The main results of the Agricultural Biotechnology Research Institute of S.Seifullin KazAU in the field of the diagnosis of invasive disease



Projects

On the scientific and technical program: "Development and use of genetically engineered and cellular technologies in medicine, agriculture, environmental protection, food and processing industry for 2009-2011":

- Development of methods for enzyme immunoassay diagnostics of opisthorchiasis.

- Development of an enzyme immunoassay system for serological rapid diagnosis of echinococcosis in farm animals.

 

Using the Lateral Flow Assay (chromatographic immunoassay) in the rapid diagnosis of infectious and parasitic diseases

Practitioners and methods for diagnosing infectious diseases, based on immunochromatography, deserve attention. So, N.A.Bizova et al. (2010) suggested immunochromatographic detection of Mycobacterium tuberculosis cells. Competitive and "sandwich" variants of immunochromatographic analysis are developed in which contact of the sample and test strips with applied immunoreagents directly initiate fluid movement through the membrane components of the test strip, immunochemical interaction and formation of visually detectable colored zones. It is shown that both competitive and "sandwich" variants of the method make it possible to determine the cells of the causative agent of tuberculosis in a concentration of up to 10 thousand cells / ml (50-100 cells in the sample). The advantage of "sandwich" analysis is the possibility of a qualitative visual diagnosis, when the coloring of the analytic zone irrespective of its intensity indicates a positive result of the analysis.

Zherdev et al. (2010) developed an immunochromatographic method for the detection of antibodies to the causative agent of tuberculosis. For the detection of antibodies, two antigenic reagents are used: the Mycobacterium tuberculosis antigen immobilized in the assay zone of the test strip and an antigen conjugated to colloidal gold particles. Due to the presence of at least two antigen-binding sites in antibodies at the contact of the test strip with the sample in the analytical zone, the formation of immune complexes consisting of immobilized on the membrane antigen molecules contained in the sample of antibodies to the antigen of Mycobacterium tuberculosis and conjugate of the agent antigen with colloidal gold particles. The complexes are determined visually or using an optical detector.

 

Prospects of application of anti-idiotype antibodies in the immunodiagnosis of infectious diseases

One promising approach to increasing the specificity of ELISA is the use of anti-idiotypic antibodies (AIAT) in this assay, i.e. "Internal images" of the original antigen. At the same time, once obtained monoclonal antibodies to a specific epitope of a particular pathogen can be used to obtain monoclonal AIAAT, which will subsequently replace the original antigen and thus eliminate the need for personnel to work with pathogenic microorganisms. AIAT are able to simulate any, including weakly immunogenic and weakly antigenic determinants of haptens, polysaccharides, lipids, often released in degraded form and, therefore, devoid of part of the epitopes inherent in the native molecule, or retaining high toxicity due to extraction features. AIAAT, acting as a protein "substitute" for a specific bacterial antigen, allows to remove such obstacles and effectively present the atoxic determinants to the cells of the immune system. That is why AIAAT is now successfully used by many researchers as a "surrogate" of the antigen or its individual determinants for the induction of a protective immune response against bacterial LPS, toxins, a number of intracellular parasites - pathogens of malaria, leishmaniasis, trypanosomiasis, bacterial and viral infections, etc . An indisputable advantage of this approach is the absence of the need for preliminary isolation of the substance being studied. Absolute specificity of MCA used to obtain AIAT, predetermines their homology to the original antigen.

Employees of KazAU named after S.Seifullin received AIAT to the idiotype of At2-beta monoclonal antibodies directed to poly-B antigen of brucella (S.Ospanova et al, 2009). One of the criteria for the characteristics of AIAT as Am2-beta is reported by several authors, is their ability to interact with xenogene antisera, specific for the original antigen, since only this idiotype, which carries the "internal image" of the antigen, can bind specific antibodies. Therefore, the interaction of monoclonal AIAATs with rabbit polyclonal

monospecific antiserum and polyclonal positive serum of cattle in ELISA. The monoclonal antibodies produced by the hybridoma strains exhibited activity against these xenogeneic sera in titres from

1: 1600-1: 3200. One of the significant properties of Am2 beta is their ability to induce the synthesis of homologous third-order antibodies (anti-AIAAT) directed against the original antigen. For this purpose, series of immunizations of linear mice with AIAAT preparations were carried out. Mouse sera were tested in indirect ELISA for the presence of third-order antibodies capable of interacting with the original poly-B Brucella antigen. As the results showed, AIAT obtained when administered to animals can stimulate the synthesis of third-order antibodies (Am3) directed against the original antigen. Thus, the researchers proved the possibility of using AIAT as an antigen in a study

Serum of animals on brucellosis in ELISA.

The phenomenon of anti-idiotypy in the diagnosis of blood-parasitic diseases (pyroplasmoses, anaplasmoses, nuttaliases, trypanosomiasis) was also studied by G.S. Shabdarbayeva et al. (2009). They found that AIAT to the antigens of various blood-parasites, obtained as a result of double immunization of laboratory animals - rabbits, are sufficiently informative, specific and can be used as diagnostics in serological tests such as RNGA and ELISA.

Immunoenzyme analysis using F (ab) 2 fragments of anti-idiotype antibodies in place of native antigens was proposed by B.B. Gnedenko et al. (2006). They carried out a determination of the serum content of autoantibodies to three groups of proteins in norm and in patients with schizophrenia using standard methods and test systems with immobilized F (ab) 2 fragments of AIA. The levels of autoantibodies to all proteins in patients with schizophrenia were higher than in the control group.

E.S. Zhilin et al. (2011) tested polyclonal anti-idiotypic immunoglobulins and their conjugates with horseradish peroxidase in ELISA for the detection of anti-rabies antibodies in animal sera. As a result of the tests, positive results were recorded with specific sera, regardless of the type of anti-rabies antibody producers with negative results with normal and heterologous sera. The sensitivity of the test system was 2-4 times higher than the indirect and competitive variant of ELISA.

 


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