RU2325655C9 - Immune-enzyme test system for hepatitis b virus surface antigen detection and method of hepatitis b virus surface antigen detection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: detection or confirmation of hepatitis B virus surface antigen (HBsAg). Immune-enzyme test system for HBsAg identification in blood serum (plasma) and blood preparations (albumin, fibrinolysin, immunoglobulins and leukocytic interferon) containing: solid phase containing immunosorbent that is mice HBsAg monoclonal antibodies fixed on tray strips; conjugate -1 - biotin with HBsAg monoclonal antibodies and conjugate-2 - streptavidine marked with horseradish peroxidase as liquid concentrates; positive control sample; negative control sample; washing solution; solution for conjugates dilution; substrate buffer solution; tetramethyl benzidine as chromogen; sulfuric acid as stop-reagent; neutralizing reagent, liquid or lyophilized that is goat HBsAg antibodies immunoglobulins; control reagent liquid or lyophilized that is goat immunoglobulins without HBsAg antibodies; weakly-positive control sample that is HBsAg lyophilized blood serum in concentrations 0.02-0.06 ME/ml. Method of hepatitis B virus surface antigen (HBsAg) detection is performed through immune-enzyme test of biological sample by means of the given test system.

EFFECT: invention allows to detect required antigen of low sensibility at high specificity level.

4 cl, 1 ex, 8 tbl

Description

The invention relates to the field of medicine, immunology and biotechnology.

Usage: detection of hepatitis B virus surface antigen (HBsAg) or confirmation of the specificity of the detection results.

Essence: obtaining an enzyme-linked immunosorbent assay system for identifying HBsAg in serum (plasma) and blood products (albumin, fibrinolysin, immunoglobulins and leukocyte interferon), which allows the detection of the desired antigen with a sensitivity of at least 0.01 IU / ml.

For the diagnosis of hepatitis B using specific tests, the detection of serum surface antigen of hepatitis B virus - HBsAg, which is the main marker of the disease, which is recorded long before the onset of clinical signs of the disease, is crucial. In the early stages of the infectious process and before disappearing from the circulation, the concentration of HBsAg can be several pg / ml.

Universal vaccination accelerated the accumulation of mutant forms of hepatitis B. The presence of mutations in the viral genome causes a change in the structure of HBsAg antigenic epigones or biological properties of the virus, for example, a decrease in virus secretion from cells. As a result, the detection of HBsAg is reduced using certified diagnostic tests [Weinberger K.M. et al., 2000].

It has been established that with co-infection with hepatitis B and C viruses or hepatitis B virus and HIV, the replication of hepatitis B virus is suppressed. This leads to the fact that the amount of HBsAg in the blood serum is below the sensitivity limit of most used test systems approved for use [Berger A . et al., 2000; Hofer M. et al., 1998; Schuttler C.G. et al., 2002].

Modern methods for detecting HBsAg can detect it in low concentrations. There are national criteria for the sensitivity and specificity of domestic drugs. So, since 2003, sensitivity of 0.1 IU / ml has been regulated for test systems for HBsAg.

However, the identification of cases of hepatitis B after transfusion of “seronegative” for HBsAg blood indicates the need for further work on the creation and implementation in practical healthcare of test systems with high sensitivity while maintaining high specificity. Therefore, the creation of an enzyme-linked immunosorbent assay system for the determination of serum HBsAg at a concentration of 0.01 IU / ml will reduce the risk of post-transfusion infection with viral hepatitis B and improve the quality of screening of donated blood.

One way to increase the sensitivity of detection of HBsAg in an enzyme-linked immunosorbent assay is to use the biotin-streptavidin immunochemical complex. In the synthesis of an antibody-biotin conjugate, up to 90 biotin molecules can be coupled to one antibody molecule. The streptavidin molecule has 4 biotin binding centers; therefore, each specific immunochemical complex at the final stage will contain not one enzyme molecule, but several tens. The use of a long spacer for biotinylation significantly reduces steric barriers in the interaction of biotin-labeled antibodies with streptavidin.

The closest technical solution (prototype) to the claimed invention in terms of features is the diagnostic kit “Enzygnost HBsAg 5.0” (Dade Behring, Germany), based on a two-stage formulation, which contains biotin and streptavidin conjugates (Medicines and Healthcare Products Regulatory Agency, 06 (2003, Evaluation Report MHRA num-ber 03044). In a first step, the HBsAg contained in the test sample binds simultaneously to the sheep polyclonal anti-HBsAg antibodies adsorbed into the wells of the plate and to the conjugate-1, which is mouse monoclonal anti-HBsAg antibodies linked to biotin. In the second stage, after washing the unbound reagents, conjugate-2 (streptavidin labeled with horseradish peroxidase) interacts with conjugate-1. After washing unreacted reagents, the enzymatic activity of the bound conjugate-2 is detected by the colored product of the interaction of the enzyme with a mixture of substrate and chromogen. Staining intensity is proportional to the concentration of HBsAg in the test sample. The total reaction time is 120 minutes The disadvantage of this kit is that its sensitivity does not exceed 0.1 IU / ml, which is the reason that impedes the achievement of the required technical result.

The objective of the present invention is to obtain a test system with which it is possible to detect surface hepatitis B virus antigen (HBsAg) in human serum (plasma) with a sensitivity of 0.01 IU / ml while maintaining high specificity.

The essence of the proposed technical solution is that the detection of HBsAg with a sensitivity of 0.01 IU / ml is carried out using a diagnostic enzyme-linked immunosorbent assay system based on the use of a pair of conjugates, one of which is monoclonal anti-HBsAg antibody associated with biotin, the second streptavidin labeled with horseradish peroxidase, and during the enzyme-linked immunosorbent assay, there is a stage of co-aging in the well of the test sample simultaneously with two conjugates.

The test system for determining the surface antigen of viral hepatitis B (HBsAg) by enzyme-linked immunosorbent assay in a biological sample contains:

- as a solid phase, an immunosorbent, which is monoclonal mouse antibodies to HBsAg adsorbed on strips of a polystyrene tablet,

- the first conjugate - (11-fold concentrate), a clear or slightly opalescent, violet-colored liquid, monoclonal antibodies to HBsAg associated with biotin,

- the second conjugate is a clear or slightly opalescent, blue-colored liquid, streptavidin labeled with horseradish peroxidase,

- a positive control sample, inactivated, transparent or slightly opalescent, raspberry-red liquid, human blood serum containing HBsAg, not containing antibodies to HIV-1,2 and hepatitis C virus and antibodies to HBsAg,

- negative control sample, inactivated, transparent or slightly opalescent, light green liquid, human serum not containing HBsAg, antibodies to HIV-1,2 and hepatitis C virus,

- weakly positive control sample, lyophilized, dry porous amorphous mass, hygroscopic, white or light yellow in color, is human blood serum containing HBsAg at a concentration of 0.02-0.06 IU / ml and not containing anti-HIV-1 antibodies, 2 and hepatitis C virus and antibodies to HBsAg,

- washing solution - a clear or slightly opalescent, colorless or light yellow liquid, a 25-fold concentrate of phosphate-saline solution containing 2.5% tween-20,

- a solution for diluting conjugate-1, an opaque green liquid,

- a solution for diluting conjugate-2, a clear yellow liquid,

conjugate dilution solutions are protein-salt solutions,

- substrate buffer solution containing hydrogen peroxide, a clear, colorless liquid,

- 3.3'5.5'-tetramethylbenzidine as a chromogen, a clear, colorless liquid,

- stop reagent - a solution of sulfuric acid 0.75 mol / l, a clear, colorless liquid.

The kit includes a neutralizing reagent, liquid or lyophilized, which is a goat immunoglobulin containing antibodies to the HBs antigen; a control reagent, liquid or lyophilized, representing goat immunoglobulins that do not contain antibodies to the HBs antigen to confirm the surface antigen of HBsAg.

As biological material, serum or blood plasma, a blood preparation, for example, selected from the group: albumin, fibrolisin, immunoglobulin, leukocyte interferon, can be examined.

The technical result achieved by the present invention is to improve the sensitivity of detection of HBsAg to 0.01 IU / ml by incubating directly with biotinylated antibodies in the well of the test sample, and then without incubation the incubation mixture is aged with horseradish streptavidin peroxidase conjugate, which leads to amplification of a specific signal.

This technical solution differs from the known ones by the presence of a phase of simultaneous joint curing in the well of the test sample with conjugates-1 and -2.

An enzyme-linked immunosorbent assay system for determining (detecting or confirming) the hepatitis B virus surface antigen ("DS-ELISA-HBsAg-0.01") can be made in the form of 2 sets.

The invention is illustrated by the following examples.

Example 1

To identify the surface antigen of hepatitis B in serum (plasma) and blood products (albumin, fibrinolysin, immunoglobulins and leukocyte interferon) set 1 ("DS-ELISA-HBsAg-0.01") is intended.

Reagents included in kit 1:

immunosorbent - mouse monoclonal antibodies to HBsAg adsorbed on strips of polystyrene plate; conjugate-1 - liquid (x11 concentrate) - antibodies to HBsAg conjugated with biotin; conjugate-2 - (x11 concentrate) liquid - streptavidin labeled with horseradish peroxidase; positive control sample (K + 1), liquid - human serum containing HBsAg, not containing antibodies to HIV-1,2 and hepatitis C virus, inactivated; weakly positive control sample (K + 2), lyophilized - HBsAg with a concentration of 0.04 ± 0.02 IU / ml in human serum, not containing antibodies to HIV-1,2 and hepatitis C virus, inactivated; negative control sample (K-), liquid - human blood serum not containing HBsAg, not containing antibodies to HIV-1,2 and hepatitis C virus, inactivated; washing solution (PR, concentrate x25), liquid; a solution for diluting conjugate-1 concentrate (RRK-1), liquid; a solution for diluting conjugate-2 concentrate (RRK-2), liquid; substrate buffer solution (SB), liquid; chromogen - tetramethylbenzidine (TMB), liquid; stop reagent - sulfuric acid, solution, 0.75 mol / L.

Set 1 is calculated.

Set 1 with an increased volume of components - for simultaneous 192 (96 × 2) determinations, including control ones (set 1 can be used on an automated ELISA analyzer).

Set 2 - for the simultaneous implementation of 192 (96 × 2) determinations, including control ones.

Set 3 with an increased volume of components - for simultaneous 96 (8 × 12) determinations, including control ones (set 3 can be used on an automated ELISA analyzer).

Set 4 - for simultaneous 96 (8 × 12) determinations, including control.

Preparation of solutions.

Weakly positive control sample. Prepare before use. Dissolve the contents of the vial with physiological saline in the volume indicated on the vial label, mix gently and let stand for 5-10 minutes until use.

Wash working solution - for washing the immunosorbent. The contents of the bottle with PR concentrate are diluted 25 times with distilled water. The resulting solution was thoroughly mixed. The prepared wash solution is stored for no more than 3 days at a temperature of 2 ° C to 8 ° C.

Conjugate-1, working solution. Prepare before use. When using the whole tablet, 0.5 ml of conjugate-1 concentrate is added to 5.0 ml of conjugate-1 concentrate solution and mixed carefully.

Conjugate-2, working solution. Prepare before use. When using a whole tablet, 0.5 ml of conjugate-2 concentrate is added to 5.0 ml of conjugate-2 concentrate solution and mixed carefully.

The shelf life of working solutions of conjugates is no more than 30 minutes in a dark place.

Substrate mixture. Prepare before use. Transfer 1.2 ml of tetramethylbenzidine to 18.0 ml of substrate buffer solution and mix the contents of the vial thoroughly. Not subject to storage.

The remaining unused reagents should be stored in vials sealed with plugs during the shelf life of the test system at a temperature of 2 ° C to 8 ° C.

Conducting IFA.

To avoid moisture condensation inside the wells, it is necessary to maintain the immunosorbent at room temperature in a closed bag for at least 30 minutes.

Before using sets 3 and 4, it is necessary to open the foil bag with immunosorbent. Remove the frame and the required number of strips from the package. Carefully seal the bag with unused strips without removing the desiccant. After the first opening of the package, the immunosorbent is stable for 1 month at a temperature of 2 ° C to 8 ° C.

Before use, rinse the immunosorbent of any kit 3 times with a working solution of PR, pouring it to the edges of the wells (from 380 to 400 μl per well), while not allowing the wash solution to be transfused over the edges of the wells, incubate for 40 s and remove the wash solution in an infected collection container material.

Then, 100 μl of control and test samples were added to the wells of a plate with adsorbed monoclonal antibodies to HBsAg. The lidded tablet is held for 30 minutes at a temperature of (42 ± 0.5) ° C in a humid chamber. Then, without deleting the contents of the wells and without washing the plate, 50 μl of a green colored conjugate-1 working solution are added to all strips. At the same time, the contents of the holes changes its color to gray. When testing plasma or serum having an acidic pH, the solution in the wells turns bright yellow. When conjugate-1 is added, one should not allow accidental introduction of a sample from one strip to another through the tips. The contents of the wells are thoroughly mixed by gently tapping the edges of the tablet, after which the coated tablet is kept for 45 minutes at a temperature of (42 ± 0.5) ° С in a humid chamber. Then, without deleting the contents of the wells and without washing the plate, 50 μl of yellow conjugate-2 working solution are added to all strips. When conjugate-2 is added, one should not allow accidental introduction of a sample from one strip to another through the tips. The contents of the wells are thoroughly mixed by gently tapping the edges of the tablet, after which the coated tablet is kept for 45 minutes at a temperature of (42 ± 0.5) ° С in a humid chamber. Next, the contents of the wells are removed into a container for collecting infected material and the plate is washed 7 times with a working wash solution. 150 μl of substrate mixture are added to all wells of the plate and incubated for 25 minutes in a dark place at a temperature of 20 ° C to 24 ° C. The reaction is stopped by adding 50 μl of stop reagent to all wells of the plate and after 2-3 minutes the results of ELISA are monitored.

Accounting for results.

The results are taken into account spectrophotometrically at two wavelengths: 450 / 620-680 nm with the instrument tuned "by air". Let us consider the results at one wavelength of 450 nm. The reaction should be taken into account if the average optical density (OD) in the wells with K is not more than 0.120, in the wells with K + 1 it is not less than 0.6. The OD values in wells with K- should not differ from the average value of OD K- by more than 30%. If one of the values of OP K- goes beyond this limit, it should be excluded from the calculation of the average value. If more than one OD K- value is to be excluded, the analysis should be repeated. The reaction is considered positive if the OD value in the well with the sample is equal to or exceeds the critical OD value (OD crit.). OP crit. calculated by the formula:

OP crit. = OP K- cf. + 0.060, where 0.060 is the coefficient determined by the statistical processing method at the manufacturer.

A weakly positive control sample containing HBsAg at a concentration of 0.04 ± 0.02 IU / ml should give a positive reaction. The total time for setting ELISA is 145 minutes.

When the enzyme-linked immunosorbent assay was performed on a shaker, control and test samples contribute 100 μl together with a working solution of conjugate-1 in an amount of 50 μl. The tablet is kept for 40 minutes on a shaker at a speed of 500 rpm and a temperature of (42 ± 0.5) ° С. Then, without removing the contents of the wells and without washing the plate, 50 μl of the conjugate-2 working solution are added to all the strip wells. The tablet is incubated for 20 minutes at 42 ° C on a shaker at a speed of 500 rpm and a temperature of (42 ± 0.5) ° C. Next, the contents of the wells are removed into a container for collecting infected material and the plate is washed 7 times with a working wash solution. 150 μl of substrate mixture are added to all wells of the plate and incubated for 25 minutes in a dark place at a temperature of 20 ° C to 24 ° C. The reaction is stopped by adding 50 μl of stop reagent to all wells of the plate and after 2-3 minutes the results of ELISA are monitored.

The total time for setting ELISA using a shaker is 85 minutes.

As another embodiment of the invention, there may be confirmation of the specificity of the results of the detection of hepatitis B virus surface antigen in serum (plasma) and blood products (albumin, fibrinolysin, immunoglobulins and leukocyte interferon) using kit 2 "DS-ELISA-HBsAg-0.01 -confirming. "

In addition to the reagents included in kit 1, kit 2 includes ANTI-HBs-PLUS (neutralizing reagent) - liquid or lyophilized, goat immunoglobulins containing antibodies to the HBs antigen; ANTI-HBs-MINUS (control reagent) - liquid or lyophilized, goat immunoglobulins that do not contain antibodies to the HBs antigen. The lyophilized contents of the vials are dissolved with distilled water in the volume indicated on the label.

Set 2 is calculated.

Set 5 with an increased volume of components - for the simultaneous conduct of 48 (4 × 12) confirmatory determinations, including control ones (set 1 can be used on an automated ELISA analyzer).

Set 6 - for the simultaneous conduct of 48 (4 × 12) confirmatory determinations, including control ones.

When stating ELISA control samples and samples to be confirmed, add each in two wells of 100 μl. Add 25 μl of the control reagent - ANTI-HBs-MINUS to the first wells with control and confirmed samples, and to the second wells of 25 μl of the neutralizing reagent - ANTI-HBs-PLUS. Cover the tablet with the lid for 30 minutes at a temperature of (42.0 ± 0.5) ° C in a humid chamber. Further ELISA, as well as setting on a shaker, is carried out similarly as described above using set 1.

The results should be taken into account spectrophotometrically at two wavelengths - 450 nm and 620-680 nm with the instrument tuned “over the air”. Let us consider the results at one wavelength of 450 nm. The reaction should be taken into account if the average OD value in wells with K- (without the addition of a control or neutralizing reagents) (OD K- cf.) is not more than 0.12, and in a well with K + 1 (without adding a control or neutralizing reagents) ( OP K +) - not less than 0.6. The OD value in the well with K + 1 after adding the neutralizing reagent (OPN K + 1) should be equal to or less than half the sum of the OD in the well with K + 1 after adding the control reagent (OPK K + 1) and OP K-Wed, t .e. OPN K + 1≤0.5 × (OPK K + 1 + OP K-cf.). The values of OD K- after the addition of neutralizing and control reagents should be lower than the critical OD value (OD crit.), I.e. OPn K- and OPK K- <OP crit. OP crit. calculated by the formula:

OP crit. = OP K- cf. + 0.06, where 0.06 is the coefficient determined by the method of statistical processing of the results of IFA production at the manufacturer.

The reaction is considered positive if the OD value in the well with the sample is equal to or greater than OD crit.

A weakly positive control sample containing HBsAg at a concentration of 0.04 ± 0.02 IU / ml should give a positive reaction.

Interpretation of Results

A sample is considered to contain HBsAg if

a) OPK ≥ OD crit.,

b) OPn ≤ 0.5 × (OPK + OD K- cf.),

where OPK is the OD of the sample with a control reagent, and OPN is the OD of the sample with a neutralizing reagent. In this case, the presence of HBsAg in the sample should be considered proven and no further verification is required.

The analytical sensitivity of the DS-IFA-HBsAg-0.01 test system was determined using the Industry Standard Sample of HBsAg Hepatitis B Virus OSO 42-28-311-03P (NPO Diagnostic Systems LLC), calibrated according to the International Standard - Second International Standard for HBsAg, subtype adw2, genotype A, NIBSC code number: 00/588, in comparison with the test system DS-IFA-HBsAg and Hepanostika HBsAg ULTRA (Biomerieux). 10 serial dilutions of CCA were prepared from 1 IU / ml to 0.01 IU / ml, with normal donor plasma containing no antibodies to HBsAg. The data obtained are presented in table 1.

The analytical sensitivity of the test system "DS-ELISA-HBsAg-0.01" amounted to 0.01 IU / ml

Assessment of the clinical sensitivity of the test system "DS-ELISA-HBsAg-0.01" was carried out using standard serum panels containing and not containing HBsAg.

Table 2 presents the results of comparative testing of a standard panel of sera containing and not containing HBsAg (CJSC Vector-Best), series 001.

A comparative characterization of the sensitivity and specificity of four test systems was carried out using a reference panel of human blood sera containing and not containing hepatitis B virus surface antigen (HBsAg) subtypes ay and ad OCO 42-28-364-04P (Avicenna Medical Center) , Boston Biomedics), series No. 1. The data obtained are shown in table 3.

Table 3 Test results of a reference panel of human blood sera containing and not containing hepatitis B virus surface antigen (HBsAg) subtypes ay and ad (OD / OD crit.) No. Subtype HBsAg The concentration of HBsAg ng / ml "DS-IFA-HBsAg-0.01" Abbott Auszyme EIA IFA-HBsAg / m (LLC NPO Diagnostic Systems, Russia) BIO-RAD Genetic systems HBsAg EIA 01 - - 0.68 0.1 0.26 Otr 02 - - 0.53 0.1 0.33 Otr 03 - - 0.49 0.1 0.13 Otr 04 - - 0.38 0.1 0.29 Otr 05 ad one 40.11 3.4 21.50 Pos 06 ad 0.6 39.73 2.2 12.50 Pos 07 ad 0.2 17.22 1,0 3.22 Pos 08 ay 0.9 39.62 3,5 12.70 Pos 09 ay 0.8 39.94 3,1 13.10 Pos 10 ay 0.6 38.31 2.1 12,20 Pos eleven ay 0.4 30.35 1.4 4.37 Pos 12 ay 0.2 17.76 0.7 3,51 Pos 13 ay 0.1 7.31 0.4 1.21 Pos fourteen Not at all. sample 2-3 42.26 8.2 23.98 Pos fifteen Not at all. sample 2-3 40.85 10.1 22.17 Pos 16 Not at all. sample 1-1.5 42.51 4.0 28.23 Pos 17 Not at all. sample 0.5-0.7 40.87 2.0 19.52 Pos eighteen Not at all.
sample 0.5-0.7 40.03 2,5 15.10 Pos 19 Not at all. sample 0.1-0.25 25.27 1,2 5.33 Pos twenty Not at all. sample 0.1-0.25 19.54 0.6 3.62 Pos

Table 4 shows the results of a comparative study of the clinical sensitivity of the DS-ELISA-HBsAg-0.01 test system using panels manufactured by BBI Diagnostics (Boston Biomedica Inc. (BBI), West Bridgewater, Mass., USA) and Zeptometrix Corporation.

A new test with identical sensitivity reveals HBsAg subtypes ad and ay with a significant advantage over the comparison test - the Abbott Auszyme EIA HBsAg (V2) system. From these tables it is seen that the test system "DS-ELISA-HBsAg-0.01" with identical sensitivity determines the HBsAg subtypes ay and ad in all positive samples presented in the panels. The positive coefficients of many HBsAg-positive samples in the test system "DS-ELISA-HBsAg-0.01" significantly exceed the positive coefficients of the same samples tested on alternative tests. In low-panel panel samples (from 0.05 to 0.6 IU / ml), the new test system also allows the detection of HBsAg with higher positivity ratios compared to alternative tests.

An analysis of seroconversion panels showed that the DS-IFA-HBsAg-0.01 test system allows HBsAg to be detected in positive samples at an earlier date of its appearance compared to foreign test systems, the data for which are given in the panel passports. According to the analysis of two panels of samples obtained from one donor in dynamics during the seroconversion period (from the moment of emergence to the moment of disappearance of HBsAg) (RNM935A (M) and RNM935 B), it follows that the new system, in comparison with alternative tests of import production, begins to detect HBsAg at an earlier date of its appearance and continues to determine at a later date as it disappears, as a result of which the HBsAg-positive period increases by 28 days compared to the Abbott Prism HBsAg ^{* system} .

It was found that the use of the DS-IFA-HBsAg-0.01 test system for the analysis of seroconversion panel samples allows the detection of HBsAg simultaneously with viral DNA (BBI-PHM933, BBI-PHM934, BBI-PHM935A (M), ZeptoMetrix Corporation Cat No. HBV6281), and in two cases (ZeptoMetrix Corporation Cat. No. HBV6277 and Cat. No. HBV6279) ahead of the time of the first DNA detection.

It is known that there is a problem of detecting HBsAg during co-infection. 473 serum samples of anti-HIV-positive individuals were analyzed. Among them, HBsAg was detected in 66 in the test "DS-ELISA-HBsAg". Additionally, 16 samples containing HBsAg at a concentration below 0.1 IU / ml were detected. Moreover, 7 of them contained antibodies to the HBcore antigen, and 9 samples did not have any other markers, except for HBsAg, detected only in a system with high sensitivity (table 5). Therefore, increasing the sensitivity of tests for detecting HBsAg will reduce the incidence of latent hepatitis B, both with the presence of an additional anti-HBc marker and in the absence of any markers.

When testing 3348 blood serum samples of donors and 381 blood serum samples of pregnant women in the test system "DS-ELISA-HBsAg-0.01" revealed 6 and 1 sample, respectively, containing HBsAg in a concentration below 0.1 IU / ml.

Due to the increased sensitivity of the new test, mutant HBsAg variants show higher reactivity compared to other systems (table 6).

Table 6 Comparative determination of mutant forms of recombinant HBsAg (ODm / OD crit.) adw2 DS-IFA-HBsAg DS-IFA-HBsAg-0.01 "Hepanostika" (Biomerieux) Rekomatgep-V-strip (Ekolab) Vectohep-HBsAg-strip Number 4 K141E 1,6 10,4 8.6 1.3 2.2 Q129H 1,2 13.0 16.6 1.4 2,3 Q129L 1,5 14.1 11.0 1.7 1,2 M133L 2,8 16.9 13.6 1,6 2.2 T126N 2.9 17.7 8.1 1,6 1.4 G145RI 1.4 9.1 5.8 1,2 1.7 G145RII 1,6 15.7 18.8 1,2 0.8 K142S 2.0 5,6 44.1 1,5 1.3 P142s 1,5 11.8 54.8 1,5 1.4 T143K 1,5 11.7 2.9 1,2 0.6 K141BI 3.6 2,5 54.6 0.9 0.9 ayw1 G145R 2,5 27.4 45.1 2.1 3.3

To assess the specificity of the test system "DS-ELISA-HBsAg-0.01" were used samples of blood serum donors; blood serum samples of patients with various infectious diseases not associated with HBV-pneumonia, tonsillitis, bronchitis, viral hepatitis C; blood serum samples of patients with various non-communicable diseases - with pathology of the cardiovascular system, gastrointestinal tract, cancer, etc. blood serum samples of pregnant women; serum samples of HIV-infected individuals. A total of 4710 samples were tested. Data on the number of false positive results in the test system "DS-ELISA-HBsAg-0.01" in the group of healthy and various groups of patients are presented in table 7.

Table 7 Assessment of the specificity of the test system "DS-IFA-HBsAg-0.01" in different categories of the population Surveyed group The number of samples Number of false positives % specificity in this group Healthy donors 3348 7 99.8 Patients with various infectious diseases not associated with viral hepatitis B 392 5 98.7 Patients with various noncommunicable diseases 95 one 98.9 Pregnant women 381 3 99,2 HIV infected 494 four 99,2 Total 4710 twenty 99.6

Thus, the studies showed that the specificity of the test system "DS-ELISA-HBsAg-0.01" is 99.6% in the study of blood serum samples from normal donors and patients with various infectious and somatic diseases.

The possibility of using a new test for the detection of HBsAg in blood products was studied. In the course of the studies, a wide range of blood preparations was analyzed by different manufacturing companies: albumin, fibrinolysin, leukocyte interferon, normal immunoglobulin for intravenous and intramuscular administration, anti-allergic immunoglobulin and anti-staphylococcal immunoglobulin. Liquid blood products are used undiluted; freeze-dried blood products are diluted before testing in the test system in accordance with the instructions for use of this drug. The specificity of the test system with respect to blood products (five different series of drugs were taken) was studied in three experimental-production series of the test system (table 8).

Table 8 The specificity of the test system "DS-IFA-HBsAg-0.01", established in the study of blood products The name of the drug OD of drugs (p.u.) average (n = 5) for each series of test system C.1 C.2 C.3 Albumen 0.017 0,025 0,032 Fibrinolysin 0.007 0.009 0.005 Immunoglobulin intravenous 0.009 0.011 0.015 Immunoglobulin Intramuscular 0.014 0.008 0.010 Interferon 0.005 0.007 0.008 OP crit. (P. E.) 0,072 p.u. 0,069 p.u. 0,075 p.u.

From the data of the table it can be seen that the OD values of all drugs were lower than OD crit. (p.u.), i.e. test system "DS-ELISA-HBsAg-0,01" showed 100% specificity when testing blood products.

The advantage of the proposed test system for detecting HBsAg "DS-ELISA-HBsAg-0.01" is the ability to detect HBsAg in serum (plasma) and blood products with a sensitivity of 0.01 IU / ml while maintaining high specificity.

Claims (4)

1. A test system for determining the surface antigen of viral hepatitis B (HBsAg) by enzyme-linked immunosorbent assay in a biological sample, characterized in that it contains a solid phase containing immunosorbent, which is a monoclonal mouse antibody adsorbed on strips of the tablet against HBsAg; conjugate-1 - biotin with monoclonal antibodies to HBsAg and conjugate-2 - streptavidin labeled with horseradish peroxidase, presented in the form of liquid concentrates; positive control sample; negative control sample; washing solution; solutions for diluting conjugates; substrate buffer solution; tetramethylbenzidine as a chromogen; sulfuric acid as a stop reagent; a neutralizing agent, liquid or lyophilized, representing goat immunoglobulins containing antibodies to HBsAg; a control reagent, liquid or lyophilized, representing goat immunoglobulins that do not contain antibodies to HBsAg; weakly positive control sample, which is serum in lyophilized form, containing HBsAg at a concentration of 0.02-0.06 IU / ml 2. The test system according to claim 1, characterized in that the biological material is serum or blood plasma. 3. The test system according to claim 1, characterized in that the biological material is a blood preparation selected from the group of albumin, fibrolizin, immunoglobulin, leukocyte interferon. 4. A method for determining the surface antigen of viral hepatitis B (HBsAg) by enzyme-linked immunosorbent assay in a biological sample using the test system according to claim 1, comprising maintaining the control and test biological samples introduced into the wells of a plate with adsorbed mouse anti-HBsAg monoclonal antibodies at temperature ( 42 ± 0.5) ° С followed by the addition of a colored conjugate solution, which is a monoclonal antibody to HBsAg, bound to biotin, and then without incubation the incubation mixture is thoroughly mixed ivayut and incubated with colored conjugate solution, which is a streptavidin-labeled horseradish peroxidase, at a temperature of (42 ± 0,5) ° C, the reaction was stopped by addition of stop reagent, and records of the results is carried out spectrophotometrically.