RU2545792C2 - Method of simultaneous detection of antibodies of class g to antigens of agents of torch-infections using immunochip - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: purpose of present invention is to provide a method of simultaneous detection of immunoglobulins of class G to specific antigens of agents of TORCH-complex infectious. The solid phase for immunosorbent is used as polystyrene microplate of low luminescent grade of plastic. The discrete microregions are applied to the bottom of a microplate well in the form of at least two microzones. The first immunospecific component is used as the mixtures of recombinant antigens. The test sample for analysis may be used as blood serum or whole blood dry spots. The second immunospecific component is used as monoclonal murine antibodies.

EFFECT: increase in accuracy, sensitivity and specificity of detection of IgG-antibodies as markers of TORCH-infections.

8 cl, 5 dwg, 7 tbl, 6 ex

Description

The invention relates to immunological methods of analysis and can be used for mass screening of infections of the TORCH group in newborns, pregnant women and women planning a pregnancy.

State of the art

Viral and bacterial infections play an important role in the pathology of pregnant women, the fetus and newborns. The relevance of the problem of infections in pre- and perinatal diagnosis in recent years has increased significantly due to adverse socio-economic changes in society, which are manifested in an increased risk of infection of women during pregnancy. Of the many intrauterine infections, a group of diseases was identified that caused the fetus to have persistent structural defects in many organs and systems, especially the central nervous system. To denote this group, the abbreviation TORCH-complex is used, combining the following intrauterine infections: toxoplasmosis, rubella, cytomegaly, as well as infections caused by the herpes simplex virus. Some researchers significantly expand this list, in particular, sexually transmitted diseases (syphilis, chlamydia, myco- and ureaplasmosis, hepatitis B and C, etc.).

The need for the diagnosis of TORCH infections in pregnant women is formulated in the order of the Ministry of Health of the Russian Federation No. 50 dated 02.10.2003 “On improving obstetric and gynecological care in outpatient facilities”. Despite this, a number of issues that are relevant for laboratory diagnosis of TORCH infections in pregnant women and newborns remain unresolved: the lack of diagnostic standards, the difficulty of interpreting the results.

Laboratory diagnosis is carried out by directly identifying the causative agents of TORCH infections in the biomaterial, or using serological methods designed to determine antibodies of class A, G and M to the antigens of pathogens of the TORCH complex. The most diagnostic value is the detection of class M immunoglobulins as an indicator of the activity of the process, which may indicate an acute disease or reinfection. However, in many cases, including immune deficiency during reinfection, the infectious process in newborns, etc., specific IgM antibodies are rarely detected. In this case, the determination of class G antibodies is crucial in the diagnostic plan. Determining the titer and avidity of IgG antibodies is advisable not only for the purpose of diagnosis, but also for predicting the course of the disease, evaluating the effectiveness of therapy and monitoring.

Due to the fact that the diagnostic methods used today have a number of drawbacks and limitations in their application, their complex use is necessary, which is a lengthy and expensive procedure.

It is also known that the cause of severe forms of lesions in children, as a rule, is not mono-infections, but associated viral and bacterial viral infections, the so-called mixed infections.

The main method of serological diagnosis is enzyme immunoassay. Test systems in the format of linear immunoblotting (IB) of various commercial companies (for example, EUROIMMUN and MIKROGEN) are used as a test to confirm the results of the study and to evaluate the reactivity of a particular class of antibodies to individual antigens of the pathogen. However, the IB method does not provide for objective instrumental recording of research results, it is used only in individual cases as a confirmatory test and, due to the high cost of analysis, cannot be used for mass examinations.

The development of modern complexes of laboratory tools for diagnosing infections of the TORCH group involves the use of methods and hardware solutions that, with minimal time and reagents, save and increase the sensitivity and specificity of the study. This is achieved by complex use, along with classical methods of analysis (ELISA - enzyme-linked immunosorbent assay, IB - immunoblotting, etc.) screening systems that allow you to determine in the test material antibodies to several pathogens simultaneously. In this case, the detection method must be adapted for mass surveys.

To date, kits of reagents for conducting an enzyme immunoassay are known to determine antibodies of one class or another (RF patent No. 134329, RF patent No. 134328) against toxoplasma, cytomegalovirus, rubella virus and herpes simplex virus types 1 and 2 in one analysis. The kits use a mixture of antigens immobilized on a solid support, simultaneously detecting antibodies to all 4 infections in one well.

The disadvantage of these kits is the impossibility of differentially identifying markers of individual pathogens.

A known diagnostic kit for multi-analytic Dot immunoassay, including a flat dense substrate with antigens deposited on its surface, is additionally blocked by non-specific natural or synthetic polymers; solutions for washing, diluting the sample and conjugate, conjugate and development system. In this case, the dense substrate is made of non-porous plastic, on the surface of which the antigens of pathogens of TORCH infections (Toxoplasma gondii, Chlamydia trachomatis, Rubella virus, Mycoplasma hominis, Ureaplasma urealyticum, Treponema pallidum, Cytomegalovirus, Herpes simplex virus are applied) antibodies to human immunoglobulins G or staphylococcal proteins A or G associated with catalytically active salts of gold or silver (RF patent 2298795, published May 10, 2007).

The disadvantages of this kit are:

1. The excess determination of the number of pathogens of eight infections of the TORCH group (according to our observations, the most common combination in the clinic is toxoplasmosis, rubella, cytomegalovirus, herpes of the first and second type);

2. The inability to use the test to determine antibodies to the herpes simplex virus of the second type;

3. The decrease in the accuracy of the analysis due to the fact that the result of the analysis is recorded only one point for each infection.

A known method for the simultaneous detection of class G and M antibodies to pathogens of the TORCH complex infections (Chekanova T.A., Markelov M.L., Pudova E.A., Kirdyashkina N.P., Sudyina A.E., Sazhin A.I. ., Shipulin GA "Immunochips - a new format of test systems for serological diagnosis of infectious diseases", "Clinical Laboratory Diagnostics", 2011, No. 10, p.3). This method allows you to simultaneously determine the content of class G and M antibodies to 23 recombinant antigen antigens of 8 pathogens of TORCH infections (rubella virus, Epstein-Barr virus, cytomegalovirus, parvovirus B19, herpes simplex virus type 1 and 2, T. Gondii and Chlamydia trachomatis) in serum blood. All 23 recombinant antigens were sorbed using nanoplotters for piezoelectric microprinting in duplicate on an activated glass slide (glass slide). The resulting complex "antigen-antibody" is detected by the interaction of the conjugate of multivariate antibodies (anti-IgG and anti-IgM) with cyanine fluorophores. Signal detection is carried out using a multi-channel fluorescence scanner.

The disadvantage of this method is the difficulty in interpreting the results of the study due to too many antigens to which antibodies are determined. In addition, in this analysis, only human blood serum can be used as a test sample, which limits the possibility of using the method.

A known method for the separate detection of IgM and IgG antibodies to pathogens of TORCH infections (Yi Liu, Fengling Yu, Haiyan Huang, Jinxiang Han, "Development of Recombinant Antigen Array for Simultaneous Detection of Viral Antibodies", "PLoSONE", vol. 8, 2013). The method is intended to detect antibodies of class G or M in samples of blood serum or cerebrospinal fluid (CSF). Recombinant and peptide antigens of cytomegalovirus (pp150), herpes simplex virus type 1 (gG-1) and type 2 (gG-2) and rubella (a mixture of E1, E2 and core antigens), as well as human immunoglobulins G or M, are applied to activated glass slides in five concentrations. The result of the analysis is defined as the emission of a fluorescent label associated with antispecies antibodies that bind to the complex formed between the immunosorbent antigens and the antibodies in the test sample. Fluorescence intensity is measured on a fluorimeter.

The disadvantage of this method is the decrease in the sensitivity of the analysis due to the use of a single antigen to determine one infection, while antibody formation occurs to several immunodominant antigens and mainly depends on the stage of the infectious process. In addition, this method does not provide for the determination of toxoplasmosis markers, which significantly limits the use of the test.

The closest analogue to the present invention is a method for detecting class G or M antibodies to immunodominant antigens of pathogens of TORCH infections (T. Bacarese-Hamilton, L. Mezzasoma, A. Ardizzoni, F. Bistoni, A. Crisanti, "Serodiagnosis of infectious diseases with antigen microarrays ", J Appl Microbiol. 2004; 96 (1), pp. 7-10). The method allows to detect antibodies to five pathogens (rubella, toxoplasma, rubella, herpes type 1 and 2). The analysis is carried out on activated slides, on the surface of which five antigens are immobilized in six repetitions, a negative control (rabbit myosin) and human immunoglobulins M or G in four concentrations deposited in duplicates. To identify antibodies G or M, Alexa 546 and Alexa 594 fluorophores, respectively, labeled with antibodies are used as tags. The results are recorded using a fluorescence scanner.

The disadvantage of this method is the use as a solid substrate of glass slides with antigens deposited on their surface, allowing sequential detection of antibodies: a study in the "one test - one analysis" mode, which does not meet the requirements of screening examinations, and also has lower efficiency and longer duration.

The purpose and essence of the invention

The aim of the present invention is to provide a method for the simultaneous detection of class G immunoglobulins to individual antigens of pathogens of the TORCH complex.

The technical result achieved by using the invention is to increase the accuracy, sensitivity and specificity of detection of IgG antibodies as markers of TORCH infections when they are simultaneously detected in comparison with known solutions.

The technical result is achieved by the fact that a polystyrene microplate from a low-luminescent grade of plastic is used as the solid phase for the immunosorbent.

The technical result is achieved by the fact that discrete microregions are deposited on the bottom of the microplate well in the form of at least two microzones.

The technical result is achieved by the fact that as the first immunospecific component, mixtures of recombinant antigens are used.

The technical result is also achieved by the fact that monoclonal murine antibodies are used as the second immunospecific component.

The technical result is also achieved by the consistent use of biotylated antibodies and conjugate of streptavidin with Pt-coproporphyrin.

The technical result is also achieved by the fact that as a test sample for analysis, both blood serum and dry spots of whole blood can be used.

The technical result is also achieved by the fact that p30 [SAG1], p29 [GRA7], p35 [GRA8] are used as antigens to detect antibodies to Toxoplasmagondii, gB, Pp150 to detect antibodies to CMV, gG-1, gD-1 to detect antibodies to HSV-1, gG-2, gD-2 for the detection of antibodies kVPG-2, E1, E2 for the detection of antibodies to rubella virus.

The authors are not aware of technical solutions with the claimed combination of features, therefore, this invention meets the criterion of novelty.

Known technical solutions used to determine IgG antibodies are based on the formation of immune complexes between an antigen or antigens of four, five or eight infectious agents immobilized on activated slides, class G antibodies of a test sample (blood serum or CSF) and biospecific components, including conjugates antibodies with various fluorescent labels, followed by detection of label emission in one way or another.

The proposed technical solution through the use of a microplate format of the study, the use of several biospecific reagents within the same microzone, as well as the sequential introduction of a biotin-streptavidin complex and a highly phosphorescent label, a method for detecting a phosphorescent signal and assessing the level of the desired analytes together with other signs determines the possibility of increasing the accuracy and sensitivity analysis with minimal time.

Therefore, the present invention is effective in relation to the known technical solutions, is new and not obvious from the prior art.

Industrial applicability

The invention can be used in health care, in particular for screening pregnant women, newborns and children in order to prevent and reduce the risk of intrauterine and congenital malformations. Therefore, the present invention meets the criterion of industrial applicability.

Description of illustrations

Figure 1 shows an immunosorbent - a microplate well sensitized with antigens and antibodies applied as microregions. Figures 2, 3, and 4 show diagrams of the various stages of the analysis of the detection of class G antibodies to antigens of five TORCH group infection pathogens, where 1 are microregions for detecting IgG antibodies to T. gondii, 2 are microregions for detecting IgG antibodies to cytomegalovirus , 3 - microregions for detecting IgG antibodies to HSV type 1, 4 - microregions for detecting IgG antibodies to HSV type 2, 5 - microregions for detecting IgG antibodies to rubella virus, 6 - human IgG (10 mg / ml), 7 - human IgG (50 mg / ml), 8 - human IgG (100 mg / ml), 9 - 96-well well ikroplansheta, 10 - the bottom of the wells 11 - Investigated antibody IgG, 12 - antispecies antibodies labeled with biotin 13 - conjugate - Streptavidin - Pt-coproporphyrin. Figure 5 shows a graph of the dependence of the phosphorescence signal on the concentration of IgG antibody immunosorbent.

The invention is as follows.

In the blood serum samples, the presence / absence of IgG antibodies to the antigens of ToRCH pathogens is simultaneously detected. A high titer of antibodies indicates an acute, subacute stage of the infectious process or reinfection. A low titer of antibodies is observed with a latent course, the initial stage of the infectious process, or is an indicator of the disease.

The detection scheme for class G antibodies is shown in Figs. 2, 3, and 4.

Discrete microregions are formed at the bottom of each well of the microplate, which are mixtures of recombinant analogues of immunodominant antigens of TORCH group infection pathogens:

Microregion 1 - a mixture of antigens (p30 [SAG1], p29 [GRA7], p35 [GRA8]) to detect antibodies to Toxoplasmagondii;

Microregion 2 - a mixture of antigens (gB, Pp150) to detect antibodies to CMC;

Microregion 3 - a mixture of antigens (gG-1, gD-1) to detect antibodies to HSV-1;

Microregion 4 - a mixture of antigens (gG-2, gD-2) for the detection of antibodies kVPG-2;

Microregion 5 - a mixture of antigens (E1, E2) to detect antibodies to rubella virus;

Microregion 6 - human immunoglobulins of class G at a concentration of 10 mg / ml;

Microregion 7 - human immunoglobulins of class G at a concentration of 50 mg / ml;

Microregion 8 - human immunoglobulins of class G at a concentration of 100 mg / ml for internal reaction control and evaluation of the analysis components (internal study calibrators).

The components of each microregion containing a mixture of antigens for an individual pathogen and human IgG in three concentrations are immobilized in two replicates (duplicates), which improves the accuracy of the analysis by reducing variability and eliminating the possibility of antigen desorption. Thus, 16 microzones with a diameter of 0.5 mm are formed at the bottom of each well of a microplate (Fig. 1.).

Glycerin is added to the sensitization solution in order to reduce the drying rate of the solution droplets within the microregion, which in turn increases the uniformity and completeness of the adsorption of biospecific components.

When analyzing blood serum, the test sample is pre-diluted 1: 100 in a solution for diluting samples and introduced into a sensitized well in a volume of 100 μl. When analyzing dry blood stains, a paper disk with a diameter of 3.2 mm is placed in a well and 100 μl of a solution is added to dilute the test samples. The analysis is carried out in three stages: 1 and 2 stages - immunospecific, 3 - detecting. At the first stage, antibodies (if they are present in the test sample) specifically bind to antigens immobilized as discrete microregions 1-10 at the bottom of the well (Fig. 2). After incubation, unbound reaction components are removed by washing the microplate.

To carry out the second stage of analysis, a reaction solution of the second immunospecific components containing biotinylated monoclonal antibodies is added to the microtiter well, forming antigen-antibody-conjugate complexes on microregions 1-10 and IgG-antibody-anti-IgG antibody complexes on microregions 11 -16. Biotinylation of anti-species antibodies is used to enhance the signal of immune complexes formed in specific microzones (Fig. 3).

For the third stage of analysis, a detection solution containing a streptavidin conjugate with Pt-coproporphyrin III (RF patent No. 1707539) is introduced into the wells of the microplate, which forms phosphorescence complexes of biotin-streptavidin on specific microregions at the bottom of the hole (Fig. 4). Then, the microplate is washed and dried, after which phosphorescence emission is detected using a phosphorescence scanner, which sequentially scans with a focused laser beam of each microregion at the bottom of the microplate well.

Based on the results of the analysis of internal calibration samples, a curve is constructed that represents a linear dependence of the phosphorescence signal level on the concentration of IgG antibodies immobilized in the form of microzones 11-16 (Fig. 5.)

If there are antibodies to the antigens of the immunosorbent in the test sample, during the first exposure they form the antigen-antibody complex, which then binds to the conjugate, and the antigen-antibody-conjugate complex is detected by phosphorescence when scanned in a biochip analyzer; the phosphorescence intensity in this case corresponds to the concentration of antibodies in the test sample.

Example 1. Detection of antibodies of class G to cytomegalovirus.

Mixtures of recombinant analogs of toxoplasma antigens, rubella, cytomegalovirus, herpes simplex virus type 1 and 2 in the form of discrete regions are immobilized on the bottom surface of the wells of a polystyrene 96-well microplate (Thermo Labsystems, Finland). To detect IgG antibodies to each of these pathogens, 2 identical microzones with a diameter of 0.5 mm are formed. In the same way, the bottom of the microplate well is sensitized with human immunoglobulins G in three concentrations, 2 microzones per concentration. Thus, 16 microregions are formed at the bottom of each well of the microplate. To do this, using a robotic chip printing device (nanoplotter), microdrops of solutions of the corresponding immunospecific components are applied to specific zones of the bottom of the bottom: human IgG antibodies (at concentrations of 10, 50 and 100 mg / ml) and antigens (at a concentration of 0.05 mg / ml) in a sensitization solution (0.01 M phosphate-saline solution, pH 7.2). To reduce the drying rate of the droplets, 5% glycerol (Sigma) is added to the sorption solution. The microplate is incubated for 24 hours at 4 ° C, then washed and treated with a blocking solution (0.05 M Tris-HCl, pH 7.75, 1% BSA) for 1 hour at 37 ° C. After that, the blocking solution is removed and the microplate is dried for 2 hours at room temperature.

For blood serum analysis, 1: 100 is preliminarily diluted in phosphate-buffered saline (0.01 M, pH 7.4, 0.5% BSA, 0.05% sodium azide, 0.01% TWIN-20) and 100 μl is added to the immunosorbent wells. When analyzing a dry blood stain, paper disks with a diameter of 3.2 mm are placed in the wells and 100 μl of the same solution, universal for all studies of the content of IgG antibodies, is added. Control samples containing and not containing class G immunoglobulins to cytomegalovirus (or to any other pathogen) are added to the wells of a microplate without preliminary dilution in a volume of 100 μl. The test samples and control samples are checked for the presence / absence of class G antibodies to cytomegalovirus using commercial monotests (IFA-CMV-IgG, CJSC ECOlab, and CMV-IgG-ELISA, Medac).

The microplate is kept for 2 hours on a shaker at 25 ° C and 720 rpm, after which it is washed 3 times with wash buffer (phosphate-buffered saline, pH 6.4, 0.0023% sodium phosphoric acid 1-substituted 2 -aqueous, 0.03331% sodium 2-substituted 12-aqueous, 0.2125% sodium chloride, 0.0125% TWIN-20; 25-fold concentrate). Then, a solution of biotinylated antibodies (Biotin N-succinimidylester, Fluka, Switzerland) in a concentration of 750 ng / ml in reaction buffer (0.05 M Tris-HCl, pH 7.75, 0.87% sodium chloride, 0, 5% BSA, 0.05 sodium azide, 0.01% TWIN-20). The microplate is kept on the plate for 1 hour at 25 ° C on a shaker (720 rpm) and then washed 3 times with wash buffer. Then, 30 μl of a solution of streptavidin conjugate with Pt-coproporphyrin at a concentration of 333 ng / ml in reaction buffer (0.05 M Tris-HCl, pH 7.75, 0.87% sodium chloride, 0.5% BSA) are added to the wells. , 0.05 sodium azide, 0.01% TWIN-20). The microplate is incubated for 15 min on a shaker (720 rpm) at 25 ° C, after which the wells are washed 3 times with wash buffer and 3 times with distilled water. The tablet is dried for 1 h at room temperature and the analysis results are recorded using a phosphorescence biochip analyzer in the excitation mode at a length of 532 nm with a linear resolution of 15-20 μm and a signal attenuation time constant of 80 μs directly from the solid phase of the bottom of the microplate wells. Signal detection is carried out by step-by-step scanning of a site with a diameter of about 1 mm. Using a computer program for each marker under investigation, the average number of photopulses for the marker-specific microregions is automatically calculated.

The obtained signal intensities from microregions containing human IgG antibodies as biospecific components are used to construct a calibration curve (Fig. 5). As can be seen from the graph, the increase in the signal from these microregions with an increase in the concentration of IgG antibodies in the composition of the microzones is linear, which indicates the adequate operation of the components and high sensitivity of the analysis when it is possible to measure the marker at low concentrations. At the same time, variations in the signal intensity values from microregions containing antigens of other pathogens do not exceed the threshold level calculated based on the formula: Cutoff = IFsr + 3d, where

Cutoff - signal intensity of microregions containing antigens;

IFRS - the average value of phosphorescence of a control sample that does not contain IgG antibodies, measured in pulses;

d is the standard deviation.

This shows the high specificity of the method, since no cross interactions of the analyte under study with other microregions are observed (Table 1). These results correlate with the results of the study using commercial enzyme-linked immunosorbent monotests (table 2).

Example 2. Detection of class G antibodies to a mixture of antigens (gD-1 and gG-1) of herpes simplex virus type 1.

In the wells of the microplate sensitized as described in Example 1, pre-prepared test samples were added in the form of blood serum or whole blood stains on paper disks, as well as control samples containing and not containing IgG antibodies to HSV type 1, in a volume of 100 μl . The test samples and control samples are checked for the presence / absence of class G antibodies to herpes simplex virus type 1 using commercial monotests (IFA-HSV-1-IgG, ZAO ECOlab, and HSV-1-IgG-ELISA, Medac).

The progress and detection of the analysis results is carried out as described in example 1. The results of the study, shown in table 3, demonstrate the receipt of specific signals from samples containing IgG antibodies to HSV type 1, and the level of their phosphorescence is significantly different from negative samples and signals from other microregions.

Example 3. Detection of class G antibodies to a mixture of antigens (gD-2 and gG-2) of herpes simplex virus type 2.

In the wells of the microplate sensitized as described in Example 1, pre-prepared test samples were added in the form of blood serum or whole blood stains on paper discs, as well as control samples containing and not containing IgG antibodies to HSV type 2, in a volume of 100 μl . Test samples and control samples are checked for the presence / absence of class G antibodies to herpes simplex virus type 1 using commercial monotests (IFA-VGGG-2-IgG, ZAO ECOlab, and HSV-2-IgG-ELISA, Medac).

The analysis stages, components and detection of the results are carried out as described in example 1.

From the results of the study, shown in table 4, it is seen that when analyzing samples containing IgG antibodies to HSV type 2, the method allows you to record the signal level from the corresponding microregions, which differs from the results of the analysis of negative samples and exceeds the threshold level, which demonstrates the high sensitivity of the method . At the same time, there is a lack of significant signals on microregions specific to other markers, which confirms the specificity of the study.

Example 4. Detection of antibodies of class G to a mixture of antigens (p29, p30, p35) Toxoplasma gondii.

In the wells of the microplate sensitized as described in Example 1, pre-prepared test samples in the form of blood serum or whole blood stains on paper discs, as well as control samples containing and not containing IgG antibodies to T. gondii, were added in a volume of 100 μl . The test samples and control samples are checked for the presence / absence of class G antibodies to toxoplasma using commercial monotests (IFA-Toxo-IgG, CJSC EKOlab, and Toxoplasma-IgG-ELISA, Medac).

The progress and detection of the analysis results is carried out as described in example 1. The results of the study, shown in table 5, demonstrate the receipt of high signals from samples containing IgG antibodies to toxoplasma, while the signal intensity from microregions corresponding to other pathogens does not exceed threshold level, which confirms the absence of cross-reactions and the specificity of the analysis.

Example 5. Detection of antibodies of class G to a mixture of rubella antigens (E1 and E2).

Pre-prepared test samples in the form of blood serum or whole blood stains on paper discs, as well as control samples containing and not containing rubella virus IgG antibodies, were added to the wells of the microplate sensitized as described in Example 1. The test samples and control samples are checked for the presence / absence of antibodies of class G to toxoplasma using commercial monotests (IFA-Rubella-IgG, CJSC ECOlab).

The analysis stages, components and detection of the results are carried out as described in example 1.

From the results of the study, shown in table 6, it is seen that when analyzing samples containing IgG antibodies to rubella virus, the method allows you to record the signal level from the corresponding microregions, which differs from the results of the analysis of negative samples and exceeds the threshold level, which demonstrates the high sensitivity of the method. At the same time, there is a lack of significant signals on microregions specific to other markers, which confirms the specificity of the study.

As can be seen from examples 1-5, the method allows to obtain high sensitivity and specificity while detecting IgG antibodies to each of the five causative agents of TORCH infections.

Example 6. Simultaneous determination of antibodies of class G to five pathogens of infections of the TORCH complex.

The method is carried out as described in examples 1-5. As the studied samples, control sera and clinical samples pre-certified by ELISA-ELISA-CMV-IgG, IFA-HSV-1-IgG, IFA-HSV-2-IgG, IFA-Toxo-IgG "," IFA-Rubella-IgG "(CJSC" EKOlab ").

The results of the study of control samples and clinical samples are shown in table 7. The correlation coefficient of the estimates of the samples during the study in ELISA and the presented method was 98%. The study of samples No. 1-10 (serum of healthy newborns) gave negative results for IgG antibodies to the causative agents of five TORCH infections. In samples No. 11-30, the presence of class G antibodies to antigens of one or another pathogen, or to antigens of several pathogens in various titers was revealed (Table 7).

Thus, the study carried out by the proposed method provided adequate results for the detection of IgG antibodies. The method allows, based on the study of one sample, to characterize it by the spectrum of class G antibodies simultaneously for five TORCH infections with a high degree of specificity and accuracy.

The application of the proposed method can significantly increase the effectiveness of prenatal screening.

Table 1 The results of the analysis of samples containing and not containing IgG antibodies to cytomegalovirus, studied by ELISA and immunochip Counter number samples and specimens IFA positivity coefficient The average intensity of phosphorescence of microregions, imp. CMV HSV type 1 HSV type 2 T. gondii Rubella K + 3.49 2256 82 21 67 60 TO- 0.12 46 35 47 71 13 one 0.38 71 69 34 25 27 2 0.65 88 22 eighteen 66 45 3 0.81 102 43 22 70 16 four 1.24 569 61 17 46 10 5 1.43 593 57 53 39 34 6 1,52 923 72 24 21 62 7 1.35 1356 36 19 26 fifteen 8 2.61 1702 29th 9 54 58 9 3.2 1940 46 42 73 26 10 3,58 2174 72 12 52 21

table 2 The results of the analysis of samples for IgG antibodies to cytomegalovirus, investigated by ELISA and immunochip Evaluation of samples in the immunochip The number of samples evaluated in ELISA as negative doubtful positive negative 7 2 3 doubtful 0 0 3 positive 0 one 157

Table 3 The results of the analysis of samples containing and not containing IgG antibodies to herpes simplex virus type 1, studied by ELISA and immunochip Counter number samples and specimens IFA positivity coefficient The average signal of the intensity of phosphorescence of microregions, imp. CMV HSV type 1 HSV type 2 T. gondii Rubella K + 3.56 42 1432 61 fourteen 54 TO- 0.09 8 35 29th eleven 12 one 0.11 16 26 44 25 24 2 0.34 23 48 51 16 45 3 0.42 31 55 12 10 26 four 1.23 22 245 17 22 19 5 1,54 fourteen 573 63 19 46 6 1.92 thirty 724 62 21 31 7 2.08 17 916 59 fourteen 16 8 2.71 24 1206 25 8 28 9 3.15 eighteen 1297 32 23 32 10 3.29 26 1488 53 9 41

Table 4 The results of the analysis of samples containing and not containing IgG antibodies to herpes simplex virus type 2, studied by ELISA and immunochip. Counter number samples and specimens IFA positivity coefficient The average signal of the intensity of phosphorescence of microregions, imp. CMV HSV type 1 HSV type 2 T. gondii Rubella K + 2.84 54 71 1064 32 fourteen TO- 0.1 12 21 9 29th eleven one 0.13 36 27 fourteen 21 29th 2 0.28 23 eighteen 34 36 42 3 0.62 40 25 32 39 34 four 1,2 13 42 174 46 19 5 1.34 24 38 453 17 55 6 1.55 36 72 627 eleven 27 7 1,64 17 67 771 53 19 8 1,69 27 69 862 34 63 9 1.77 19 17 935 23 32 10 1.92 27 58 1149 39 34

Table 5 The results of the analysis of samples containing and not containing IgG antibodies to Toxoplasmagondii, studied by ELISA and immunochip Counter number samples and specimens IFA positivity coefficient The average signal of the intensity of phosphorescence of microregions, imp. CMV HSV 1
of type HSV type 2 T. gondii Rubella K + 2.98 24 13 17 1750 24 TO- 0.09 10 24 8 39 32 one 0.12 eighteen 22 17 21 24 2 0.26 23 28 twenty 46 fourteen 3 0.43 46 fifteen 31 49 31 four 1.3 52 48 41 321 19 5 1.45 28 63 32 625 25 6 1,62 31 12 27 430 19 7 1.7 9 27 fourteen 792 13 8 1.93 47 49 22 1348 43 9 2.04 eighteen 34 35 1632 36 10 2.27 17 52 eleven 1824 22

Table 6 The results of the analysis of samples containing and not containing IgG antibodies to rubella virus, studied by ELISA and immunochip. Counter number samples and specimens IFA positivity coefficient The average signal of the intensity of phosphorescence of microregions, imp. CMV HSV type 1 HSV type 2 T. gondii Rubella K + 4.14 8 62 21 37 2628 TO- 0.13 13 56 13 39 62 one 0.14 eleven 27 9 eighteen 64 2 0.29 23 8 27 44 77 3 0.7 37 fifteen thirty 29th 91 four 1.32 12 58 fifteen 21 265 5 1,5 28 23 eighteen 25 512 6 1,67 39 45 28 43 903 7 1.89 43 34 42 12 877 8 2.16 47 29th 29th 13 1435 9 2.41 fifteen fifty 35 32 2100 10 2.83 24 52 31 24 2552

Table 7 The results of the simultaneous detection of class G antibodies to CMV, Toxoplasma, HSV types 1 and 2 and rubella virus. Sample No. The positive coefficient of the sample in ELISA The average signal of the intensity of phosphorescence of microregions, imp. CMV HSV type 1 HSV type 2 T. gondii Rubella CMV HSV type 1 HSV type 2 T. gondii Rubella one 0.11 0.13 0.09 OD 0.11 32 10 eleven 19 13 2 0.46 0.29 0.11 0.1 0.2 36 45 23 33 22 3 0.09 0.17 0.12 0.19 0.21 31 23 9 21 44 four 0.21 0.34 0.16 0.23 0.26 54 fourteen twenty eighteen 28 5 0.57 0.35 0.22 0.28 0.34 42 36 fourteen 10 35 6 0.45 0.56 0.28 0.33 0.43 68 8 37 29th 41 7 0.66 0.61 0.52 0.36 0.49 twenty 36 12 45 52 8 0.72 0.68 0.46 0.4 0.65 65 48 38 48 57 9 0.73 0.77 0.6 0.54 0.74 73 52 26 63 73 10 0.8 0.79 0.62 0.67 0.75 77 57 45 71 64 eleven 0.67 1.34 0.23 0.54 1.17 68 94 44 58 612 12 0.5 1,66 0.18 0.62 1.25 45 1116 17 60 289 13 0.32 1.9 0.3 0.78 1.36 28 1856 64 69 741 fourteen 0.64 2.54 0.31 0.83 1.39 219 2374 37 43 168 fifteen 0.78 2.17 0.38 0.84 1.43 103 1986 fifty 42 996 16 0.82 2.78 0.42 1,2 1,57 83 1943 39 457 874 17 1.87 1.4 0.44 1.34 1.65 968 922 61 79 848 eighteen 1.25 2.07 0.51 1.45 1.74 875 1608 48 846 1007 19 2,32 3.42 0.83 1.51 1.88 1289 2671 56 1175 1153 twenty 1,64 3,5 0.16 1,6 1.86 1219 2833 23 1452 1266 21 2.78 1,64 1.31 1,63 1.92 2016 1439 557 774 949 22 3,1 1.78 1.65 1.76 1.97 2443 1852 916 1189 975 23 1,56 2,32 1.28 1.82 2D 1035 1989 855 1395 1390 24 1.98 2.84 1.47 1.82 2,34 1673 2304 1068 1618 1617 25 1.3 1.96 1.73 1.89 2.46 426 2478 1184 1709 1594 26 2.14 3.03 1.76 1.94 2.49 2250 2604 1395 2226 1707 27 2.84 2.97 1.89 1.97 2,53 2137 2035 1216 1930 2004 28 3.27 1.43 2.05 2.01 2.7 2588 1441 1836 2004 1986 29th 3.46 2.19 2.2 2,56 2.78 2542 1947 1738 2278 2058 thirty 3.65 1.77 2.67 2.88 3.04 2736 2113 1992 2392 2231

Claims (8)

1. A method for the simultaneous detection of class G antibodies to antigens of causative agents of TORCH infections using an immunochip, including the immobilization of immunospecific components in the form of mixtures of recombinant rubella antigens, toxoplasma, cytomegalovirus, herpes simplex virus type 1 and 2, and internal control represented by human IgG antibodies, introducing test samples into the well, conducting an immune reaction with biotinylated antibodies, adding streptavidin conjugate with a phosphorescent label followed by detection of phosphorescence emission of the label associated with the formed complexes, characterized in that in the well of the microplate used as a solid phase, immunospecific components are immobilized in the form of internal calibrators and a mixture of antigens for each of the five pathogens at the bottom of the well, then into the well add a reaction solution of the second immunospecific components containing biotinylated monoclonal antiviral antibodies to obtain immune complexes, echennyh discrete microregions biotin, streptavidin is added with Pt-coproporphyrin conjugate solution to form a phosphorescent biotin-streptavidin complexes microregions at the bottom of a microplate well, thus detecting the emission of phosphorescence label sequential scanning each discrete microregions focused laser beam. 2. The method according to claim 1, characterized in that a polystyrene microplate from a low-luminescent grade of plastic is used as the solid phase of the immunosorbent. 3. The method according to claim 1, characterized in that the discrete microregions are applied to the bottom of the wells of the microplate in the form of at least two microzones. 4. The method according to claim 1, characterized in that the discrete microregions are applied to the bottom of the microtiter well in the form of 16 microzones. 5. The method according to claim 1, characterized in that mixtures of recombinant antigens are used as the first immunospecific component. 6. The method according to claim 1, characterized in that as the test sample for analysis, both blood serum and dry spots of whole blood can be used. 7. The method according to claim 1, characterized in that monoclonal murine antibodies are used as the second immunospecific component. 8. The method according to claim 1, characterized in that p30 [SAG1], p29 [GRA7], p35 [GRA8] are used as antigens to detect antibodies to Toxoplasmagondii, gB, PP150 to detect antibodies to CMV, gG-1, gD -1 to detect antibodies to HSV-1, gG-2, gD-2 to detect antibodies to HSV-2, E1, E2 to detect antibodies to rubella virus.

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