RU2206095C1 - Test system for detecting antibodies to hbs antigen and test system blocker - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves using test system having stripped palette sensibilized with recombinant yeast HbsAg of ay and/or ad serologic types, rinsing solution, biotin HbsAg conjugate, avidine-pyroxidase or streptavidine-peroxydase, substrate buffer with hydrogen peroxide, chromogen, stop-reagent, sample of positive and negative control as blood serum obtained by immunizing healthy donors with recombinant yeast vaccine against hepatitis B, titrated according to international standard of anti-Hbs contents or branch standard sample of human immunoglobulin against hepatitis B, blocker in the form of 5% sodium caseinate solution in sodium-borate buffer having pH equal to 8.3, reference-standard for quantitatively determining antibodies to HBs-antigen as blood serum produced by immunizing healthy donors with recombinant yeast vaccine against hepatitis B, titrated according to international standard of anti-Hbs contents or branch standard sample of human immunoglobulin against hepatitis B and logarithm table for calculating quantitative contents of antibodies to HBs-antigen. EFFECT: high specificity and safety of analysis. 2 cl, 1 dwg, 4 tbl

Description

 The invention relates to virology, biotechnology and immunology, and for the development of an enzyme-linked immunosorbent assay system for determining antibodies to the hepatitis B virus surface antigen (HBsAg).

 A known test system designed to detect antibodies to HBsAg in human serum (plasma). Test system "Vekto HBsAg - antibodies-strip". CJSC Vector - Best, Russia. Instructions for use. 04/03/2000

 A well-known test system can be used to monitor the level of specific immune response during hepatitis B vaccination, is a set of reagents for detecting antibodies to HBs antigen by enzyme-linked immunosorbent assay and is designed for 96 analyzes, including controls.

Set Composition:
a stripped plate with immobilized HBsAg subtypes ad and ay - 1 pc;
control positive serum (K ⁺ ) - 1 vial, 1.3 ml;
control negative serum (K ^- ) - 1 vial, 2.5 ml;
conjugate (HBs antigen labeled with horseradish peroxidase) - 1 vial, 1.3 ml;
concentrate of phosphate-saline buffer solution with tween (25-fold) - 1 vial, 20 ml;
citrate-phosphate buffer - 1 vial, 12 ml;
tetramethylbenzidine (concentrate) - 1 vial, 1 ml;
stop reagent - 1 vial, 6 ml.

 In the known test system, a positive control is represented by the blood serum of animals immunized with HBsAg. In this case, the presence of biological material carries the risk of infection, and a similar reagent can be attributed to those that require precautionary measures, while the blood serum of animals may contain other infectious agents.

 The test system is intended only to detect antibodies to the HBs antigen and does not allow determination of their quantitative content in the sample.

 As the closest analogue, the known test system for determining antibodies to the hepatitis B pathogen surface antigen is considered.

 "Monolisa anti - HBs 3.0". Code 72400. 96 tests. Franch National Center for Blood Transfusion, 2000.

 The composition of the known set is given in table. 1.

 In the preparation of the known test system, highly pathogenic material is used - the blood serum of people infected with hepatitis B. There is no complete guarantee that there are no pathogens of other infectious agents. Reagents of "human" origin are considered to be potentially infected and appropriate precautions must be observed when working with them.

 The objective of the invention is the creation of a test system for determining antibodies to HBsAg, which allows high-quality and safe analysis of qualitative and quantitative analysis in various biological samples - blood serum, plasma.

 The technical result of the invention is to increase the specificity of the analyzes using the developed test system, which is ensured by the use of reagents that are not substitutes for biological agents, and also by the fact that when working with this test system, the best washing of the antigen-antibody complex from non-specific components of the studied biological sample.

 The problem is solved using the proposed new test system.

 The invention consists in the following.

 The test system is a set of reagents necessary for the qualitative and quantitative determination of antibodies to the hepatitis B pathogen surface antigen by ELISA in blood serum or plasma.

 The test system additionally includes a reagent-blocker (code name) developed by the inventors, which is a protein-salt solution.

 The use of a new reagent in the performance of ELISA provides a reduction in non-specific reactions, which ultimately increases the accuracy of the method.

 The test system is designed for 96 analyzes, including controls.

Set Composition
1. 1 microplate (12 strips of 8 holes in a frame), sensitized with yeast recombinant HBsAg serotypes ay and / or ad. Marking MP.

2. Washing solution:
0.01 mol / L phosphate-buffered saline pH 7.2 ± 0.2 - 24 ml
Twin 20 - 1.25%
Sodium merthiolate (preservative) - 0.01%
1 bottle contains 24 ml.

 Marking FSB-T.

3. Blocker - protein-salt solution, freeze-dried:
Sodium Caseinate - 0.25 g (5%)
Sodium borate buffer pH 8.3 - 5 ml
3 vials.

 Marking B.

 4. Negative control sample.

 Monitored for the absence of HBs antibodies.

Human serum that does not contain antibodies to the surface antigen of hepatitis B virus - 7 ml
Sodium azide (preservative) - 0.1%
1 vial containing 7 ml. Marking To.

 5. Positive control sample.

Blood serum of donors immunized with recombinant hepatitis B hepatitis B vaccine serotypes ay and / or ad - 0.2 ml
Sodium azide (preservative) - 0.1%
Marking K +.

Calibrated in mIU / ml according to the international standard anti-HBsAg
1 vial containing 0.2 ml, or an industry standard sample of human immunoglobulin against hepatitis B virus OCO 42-28-320-00.

It is a protein solution of an immunologically active fraction of immunoglobulin isolated by fractionation with ethyl alcohol at a temperature below 0 ^o C from the plasma (serum) of blood from donors immunized with a recombinant yeast liquid hepatitis B vaccine. Sterile immunoglobulin contains a 50% sucrose solution with a concentration of 2% in the final product. Lyophilized in ampoules.

 Ampoule 0.2 ml.

 6. Biotin-HBsAg-conjugate.

Biotin-labeled yeast recombinant HBsAg serotypes ai and / or ad - 0.6 ml
Bovine Serum Albumin - 1%
Sodium merthiolate (preservative) - 0.01%
1 vial containing 0.6 ml of solution. Marking Kt-1.

 7. Conjugate avidin peroxidase or streptavidin peroxidase.

Avidin or Streptavidin-labeled peroxidase - 0.6 ml
Bovine Serum Albumin - 1%
Sodium merthiolate (preservative) - 0.01%
1 vial containing 0.6 ml of solution.

 Marking Kt-2.

 8. The substrate buffer.

Citrate-phosphate buffer pH 5.2 ± 0.2 - 39 ml
Hydrogen Peroxide - 0.033%
1 bottle contains 39 ml of solution. Marking Sat.

 9. Chromogen.

 Orthophenylenediamine (CRF content in a 6 mg tablet). 1 vial, contains 3 tablets. Marking OFD.

 10. Stop reagent.

 5% sulfuric acid solution.

 1 bottle contains 12 ml.

 Marking CP.

 11. Reference standard.

 It is a blood serum obtained by immunizing healthy (non-painful) donors with a recombinant yeast vaccine against hepatitis B, titrated according to the international standard for the content of anti-HBs.

1 bottle
or an industry standard human immunoglobulin sample against hepatitis B virus OCO 42-28-320-00.

 Ampoule 0.2 ml.

The test system is equipped with reagents in various versions, which allows the consumer, depending on the task, to use one of the 3 proposed sets:
1. The test system contains reagents for the determination of antibodies to HBsAg serotype ay.

 2. Test system for determining antibodies to HBsAg serotype ad.

 3. Test system for determining antibodies to HBsAg serotypes ay and ad.

 The test system does not contain as reagents any samples isolated from the blood of infected patients or animals, which ensures the complete elimination of the risk of infection when working with this test system and does not require special precautions. This circumstance makes the system convenient and safe to use.

 1. Preparation of immunosorbent.

Recombinant HBsAg, serotypes ay and / or ad, diluted in sterile 0.01 mol / L phosphate-buffered saline (pH 7.2 ± 7.4) to a protein concentration of 1-2 μg / ml, contribute to each well 96- hollow solid or collapsible polystyrene tablet company "Costar" (USA, cat. 92592) or similar quality of other companies. The tablet is sealed and held for 18-20 hours at a temperature of 4-10 ^o C. After this, the contents of the wells are removed by strong shaking and the tablet is washed three times with sterile phosphate-saline buffer solution (pH 7.2 ± 0.2) containing 0.5% Tween-20. The washed tablet is placed in a bag of plastic film, lay a desiccant and hermetically sealed.

Tablets with immobilized HBsAg stored at a temperature of 4-10 ^o C.

 2. Preparation of the conjugate (HBsAg-biotin).

 2.1. Dissolve the biotin N-hydroxysuccinimide ester in dimethyl sulfoxide to a protein concentration of 3 mg / ml.

2.2. Recombinant HBsAg serotypes ay and / or ad are dissolved in 0.1 mol / L carbonate-bicarbonate buffer pH 8.6 to a protein concentration of 1 mg / ml and dialyzed against several changes of this buffer for 12 hours at a temperature of 4 ^o C.

2.3. Two solutions are mixed in a volume ratio of 1:10 (activated biotin: recombinant HBsAg) and incubated at a temperature of 20-22 ^o C for 4 hours.

2.4. The resulting HBsAg-biotin conjugate is dialyzed against several shifts of 0.05 mol / L Tris-HCl buffer solution pH 7.4 at a temperature of 4-10 ^{° C.} for 12 hours in order to remove the unbound biotin derivative.

2.5. After removal from dialysis, bovine serum albumin and sodium thiolate are added to the conjugate to a final concentration in the solution of 1 and 0.01%, respectively. The finished conjugate is stored at a temperature of 4-10 ^o C.

 3. Preparation of the HBsAg-Biotin-Avidin-Peroxidase (or HBsAg-Biotin-Streptavidin-Peroxidase) Conjugate.

 Prepared immediately before use: Avidin-peroxidase conjugate (streptavidin-peroxidase) in working dilutions is added to biotinylated HBsAg.

 The conjugate avidin peroxidase (streptavidin peroxidase) - peroxidase labeled with avidin (streptavidin) - commercial preparations of the company "Sigma" (cat. 3151, 9420) or similar quality from other companies.

 4. Preparation of a positive and negative control sample.

 For the preparation of a positive control sample, the serum of donors immunized with a recombinant yeast liquid hepatitis B vaccine is used. To detect antibodies to the ad subtype, a recombinant vaccine containing ad-HBsAg is vaccinated. A positive control sample is calibrated in mIU / ml relative to the World Health Organization standard.

 To prepare a negative control sample, use the serum of donors who were not immunized with the hepatitis B vaccine, who did not have hepatitis B, and were monitored for the absence of HBs antibodies.

 All sera used for the preparation of standard samples are monitored for the absence of antibodies to human immunodeficiency viruses HIV-1, HIV-2, hepatitis C virus and the absence of hepatitis B virus surface antigen (HBsAg).

 5. Preparation of a protein-salt solution (5% casein solution in sodium borate buffer solution (pH 8.3)). (Blocker).

Preparation of a solution of boric acid with a concentration of 0.5 mol / L. 31 g of boric acid (H ₃ BO ₃ ) are weighed, dissolved in distilled water and the volume is adjusted to 1 liter.

 Preparation of a solution of sodium hydroxide concentration of 0.5 mol / L. 20 g of sodium hydroxide (NaOH) are weighed, dissolved in distilled water and the volume is adjusted to 1 liter.

 Preparation of sodium borate buffer solution (pH 8.3). Sodium hydroxide solution is added to the boric acid solution until the pH of the solution reaches 8.3.

25 g of casein are weighed, transferred to a 500 ml conical flask and 250 ml of sodium borate buffer solution are added. The flask was placed on a magnetic stirrer, the solution was stirred for 30 minutes, then, while continuing to mix, the flask was placed in a water bath and heated to 40 ^{° C.} until casein was completely dissolved. The solution from the conical flask was transferred to a volumetric flask with a capacity of 500 ml, cooled to 20 ^° C and the volume was adjusted to 500 ml with sodium borate buffer solution.

 The resulting casein solution is poured into 5 ml into 15 ml vials, freeze-dried and sealed. Protein-saline solution is added to FSB-T in order to reduce non-specific reactions during ELISA.

 6. Preparation of citrate-phosphate buffer solution pH 5.0-5.5, containing 0.033% hydrogen peroxide (TSFBR).

Raw materials:
- distilled water - FS-42-2619-97;
- citric acid - GOST 3652-89;
- sodium phosphate disubstituted - GOST 4172-76;
- hydrogen peroxide, 33% solution - GOST 177-88.

Description of the technological process:
4.1 g of citric acid (C ₆ H ₈ O ₇ x ₃ H ₂ O) and 11.15 g of sodium phosphate disubstituted (Na ₂ HPO ₄ x ₃ H ₂ O) are weighed, dissolved in distilled water and the volume is adjusted to 1 liter. To the resulting solution was added 1.0 ml of 33% hydrogen peroxide (H ₂ O ₂ ).

 Citrate-phosphate buffer solution with hydrogen peroxide is poured into 39 ml glass graduated pipettes into bottles. The vials are closed with rubber stoppers and sealed with aluminum caps.

 Each bottle with CFRD is completely wrapped in metal foil.

 7. Chromogen.

 Tablets OFD - TU 6-14-113-70-95.

 8. Tab. 4 (the logarithm table) is included in the instructions for use of the test system for the detection of antibodies to the surface hepatitis B virus.

9. Phosphate-saline buffer solution:
- distilled water - FS-42-2619-97;
- potassium phosphate disubstituted (K ₂ НРО ₄ х3Н ₂ О) - GOST 2493-75;
- monosubstituted potassium phosphate (KH ₂ PO ₄ ) - GOST 4198-75;
- sodium chloride (NaCl) - GOST 4233-77;
- Tween-20 - imported ("Pharmacia", Sweden).

 Preparation of a 25-fold concentrate of phosphate-buffered saline pH (7.2 ± 0.2) containing 1.25% Tween-20 (FSB-T).

228 g of potassium phosphate disubstituted (K ₂ НPO ₄ х 3Н ₂ О) are weighed, dissolved in distilled water and the volume is adjusted to 1 liter. 136 g of potassium phosphate monosubstituted (KH ₂ PO ₄ ) are weighed, dissolved in distilled water and the volume is adjusted to 1 liter. 215 ml of a solution of K ₂ HPO ₄ × 3H ₂ O and 35 ml of a solution of KH ₂ PO ₄ prepared as described above are mixed, 12.5 ml of Tween-20 are added and the volume is adjusted to 1 liter with distilled water. To the resulting solution was added 215.5 g of sodium chloride (NaCl).

 Phosphate-saline buffer solution with tween is poured into 24 ml glass graduated pipettes into bottles. The vials are closed with rubber stoppers and sealed with aluminum caps.

 10. As a standard calibrator for constructing a calibration graph, a positive control sample is used. The graph is based on the measurement results of OP for five dilutions of K +, Fig.1.

 The positive control for the ad-test system is based on ad Ig.

 When preparing an immunosorbent for sensitization, for example, yeast recombinant HBsAg serotype au can be obtained by culturing cells of the Saccharomyces cerevisiae strain VKPM Y-2203 (RF Patent 2088664) or KBT-98p HB-50 / VKPM - Y2412. (ad) (FSP 42-0029-1385-01). The same known antigens are also used in the preparation of the biotin-HBsAg conjugate.

 In the case of obtaining a positive control sample and reference standard, for example, a well-known hepatitis B vaccine is used containing an effective amount (20 μg) of hepatitis B virus surface antigen obtained by culturing transformed cells of the Saccharomyces cerevisiae strain VKPM Y-2203, 0.5 mg gel aluminum hydroxide (in terms of Al), PBS buffer (50 mM Na-phosphate, pH 6.8 + 0.13 M Nad), 0.005% sodium merthiolate.

 RF patent 2088664.

 Immunization of donors is carried out according to known schemes developed for the introduction of the indicated vaccines, for example, three times / m in the deltoid muscle.

Mode of application
Serum or plasma may be used in the study. To exclude false results, test samples must be taken and stored under conditions excluding bacterial growth.

 The presence of precipitates, blood clots, blood cells can lead to false positive results. Therefore, insoluble particles must be removed before staging the reaction by centrifugation. Chylous serum may cause false negative results. Therefore, lipids must be removed before staging the reaction by centrifugation.

The studied samples of serum (plasma) of human blood are stored at a temperature of (6 ± 2) ^o C no more than 24 hours. Longer storage is allowed at a temperature not exceeding -20 ^o With the exception of repeated thawing - freezing (no more than 1 time).

 Samples with severe hemolysis, strongly opalescent or contaminated, cannot be analyzed.

Reagent Preparation
All components used in the formulation of the reaction must be maintained for at least 30 minutes at a temperature of (22 ± 2) ^o C.

 Preparation of a solution for washing the tablets and diluting the conjugate (solution 1).

 Measure the required amount of FSB-T concentrate, dilute 25 times with distilled water. To stir thoroughly.

After storage in the FSB-T concentrate after storage at a temperature of 2-8 ^o C, a crystalline precipitate of salts may form. In this case, before diluting FSB-T, it is necessary to heat it at a temperature of (37 ± 1) ^o С until the precipitate is completely dissolved.

 Dissolve the contents of the vial with the blocker in 5.0 ml of the prepared FSB-T. Add the resulting solution to a container with a solution of FSB-T (according to Table 2) and mix thoroughly.

 Preparation of a solution for diluting K + and test samples (solution 2).

 Measure the required amount of negative control sample (K-) and dilute it with solution 1 10 times. To stir thoroughly.

 Preparation of control samples (K + and K-).

 K- is ready for use and does not require additional dilution.

 Dilute the positive control sample in a clean glass vial with solution 2 to a content of 100 mIU / ml of antibodies to the surface hepatitis B virus. For example, if the titer is K + 2000 mIU / ml, the first dilution should be 1: 20. Subsequent two-fold dilutions (1:40 , 1:80, etc.) respectively contain 50; 25; 12.5; 6.25 mIU / ml.

 Preparation of conjugate solution.

 Cook immediately before use. Measure 4.8 ml of solution 1 into a clean glass bottle, mix with 0.2 ml of CT-1. Measure 4.8 ml of solution 1 into another bottle, mix with 0.2 ml of CT-2. To the solution of Kt-1 add a solution of Kt-2, mix thoroughly. The prepared solution is not subject to storage.

 Preparation of the CRF solution.

 Cook immediately before use in a dark place. Dissolve 1 tablet of CRF in 13 ml of substrate buffer, mix thoroughly (until the tablet is completely dissolved). The prepared solution is not subject to storage.

Enzyme immunoassay
Flushing. Before use, rinse the tablet three times with solution 1, adding 0.3 ml of solution to each well of the tablet. After washing, it is necessary to carefully remove moisture from the wells of the tablet by tapping on filter paper folded into several layers. Do not allow the tablet to dry between separate operations when setting up the reaction.

 The introduction of control and test samples. Dilutions of the positive control sample (K +) add 0.1 ml per well: C1 - 100 mIU / ml, D1 - 50 mIU / ml, E1 - 25 mIU / ml, F1 - 12.5 mIU / ml, G1 - 6 25 mIU / ml.

 Negative control sample (K-) add 0.1 ml to wells A1 and B1.

 To control the conjugate, add 0.1 ml of solution 1 (“blank”) to the HI well.

 The test samples of blood serum (plasma) are whole or diluted (diluted with solution 2 on a separate tablet), add 0.1 ml to the remaining wells of the tablet (A2, B2, C2, etc.).

The tablet is sealed and incubated for (60 ± 5) minutes at a temperature of (37 ± 1) ^o C.

 At the end of the incubation, collect the contents of the wells in a vessel with a disinfectant solution, rinse the tablet 5 times with solution 1 and remove moisture by tapping on filter paper folded into several layers.

The introduction of the conjugate. In addition to H1 (blank), add 0.1 ml of conjugate to each well of the plate. The tablet is sealed and incubated for (60 ± 5) minutes at a temperature of (37 ± 1) ^o C.

 At the end of the incubation, collect the contents of the wells in a vessel with a disinfecting solution, rinse the tablet 5 times with solution 1 and remove the remaining moisture by tapping on filter paper folded into several layers.

 The introduction of the CRF solution. Pipette 0.2 ml of CRF solution into each well of the plate.

The tablet should be kept at a temperature of (20 ± 2) ^o С for 30 minutes in a dark place.

 The introduction of a stop reagent. The reaction is stopped by adding 0.1 ml of a stop reagent (CP) to each well and the optical density (OD) is immediately measured.

Registration and evaluation of results
The ELISA results are recorded using a spectrophotometer, measuring the optical density (OD) at a wavelength of 492 nm. Bringing the spectrophotometer to the zero level ("blank") is carried out in the well of the H1 plate. The results of the studies are evaluated if the average OD value in the wells with K- is not more than 0.1, and the OD value in the well with K + 100 mIU / ml is not less than 0.80 optical units.

The test sample is regarded as positive if the corresponding OD value exceeds the critical OD value (OD _crit ), which is calculated by the formula
OD _crit = OD K- _cf + 0.05,
where OP K- _cf is the average value of OP K-.

The determination of the level of specific antibodies in positive serum samples is carried out according to a calibration graph constructed in the coordinates: y - log OP K +, x - log ₂ of its dilution ratio (see graph) of FIG. The graph is based on the results of measuring the OD for five dilutions of K +.

Knowing the OD of the test sample (for example, 0.371 - undiluted), find the lg of this number (lg 0.371 = -0.43) and draw a line parallel to the abscissa through the point on the ordinate axis corresponding to its value (-0.43). Perpendicular to the abscissa axis is lowered through the point of intersection of the straight and linear sections of the graph and the log ₂ multiples of dilution of the positive control sample (X = 2.8) are determined, which corresponds to the OD value of this dilution of the test sample.

 Then, according to the table. 3 find the multiplicity of dilution of the control positive sample corresponding to this logarithm (A = 6.96).

где Т - титр исследуемого образца, мМЕ/мл; T₁ - титр положительного контрольного образца, откалиброванного в мМЕ/мл по международному стандарту анти-HBs; А - кратность разведения положительного контрольного образца, найденная по табл. 3; A х 10 - разведение положительного контрольного образца; В - разведение исследуемой сыворотки, в котором определялась оптическая плотность.After that, calculate the titer of antibodies in the test sample according to the formula

where T is the titer of the test sample, mIU / ml; T ₁ is the titer of the positive control sample calibrated in mMU / ml according to the international standard anti-HBs; And - the multiplicity of dilution of the positive control sample, found in table. 3; A x 10 - breeding of a positive control sample; B - dilution of the test serum, in which the optical density was determined.

Предлагаемая тест-система выявляет анти-HBs в сыворотке крови с чувствительностью не менее 10 мМЕ/мл.Calculation example (Table 4): if the titer of the positive control sample is 2000 mI ml, then the titer of the test serum in our example

The proposed test system detects anti-HBs in serum with a sensitivity of at least 10 mIU / ml.

 The characteristics of the developed test system were determined using random samples of sera, samples from vaccinated individuals, samples from patients with viral hepatitis B, serum of vaccinated animals and anti-hepatitis B immunoglobulin. The level of anti-HBs was determined using the developed test system and Abbott kits. and "Monolisa".

 Qualitative and quantitative analysis was performed on 322 positive samples: 155 vaccinated people, 42 patients with viral hepatitis B, 114 sera of vaccinated animals (white mice and guinea pigs), 11 series of hepatitis B immunoglobulin.

 Comparability of the results of the determination of anti-HBs using the developed test system and test kits of Abbott and Monolisa firms was established: the correlation coefficient, respectively, was 0.90-0.95.

 The specificity of the test kit, estimated in a population of 553 unselected blood donors, was 99.9%.

 Analysis of 34 samples of patients with pathology unrelated to hepatitis B did not allow to establish the presence of nonspecific reactions.

The developed test system can be used for:
- determining the immune status of the population before vaccination and evaluating the effectiveness of vaccination;
- determining the severity and prognosis of the disease;
- selection of donor plasma with a high content of antibodies to HBsAg for the production of specific immunoglobulin;
- determination of the concentration of antibodies to hepatitis B virus in a specific immunoglobulin preparation;
- to assess the immunogenicity of vaccines in animals.

Claims (1)

1. Blocker in the test system for determining anti-HBs in a biological sample by enzyme immunoassay, containing sodium caseinate and sodium borate buffer lyophilized, dried in the following ratio of components:
Sodium Caseinate - 0.25 g
Sodium borate buffer pH 8.3 - 5 ml
2. Test system for determining anti-HBs in a biological sample by enzyme immunoassay, which contains a strip plate, sensitized HBsAg, washing solution, biotin-HBsAg conjugate, streptavidin-peroxidase conjugate, hydrogen peroxide substrate buffer, chromogen, stop reagent, positive a control sample and a negative control sample, characterized in that the yeast recombinant HBsAg serotypes ay and / or ad are used as antigens for sensitization of the tablet and in the biotin-HBsAg conjugate, and as of a false control sample, use blood serum obtained by immunizing healthy donors with a recombinant hepatitis B yeast vaccine, titrated according to the international anti-HBs standard, or an industry standard human immunoglobulin sample against hepatitis B virus, and the test system further comprises a blocker according to claim 1 , reference standard for the quantification of antibodies to HBs antigen in the form of blood serum obtained by immunizing healthy donors with a recombinant yeast vaccine against hepatitis B, titrated according to the international standard for the content of anti-HBs or industry standard sample of human immunoglobulin against hepatitis B virus and a logarithm table for calculating the quantitative content of antibodies to HBs antigen.

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