel filtration.

7. Ultrafiltration.

Raw materials for the OCO-HBsAg preparation can be both commercial and in-house production.

The purified plasma or recombinant HBsAg was titrated in a dilution solution simultaneously with the International Standard (MS) on one tablet of the DS-ELISA-HBsAg test system. Joint processing of the titration curves of HBsAg and MS allows you to select a dilution factor to obtain HBsAg at a concentration of 20 IU / ml. If the concentration obtained does not correspond to the preset one, an adjustment is possible in the direction of increasing by introducing an additional amount of HBsAg, or in the direction of decreasing using a diluting solution. Then the finished solution was poured into bottles with a capacity of 3 cm ³ .

Reagent 1 was prepared in the following way: selected serum (plasma) of blood that did not contain HBsAg, antibodies to HBsAg; antibodies to HIV-1,2; Treponema pallidum, HCV. The selection of the studied sera was performed using enzyme-linked immunosorbent assay systems. The selection method allows you to select only those serums for pooling that adequately interact with the antigen, i.e. do not give reaction "quenching", suppression of HBsAg.

Example 2

Each serum was introduced into two wells of the immunosorbent of the DS-ELISA-HBsAg test system, then 10 μl of the OCO-HBsAg preparation (20 IU / ml) was added to one of the wells, and further formulation was carried out in accordance with the instructions for use. The result of the analysis was evaluated by the serum OD in the well (without the addition of the CCA-HBsAg preparation) and by the serum OD (with the added CCA-HBsAg preparation). Serum selection criteria:

1. The OD in the well with serum without the addition of OCO-HBsAg should not exceed twice the OD value of the K-test system.

2. The OD in the well of each serum with the added OSO-HBsAg preparation should correspond to the average OD (of the studied sera) ± 20%; or ± 20% of 1 IU / ml OCO-HBsAg.

Selected sera were pooled, centrifuged, thimerosal was added to 0.01%. 20-30% (of the total volume of the prepared pool) of the mixture for dilution was added to the treated pool. To prepare 1 l of the mixture for dilution of the pool you will need: 23.36 g (0.4 M) NaCl; 0.16 g (0.002 M) KCl; 0.16 g (0.001 M) KH ₂ PO ₄ ; 17.28 g (0.12 M) of Na ₂ HPO ₄ · 12H ₂ O; 10 g (1%) of bovine serum albumin; 0.5 ml (0.05%) EDTA - ethylenediaminetetraacetic acid; 0.1 ml of 10% thimerosal solution. Then the finished solution was poured into 1.0 ml into bottles with a capacity of 3 cm ³ (filling error not more than 1%) and lyophilized (residual moisture content not more than 3%).

The set (set) of "OCO-HBsAg" includes:

1. OCO-HBsAg 20 IU / ml, freeze dried - 1 vial.

2. Reagent 1 - human serum is negative, freeze-dried - 9 bottles.

New in comparison with the prototype is:

- the use of both plasma and recombinant HBsAg;

- introduction to the composition of the kit dilution solution (Reagent 1) for the preparation of dilutions.

The main certified characteristics of the CCA are: specific activity, uniformity, stability and shelf life.

According to the basic principle of WHO, the activity of biological preparations cannot be adequately measured by physical methods. This activity is determined only in comparison with the standard. A quantitative assessment of the tested CCA was carried out by the parallel line method in accordance with the methodological recommendations of GISK named after L.A. Tarasovich “Statistical processing of the results of quantitative ELISA (linearity, parallelism, determination of drug activity and confidence intervals of activity” [V. G. Petukhov, “Biological product”, No. 1 (13), pp. 19-23, 2004] ) Due to the complexity of the calculations and to avoid errors in the calculations, a computer program was used. In this particular case, the concentration of the HBsAg CCA preparation was determined in comparison with the international standard (Second International Standard for HBsAg, subtype adw2, genotype A, "NIBSC", United Kingdom, cod number 00/588) - 20 ± 0.5 IU / ml at confidence probability of 0.95.

The activity of the candidate for CCA HBsAg (p.5) was determined in comparison with the international standard containing 33IU / vial. For this, we prepared five dilutions of the international standard and the test candidate for CCA p.5 in triplicate:

Preparation of dilutions of international standard:

Dose Breeding preparation 1.6 me 1 / 20.6 times 0.8 me 1 / 41.2 times 0.4 me 1 / 82.5 times 0.2 me 1/165 times 0.1 me 1/330 times

Each sample was investigated in the test system "ELISA HBsAg / M" in accordance with the instructions for use. Using the Paraline computer program, the presence of linearity and parallelism was determined. Then, the equation of the dependence of the logarithms of OD cf on the logarithms of the corresponding concentrations of HBsAg was constructed.

Dependence of LN (OD) on LN activity of the international standard:

- 1.98 -2.3 - 1.27 -1.6 - 0.57 -0.91 0.124 -0,223 0.813 0.47

The dependence of LN (OP) on the dilution of the candidate in CCA p.5 (Figure 1):

Intelligence. OP Cand LN (OP Cand) Ln (soda HBsAg) soda HBsAg (IU) / ml 1 \ 12.5 2,271 0.82 0.47142 1,601915339 1 \ 25 1,148 0.138 -0.204442 0.815462371 1 \ 50 0.576 -0.551 -0.887241 0.41188957 1 \ 100 0.283 -1.26 -1.58986 0.204129639 1 \ 200 0.139 -1.97 -2.29347 0,100963118 y = 0.991X-0.3412

Based on the obtained HBsAg (ME) content in the prepared dilutions of the candidate in CCA p.5, the content of HBsAg in the vial of the study drug was determined:

Intelligence. soda HBsAd (IU) / ml Contents HBsAg (IU) / ml 1 \ 12.5 1,601915339 20,114 1 \ 25 0.815462371 20,356 1 \ 50 0.41188957 20.47 1 \ 100 0.204129639 20,201 1 \ 200 0,100963118 20,017 20,237 average

As a result of comparative studies of CCA-HBsAg s.5 and the international standard, it was found that the content of HBsAg in the bottle of CCA s.5 corresponds to 20 ± 0.5 IU / ml.

The CCA-HBsAg preparation was examined for homogeneity, the coefficient of variation was 11% (the number of tested bottles was 1% of the series). The method is as follows.

For the study of homogeneity, a certain number of vials was selected (1% of the series), one concentration was prepared from each vial of CCA-HBsAg (for example, 1 IU). Then on one tablet using ELISA examined each bottle of this concentration in three repetitions. Having obtained the result, we calculated the X mean, sigma, coefficient of variation.

After quality control of the freeze-dried components, the stability of OCO-HBsAg was determined by the accelerated test method in accordance with the requirements of the GISK them. L. Tarasovich “Determination of the stability of industry standard samples (CCA) and other MIBP accelerated method” (Author - V. G. Petukhov, Moscow, 2002). Shelf life of CCA is 3 years.

Example 3

CCA-HBsAg p. 5 concentration of 0.5 ME Time (day) 65 ° C 55 ° C 44 ° C 0 one hundred% one hundred% one hundred% 2 98% 6 89% 9 73% 92% 16 65% 89% 23 59% 81% 28 68% 99% 35 96% 45 92% fifty 89%

Logarithms of the fall in activity (Figure 2):

0 4.6 4.6 4.6 2 4,585 6 4,4886 9 4,2905 4,5218 16 4.1744 4,4886 23 4.0775 4.3944 28 4,2195 4,5951 35 4,5643 45 4,5218 fifty 4,4886

Decontamination rate constants at selected temperatures (Figure 3):

Conclusion: the shelf life of CCA HBsAg s.5 is 3 years.

Preparation and conditions of use of the standard sample

When working with OCO-HBsAg, all measuring instruments and devices must be certified and verified.

Preparation of rehydrated Reagent 1: In a bottle with dry Reagent 1 add 1.0 ml of distilled water and mix thoroughly until the contents of the bottle are completely dissolved. The rehydrated solution of Reagent 1 is a clear, colorless or light yellow solution, without precipitate, opalescence is allowed. Store at (6 ± 2) ° C for not more than 1 week.

Preparation of the CCA-HBsAg solution, concentration of 20 IU / ml: add 0.8 ml of the working solution of Reagent 1 into the vial of CCA-HBsAg and mix thoroughly until the contents of the vial are completely dissolved. The rehydrated OCO-HBsAg preparation is a clear, colorless or yellowish solution, without precipitate, opalescence is allowed. It is possible to store the OCO-HBsAg solution, previously poured in aliquots, at a temperature not exceeding minus 20 ° C for 1 month or at minus 70 ° C until the end of the shelf life of the drug. A single thawing-freezing of the drug is allowed.

Preparation of OCO-HBsAg dilutions: to determine the sensitivity of diagnostic kits designed to detect hepatitis B virus surface antigen or the quantitative content of HBsAg in test samples, a series of dilutions of CCA-HBsAg are prepared on rehydrated Reagent 1 with antigen concentration, for example, 0.05; 0.1; 0.125; 0.25; 0.5; 1.0 IU / ml. Such dilutions are prepared at least 4 and each dilution in at least 3 replicates.

Preparation of dilutions of the test sample: to determine the quantitative content of HBsAg, the optical density of the sample (OD) at the used wavelengths should be strictly in the range from 0.1 to 2.5 optical units (pu). If the OD of the test sample is outside the specified range, select empirically by diluting the sample, for example 1:10; 1: 100; 1: 1000; 1: 10000.

Serial dilutions are prepared so that the optical densities of the prepared dilutions (at least 3 replicates) lie in the linear region of this device.

Each dilution of a standard / test sample should be prepared in a separate vial or tube. In this case, each repetition is prepared separately from the original standard sample in order to assess the spread of the analysis results when diluting the standard / test sample.

Immunological tests

When determining the sensitivity of the enzyme immunoassay system and the amount of HBsAg in the test sample, ELISA is carried out in accordance with the requirements set forth in the instructions for use of the test system used.

To determine the sensitivity of the enzyme-linked immunosorbent assay dilution system, CCA-HBsAg is added to at least 3 wells of the immunosorbent.

To assess the quantitative content of HBsAg in the sample, the studied sample must be added to the wells of the immunosorbent in at least 3 replicates; on the same tablet, each dilution of CCA-HBsAg is studied in parallel.

Accounting and processing of results

When assessing the sensitivity of the test system, this indicator corresponds to the average least amount of HBsAg, which is determined by this test system.

For a rough estimate of the quantitative content of HBsAg, a graph is plotted on the abscissa axis of which the logarithms of CCA-HBsAg concentrations are plotted, on the ordinate axis, the logarithms of Oscr corresponding to these HBsAg concentrations, and a curve is obtained from which the HBsAg concentration in the test sample is calculated based on the value the logarithm of its optical density. If the sample was previously diluted, then in this case, the concentration value obtained on the abscissa axis is multiplied by the dilution factor. It should be borne in mind that the error of this estimate can reach significant values (up to 20% or more).

For accurate quantitative determination of the concentration of HBsAg in the studied images, it is necessary to use the method of serial dilutions of these samples in parallel with serial dilutions of CCA. Moreover, to calculate the concentration of HBsAg and its 95% confidence interval, the method of analysis of variance should be used [V. G. Petukhov, “Biological product”, No. 1 (13), pp. 19-23, 2004]. Due to the complexity of the calculations and to avoid errors in the calculations, it is recommended to use a computer program.

Claims (2)

1. A method of manufacturing an industry standard sample (CCA) of hepatitis B virus surface antigen (HBsAg) for quality control of test systems, as well as for determining the quantitative content of antigen in serum (plasma) and human blood products by ELISA, characterized in that in 1 % aqueous solution of bovine serum albumin containing 0.75% glycine, 0.01% thimerosal, add plasma or recombinant HBsAg hepatitis B virus surface antigen to a concentration of 20 IU / ml, mix, avoiding foaming, for 30-60 min and lyophilized. 2. A set for the quality control of test systems used to detect the surface antigen of hepatitis B virus, as well as to determine the quantitative content of HBsAg antigen in serum (plasma) and human blood products by ELISA, characterized in that it includes an industry standard sample (CCA) ) hepatitis B virus surface antigen (HBsAg) obtained according to claim 1, and reagent 1, which is a negative human blood serum, freeze-dried, prepared by centrifugation of selected negative blood serums person, pooling, adding thimerosal up to 0.01%, moreover, 20-30% of its total volume of the mixture for dilution, including 23.36 g (0.4 M) NaCl, was added to the treated pool; 0.16 g (0.002 M) KCl; 0.16 g (0.001 M) KH ₂ PO ₄ ; 17.28 g (0.12 M) of Na ₂ HPO ₄ · 12H ₂ O; 10 g (1%) of bovine serum albumin; 0.5 ml (0.05%) of EDTA-ethylenediaminetetraacetic acid; 0.1 ml of a 10% thimerosal solution, followed by lyophilization of the finished solution to a residual moisture content of not more than 3%.

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