RU2380711C2 - Method for making operational standard of serums containing antibodies to hepatitis c virus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method includes certification of serum samples for observed antibodies to hepatitis C virus to assess availability thereof as an operational standard. The serum samples are certified by immunobiological markers of HBs antigen, hepatitis C virus antibodies, HIV types 1, 2 and anti-HBs, anti-HBc, anti-HBe antibodies, and by the biochemical properties: enzyme level, total protein, pH. The immune-specific samples are serums containing RNA of hepatitis C virus and positive results of the immune-enzyme assay ("ОП<ОПкр."), negative plasma samples of "ОТ" - PCR with "ОП<ОПкр." The operational standard represents a complete set of negative and a number of positive serums containing various types of antibodies to hepatitis C virus.

EFFECT: according to the invention, the methods allows making the operational standard containing serums close to the human native serums, and also assessing the quality of each antigen: core, NS3, NS4 and NS5, used in development of the immune-enzyme test systems for detecting antibodies to various proteins of hepatitis C virus, storing and handling the operational standard at higher temperatures.

4 cl, 5 tbl

Description

The invention relates to the field of biotechnology and immunology and can be used in the manufacture of working standards containing antibodies to causative agents of dangerous viral diseases. In order to evaluate the specific characteristics of test systems and evaluate the characteristics of various test systems, it is very important to have well-certified serum samples with known IgG concentration as a standard.

The need for speedy unification of test systems from different manufacturers is due, in particular, to the increase in the spread of such dangerous infections as HIV, HCV, HBV, and the main increase in newly infected people comes from risk groups. As a result of an injection with a dirty needle, 2 million people become infected with type C viral hepatitis every year, and 5% of all new HIV infections are related to the reuse of syringes. The solution to this problem is complicated by the fact that standards for anti-HCV and anti-HIV have not yet been approved by WHO. The first step towards the development of national reference materials for the diagnosis of HCV is the development and certification of working standards for anti-HCV.

The development of serum panels containing certified amounts of specific antibodies to socially dangerous viral infections of various genotypes is of the highest priority for assessing the quality of serological tests. The lack of evidence-based criteria for the formation of serum panels impedes certification of reference anti-HCV and anti-HIV panels as international standards. The current state of the serum panels, their development and application, and the problems associated with their certification and control were discussed in detail in an expert report of the WHO Committee for Biological Standardization on the assessment of the quality of diagnostic kits for the detection of anti-HCV antibodies [7].

Known panels of serum BBI company (Boston, USA). These PHA panels are composed of individual sera containing anti-HCV antibodies in different concentrations [1].

Similar panels of anti-HCV antibodies were developed in Russia [2, 4].

The disadvantages of such panels are mainly associated with incomplete certification of samples according to biochemical parameters, as well as the fact that not native but diluted serums are used for sample preparation.

The closest analogue (prototype) is the British Working Standard (anti-HCV) (The British Working Standard for anti-HCV) [3]. BRS is a diluted pool of anti-HCV positive sera confirmed in immunoblot (Lia HCV, RIBA HCV 3.0). The pool is made up of individual sera, each of which is diluted to an antibody concentration 30 times higher than the OPcrit value of the 2nd generation test system. Equal volumes of each serum are mixed and aliquots stored at -20 ° C. A working standard is prepared from these aliquots by diluting 30 times in 5% human serum albumin (HSA) in FSB containing 0.05% sodium azide.

The main disadvantages of the prototype are:

1. Use as a diluting solution of 5% HSA in the FSB. With this manufacturing technology, PC has a matrix that is significantly different from native human serum.

2. Storage conditions for PC - in a liquid state, PC is stored only at very low temperatures (-70 ° C). This significantly limits the range of states that can use the British PC, only developed countries with cold chains.

3. The use of a pool of serums as a standard makes it impossible to assess the quality of each of the antigens: core, NS3, NS4 and NS5 used in the creation of enzyme-linked immunosorbent assays to detect antibodies to various HCV proteins.

The technical result of the proposed technical solution is the creation of such a method of manufacturing a working standard of sera containing antibodies to HCV, which would allow to produce working standards close to native human serums, to evaluate the quality of each of the antigens: core, NS3, NS4 and NS5 used to create enzyme-linked immunosorbent test systems to detect antibodies to various HCV proteins, and to store and transport operating standards at higher temperatures.

The specified technical result is achieved by the fact that in the method of manufacturing a working standard (PC) of sera containing antibodies to hepatitis C virus, including the certification of serum samples for the presence of antibodies to hepatitis C virus to assess the possibility of using them as working standards, according to the invention, serum samples are certified by immunobiological markers for the content of HBs antigen, antibodies to hepatitis C virus, HIV-1,2 types and anti-HBs, anti-HBc, anti-HBe antibodies, and biochemical properties: level of enzymes, total Protein holding pH; as immunospecific samples for preparation of the working standards panel, sera containing hepatitis C virus RNA and positive according to the results of enzyme-linked immunosorbent assay (OD> OPcr) are selected, negative plasma samples according to RT-PCR having OD <OPcr, and use as the panel of working standards a set of negative and several positive sera containing various types of antibodies to hepatitis C.

It is most optimal to use 4 positive sera containing antibodies to HCV proteins anti-Core, anti-NS3, anti-NS4 and anti-NS5 and 1 negative serum in the working standard of sera. In the manufacture of each positive sample in the formation of working standards, a pool of 3-4 sera is used with an identical spectrum of antibodies, each of which is diluted to a titer of 1: 5. Lyophilized dried whey panels have a residual moisture content of not more than 1.0 wt.%.

When storing lyophilic PC sera at a temperature of 4 ° C, they retain their specific characteristics for more than 2 years.

Given the unique nature of hepatitis C infection, anti-HCV PC was designed as a set of samples containing antibodies to various HCV proteins, namely anti-Core, anti-NS3, anti-NS4 and anti-NS5. The kit includes samples containing antibodies only to individual HCV proteins and samples with antibodies to two or more proteins.

The kit includes 4 positive sera and 1 negative serum as an internal standard. The composition of PC sera is determined using the Spectrum test from Vector Best CJSC and Diagnostic Systems CJSC proteins (Table 1).

1. Plasma samples for the manufacture of PC

Serum for the manufacture of PC is harvested in infectious diseases hospitals and SEC by plasmapheresis in accordance with ethical standards and Sanitary rules. Each plasma sample is certified for 10 immunobiological and biochemical parameters.

2. Reconstitution solution

For titration of positive plasma samples and manufacturing of PC samples, a dilution solution developed for the manufacture of reference preparations of infectious diseases is used [4]. Merthiolate and gentamicin are added as bactericides in an amount of 0.015%.

As a phosphate-salt buffer, pH 7.1 - 7.6 - use the solution proposed [5] (SOP MED 84, NIBSC),

NaCl 80 +/- 1 g Kcl 2.0 +/- 0.1 g CaCl ₂ * 2H ₂ O 1.3 g MgCl ₂ * 6H ₂ O 1.0 g Na ₂ HPO ₄ * 12H ₂ O 29.0 +/- 0.1 g KH ₂ PO ₄ 2.0

100 l dist. H ₂ O

3. Certification of plasma samples

The criterion for determining the positivity of a plasma sample is the result of RT-PCR of RNA HCV. Positive plasma samples should contain HCV RNA and be positive by ELISA, i.e. have OP> OPcr. Negative plasma samples should be negative in RT-PCR and have OD <OPcr in ELISA.

Candidates for PC a-HCV titration initially with step 5, starting with a 1:10 dilution in 2 test systems "Best - anti-HCV" (Vector Best) and "Vector anti-HCV" (Vector). 10 candidates with OP / OPcr values from 2.5 to 8.0 are selected.

At the 2nd stage, all these samples are analyzed in the tests of “Recombest Best Anti-HCV Spectrum” CJSC VectorBest and “Spectrum Anti-HCV”, built on the proteins of ZAO “Diagnostic Systems”, i.e. determine the titer of antibodies to individual antigens.

Of the 10 candidates, 5 are selected that have the desired anti-HCV antibody titer.

At the 3rd stage, 5 samples are re-titrated in the 2 indicated test systems with step 2 in order to determine the dilution range in which the dependence OD = f (recon) is linear. The research results are presented in tables 1-4.

Table 1 shows the results of the titration of candidates for PC. Titration of samples V-1, V-3, V-5, V-6 and samples 1, 3, 5, 6. First dilution 1: 5, then titration in steps of 3.

Test system IM DI. (37US, rocking chair). OPcr = 0.205

Table 1
Antibody candidate standards titration results V-1 V-3 V-5 V-6 one 3 5 6 K + K- titer BUT 2,806 2,794 0.690 2,782 2,760 2,739 0.659 2,782 2,871 5 AT 2,761 2,703 0,069 2,731 2,782 2,771 0,067 2,751 2,804 fifteen FROM 2,575 2,633 0,049 2,693 2,618 2,693 0,048 2,740 0.051 45 D 2,713 2,566 0,044 2,600 2,783 2,783 0,047 2,732 0.051 135 E 2,852 2,785 0,045 2,840 2,755 2,785 0,057 2,852 0,046 405 F 2,370 0.834 0,049 2,988 2,875 0.833 0,054 3,039 0,048 1215 G 0.474 0.104 0.051 2,491 0,993 0.116 0,053 2,627 3645 N 0,094 0.056 0,055 0.434 0.186 0.058 0,046 0.470 10935

table 2
Certification of plasma samples taken for the production of anti-HCV working standards (ZAO DS, 10.11.07) Sample No. OD for anti-HCV PC kit sera Core NS3 NS4 NS5 Attribution one 0.496 0,101 0,093 0.105 Core 2 1,665 0,086 1.98 0,070 Core + NS4 3 0.214 0.152 2,444 0.170 NS4 four 2,393 0.871 0.280 0.287 Core + NS3 + NS5 5 0.141 0.138 2,672 0.672 NS4 + NS5 6 0.145 0.118 0,099 0,097 Negatives. 7 0.116 0,126 0.109 0,096 Negatives. 8 0.138 0.122 0,090 0,100 Negatives. With 0.250 0.254 0.236 0.264

Table 3
Certification of plasma samples taken for the production of anti-HCV working standards (CJSC V-Best, 11/15/07) Sample No. OD for anti-HCV PC kit sera Core NS3 NS4 NS5 Attribution one 1,934 0.352 0.265 0.353 Core 2 2,249 0,073 1,463 0.146 Core + NS4 3 0,020 0,021 1,137 0,033 NS4 four 1,517 0.705 0,07 1.004 Core + NS3 + NS5 5 0,027 3,532 0.360 0.641 NS3 + NS5 6 0.024 0,028 0.019 0,029 Negatives. 7 0,026 0,023 0,020 0,026 Negatives. 8 0,025 0.017 0.014 0.019 Negatives. With 0.212 0.211 0.217 0.209

Table 4
Candidate antibody titration to form PC panels The control Titer OD for anti-HCV PC kit sera one 2 3 four 5 3.6 1: 5 2,589 2,967 2,983 3,045 3,063 3,5 1:10 0.647 0.578 1.002 2,977 3,099 0.019 twenty 0.158 0.134 0.235 2,977 2,892 0.009 40 0.121 0,069 0.09 1,214 0.862 0.011 80 0,081 - 0,071 0.273 0.193 0.012 160 - - - 0,112 0,079 0.017 320 - - - 0,075 - 0.015 640 - - - - -

Of the 5 candidates for positive sera and 3 candidates for negative sera, 4 positive and 1 negative serum are selected according to the results of a study of the spectrum of antibodies (Table 4).

When considering the results of titration and the spectral repertoire of antibodies from 8 candidates, positive samples No. 1, 2, 3, 5 and a negative sample No. 6 (donor plasma) are selected as part of the PC.

Thus, PC anti-HCV is a set of 4 positive sera and 1 negative serum, which acts as an internal standard for monitoring the correct operation of the diagnostic kit. Each positive serum kit has its own characteristic spectrum of antibodies. This allows you to check the test system for the quality of the diagnostic antigen composed of 4 proteins: Core, NS3, NS4 and NS5.

4. Formation of candidate pools of the antibody panel

The titer values of individual plasma samples are used to select dilution coefficients (RS) before pooling plasma samples. With this approach, antibodies to HCV from each sample will be presented in the pool in equal amounts. KR corresponds to the dilution of the sample, on average, to a titer of -1: 30.

A specific example of the implementation of the proposed method.

Serum intake from healthy donors

Donor plasma is delivered to production in 250 ml CPDA hemacons, hermetically sealed.

Each donor serum entering the production for the production of an antibody panel must have a passport with the characteristics - ID (individual number) of the donor, serum volume, date of blood sampling, the manufacturer, and also meet the following requirements: without signs of hemolysis and hyperlipidemia, transparent, color - amber or light yellow, extraneous mechanical impurities, suspension, sediment are absent.

Inactivation of healthy donor sera

Vials with donor serum are placed in a thermostat at a temperature of 56 ° C. The inactivation start time is recorded. Incubation of serum spend 3 hours. After this time, the serum is removed from the thermostat, the time of the end of inactivation is recorded. The date of inactivation is recorded in the "Journal of registration of the movement of material."

For long-term storage, serums are stored in the medical freezer MDF 436 (Sanyo) freezer at -35 ° C before use. This ensures the preservation of serum properties for 5-7 years.

Adding a preservative solution to serum

Stabilizer components are added to each donor plasma vial [2], a solution of thiolate at the rate of 0.01-0.02 g per 100 ml of serum and a solution of calcium chloride to precipitate ballast proteins. Serum is thoroughly mixed.

Packing healthy donor sera

From each hemacone in a sterile box in a laminar, 5 aliquots of (1 ± 0.05) ml of plasma are automatically pipetted into 1.5 ml Eppendorf plastic tubes to control biochemical parameters and to study viral markers in PCR and ELISA. The tubes are closed, on each tube with a marker write the code number of the plasma sample.

The remaining volume of serum is Packed in sterile plastic bottles with a capacity of 25-30 ml.

Storage of healthy donor sera

Hemacacons with donor plasma and their aliquots are stored in freezers at a temperature of minus 35 ° С. Gemacons can be stored under these conditions for 5-7 years before processing. Serum freezing and thawing are allowed.

Monitoring of biochemical parameters of healthy donor sera

2 ml of each donor serum obtained in the previous stage is transferred to a biochemical laboratory to control biochemical parameters (enzyme level, total protein content, pH). Protein content is determined by the Lowry micro method.

Sera that do not meet physiological standards for biochemical parameters are discarded and disposed of.

ELISA of healthy donor sera by ELISA

A second aliquot of each donor serum is transferred to an ELISA laboratory for testing sera for the content of HBs antigen, antibodies to HCV, HIV-1,2 types and anti-HBs, anti-HBc, anti-HBe antibodies. IFA analysis is carried out on certified GISK them. L.A. Tarasevich enzyme immunoassay systems.

Sera that do not meet the requirements for the quality of the serum of healthy donors (i.e. containing HBs antigen, anti-HIV-1,2 and anti-HBV antibodies) are discarded and placed in a serum bank for research purposes.

Production Standards

For the manufacture of PCs, all 4 positive sera are diluted in a stabilized NPP to approximately 30–40 titer. The estimated amount of each serum sample, which must be diluted in a pool of donor sera to obtain an antibody level corresponding to a titer of 1: 30-1: 40, is determined from the preliminary titration curves of positive sera performed in step 2.

Bottling and freeze drying

Each candidate is poured into 0.3 ml into bottles, the coefficient of variation of the filling error is 0.2%. Vials in cartridges are loaded into the freezer at night - 60 ° C. In the morning, frozen bottles are placed evenly on the shelves of the freeze chamber, pre-cooled to minus 35 ° C. At this temperature, the shelves of the freeze chamber can withstand more than 3 hours. The condenser temperature does not exceed - 70 ° C. Within 3 hours, the vacuum reaches its minimum. And the operator includes a program for heating shelves from -35 ° C to + 20 ° C for 20 hours. At the final stage, at room temperature, the samples are thermostated with pumping for 6 hours. Then the pressure plates are lowered and the plugs are pressed into the bottles. Bottles are removed from the freeze chamber, rolled up with aluminum caps and placed in storage at - 35 ° C.

After lyophilization, the specific activity of the sera is monitored by ELISA.

Stability tests

The stability of sera under various storage conditions in the dark in vials is determined by the results of the thermal degradation test at temperatures: -20 ° C, + 4 ° C, + 37 ° C and 45 ° C [6].

A pooled sample of lyophilized anti-HCV panel candidates is tested in an accelerated thermal degradation test. Samples are thermostated under controlled temperature conditions, removed at regular intervals, dissolved in double-distilled water and analyzed in duplicates in the test systems “RecombiBest anti-HCV spectrum” and “Best anti-HCV” (CJSC VectorBest). No experimentally significant loss of antibody activity was noted in the experiment. The average values of the calculated percent loss of activity for the month and for the year are shown in table 2. Samples demonstrate high stability. The predicted loss of activity for the year at + 4 ° C is about 0.3% for candidates.

Table 5
Predict the activity of antibody panel samples based on accelerated trials 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

The predicted loss of activity per month is approximately 4% at 37 ° C, which allows for short-term storage of serum panels at elevated temperatures, for example, when transporting samples.

Thus, when storing lyophilic anti-HCV PC sera at 4 ° C, they retain their specific characteristics for more than 2 years.

Diluted PC antibody samples are recommended for storage at 4 ° C for a month. The stability of the liquid panel samples is tested in the test freezing (-35 ° C) - thawing (37 ° C). The test results show that the specific properties of the candidates do not change during 4 cycles of freezing and thawing.

The main goal of national PCs is the daily and long-term monitoring of clinical diagnostic laboratories (CDL). The use of PCs as “yes / no” controls requires that their indicators be recorded daily for each tablet and every day.

A separate problem that can be solved using these materials is a centralized comparison, analysis and accumulation of results from users. This implies comparing the PC indicators for the selected analytical center with respect to the results from other analytical centers that use the same test systems and, based on national quality experts (GISK named after L.A. Tarasevich), select the CDLs that are most successful in analyzing PC and transfer the methodology of these CDLs to laboratories that are less successful in PC analysis.

Information sources

1. Product catalog 2003/2004. Quality control products for infectious disease testing. Boston, USA

2. Kanev A.N., Netesov S.V., Maksuytov A.Z. et al. Razrabotka reference panelei syvorotok dla kontrola kachestva immunofermentnyx test-systems in the Russia. Vestnik Akad. Med. Nauk, 1998; 43 (3): 37-41. Russian

3. WHO working group On international reference preparations for testing diagnostic kits used for the detection of HBsAg and anti-HCV antibodies. (WHO Headquarters, Geneva, 6-7 October 2003).

4. Kanev A.H., Vorobyova M.C., Shalunova H.B. et al. Design and production of serum reference panels for quality control of test systems in Russia. // Bulletin of the Academy of Medical Sciences 1998, No. 3, 47-52.

5. SOP GISK, MP “Industry standard samples. The main provisions, the procedure for the development, manufacture, certification, approval and registration ”./ Ed. Petukhov V.G., GISK im. L.A. Tarasevich, M., May 2003.

6. Kirkwood T.B.L Principles for the preparation and approval of international and other standards and reference materials for biological preparations. Ser.techn. doc. No. 37. WHO, Geneva, 1990. 14a // J. biol. Standardization. - 1984. - Vol. 12. - P.207-224.

7. Ferguson M., Holmes H., Sands ED. UK blood transfusion service working standards for: HBsAg, anti - HCV and anti - HIV - l.//Vox Sang. 2001, 80: 205-210.

Claims (4)

1. A method of manufacturing a working standard of sera containing antibodies to hepatitis C virus, including the certification of serum samples for the presence of antibodies to hepatitis C virus to assess the possibility of using them as a working standard, characterized in that the serum samples are certified by immunobiological markers for the content of HBs antigen antibodies to hepatitis C virus, HIV-1,2 types and anti-HBs, anti-HBc, anti-HBe antibodies, and biochemical properties: enzyme level, total protein content, pH, as an immunospecific image To prepare a working standard, sera containing hepatitis C virus RNA and positive according to the results of an enzyme immunoassay (OD> OPcr) are selected, negative plasma samples according to RT-PCR with OD <OPcr, and a set of negative and several positive sera containing various types of antibodies to hepatitis C. 2. The method according to claim 1, characterized in that the working standard uses 4 positive sera containing antibodies to hepatitis C virus proteins: anti-Core, airra-NS3, anti-MS4 and anti-NS5 and 1 negative serum. 3. The method according to claim 1, characterized in that in the manufacture of each positive sample in the formation of the working standard, a pool of 3-4 sera with an identical spectrum of antibodies is used, each of which is diluted to a titer of 1: 5. 4. The method according to claim 1, characterized in that the lyophilized dried serum of the working standard have a residual moisture content of not more than 1.0 wt.%.