[THE APPLICATION OF DOT-TECHNIQUE FOR DETECTING ANTIGENS OF ADENOVIRUS IN CLINICAL SAMPLES].

The article substantiates possibility of application of point enzyme-linked immunosorbent assay (dot-technique) for detecting viral antigens in samples from patients. To diagnose adenovirus infection conjugate of virus-specific monoclonal antibodies and peroxidase of horse-radish were used The chromatographic rectification of conjugate from free peroxidase permits diminishing background coloring of nitrocellulose membrane and therefore to increase sensitivity. The application of direct conjugates on the basis of virus-specific monoclonal antibodies increases specifcity of dot-technique and significantly shortens time period of analysis. As in case of application of direct conjugates on the basis of polyclonal serum, samples from patients require preliminary processing with detergent for preventing non-specific reactions. The dot-technique demonstrates good coincidence with data of polymerase chain reaction and after clinical trials it can be used in diagnostic of human viral infections.

[The development of immune enzyme test-systems for detecting potentially pandemic influenza viruses subtype A(H2), A(H5), A(H7) and A(H9).]

PURPOSE OF STUDY: To develop diagnostic test-systems constructed according principle of "sandwich" enzyme-linked immunosorbent assay to detect antigens of influenza viruses with pandemic potential: A(H2), A(H5), A(H7), A(H9).The panels of subtype-specific monoclonal antibodies interacting with molecule of hemagglutinin of potentially pandemic viruses of influenza are obtained and characterized. The viruses of every sub-type were supplied with selected pairs monoclonal antibodies/monoclonal antibodies' conjugate with peroxidase of horse-radish, interacting most effectively with virus-immunogen and lacking non-specific activity related to influenza viruses of geterologic types. Considering established threshold values, the sensitivity of test-systems varied depending on sub-type and strain of viruses within limits of 4 - 30 ng/ml at evaluation of viral purified concentrates and 0.3 - 8 hemagglutinin units at analysis of virus-containing allantoic fluid. The specificity of immune-enzyme test-system manifested in conditions of absence of non-specific interactions with both seasonal and potentially pandemic geterologic viruses. The technique can be applied for fast identification of viruses of influenza non-typeable in conditions of routine functioning of practical laboratories.

[THE COMPARATIVE ANALYSIS OF EFFECTIVENESS OF QUICK TESTS IN DIAGNOSTIC OF INFLUENZA AND RESPIRATORY SYNCYTIAL VIRAL INFECTION IN CHILDREN].

The analysis was implemented concerning diagnostic parameters of commercial quick tests (immune chromatographic tests BinaxNOW Influenza A&B and BinaxNow RSV Alere, Scarborough Inc., USA) under detection of antigens of influenza virus A and respiratory syncytial virus in clinical materials. The polymerase chain reaction in real-time and isolation ofviruses in cell cultures. The analysis of naso-pharyngeal smears from 116 children demonstrated that sensitivity and specifcity of detection of influenza virus A using device mariPOC in comparison with polymerase chain reaction made up to 93.8% and 99.0% correspondingly at total concordance of results of both techniques as 98.3%. At diagnosing of respiratory syncytial virus using device mariPOC parameters made up to 77.3%, 98.9% and 862% as compared with polymerase chain reaction. The sensitivity, specificity and total concordance of results of immune chromatographic tests BinaxNOW in comparison ofpolymerase chain reaction made up to 86.7%, 100% and 96.2% correspondingly at detection of influenza virus A and 80.9%, 97.4% and 91.6% correspondingly at detection of respiratory syncytial virus. In comparison with isolation technique in cell cultures sensitivity of system mariPOC and immune chromatographic tests proved to be in 1.3-1.4 times higher at detection of influenza virus A and in 1.7-2 times higher in case of isolation of respiratory syncytial virus. There is no statistically significant differences between diagnostic parameters received for mariPOC and immune chromatographic tests at diagnosing influenza virus A and respiratory syncytial viral infection.

[Development of competitive enzyme immunoassay for the detection of influenza A(H5) virus antibodies].

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.