New approachesto improve thediagnostic value of theELISA
One promising approach to increasing the specificity of ELISA is the use of anti-idiotypic antibodies (AIAB) 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 produce monoclonal AIAB, which will subsequently replace the original antigen and thus eliminate the need for the work of personnel with pathogenic microorganisms. AIAB 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. AIAB, 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 AIAB 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.
The main results of the Agricultural Biotechnology Research Institute of S.Seifullin KazAU in the field of the effective diagnosis of infectious diseases
At present, methods based on the use of ELISA are widely used in the improvement of serological diagnosis of infectious diseases. This is understandable: ELISA is a highly sensitive test that allows for a short time to examine a large number of serum samples. However, high sensitivity of the test, in the first place, requires the use of an antigen specific for the pathogen in it. Otherwise, the high sensitivity of ELISA is at the expense of its diagnostic value, showing false positive results due to the presence of the pathogen and some microorganisms of common antigenic determinants.
Brucellosis of animals remains one of the main problems of veterinary medicine in Kazakhstan. In ELISA kits used recently for serological diagnosis of infection, lipopolysaccharides (LPS) of the brucella molecule act as antigen. It is known that LSC Brucella is almost identical to the analogous antigen obtained from Yersinia enterocolitica 0: 9, and have homologous epitopes with many gram-negative microorganisms to which antibodies are present in the body of animals. In addition, the immune system of the vaccinated organism is more readily available LPS, located superficially, and therefore antibodies are mainly produced by the determinants of the polysaccharide molecule. In this connection, there is no doubt that the use of this antigen in ELISA leads to the unjustified slaughter of a certain number of healthy animals that have developed antibodies as a result of immunizing subinfection or vaccination.
In the Scientific Research Institute of Biotechnology KazATU.S.Seifullin (AK Bulashev et al.)Carried out work on determining the diagnostic value of outer membrane proteins (BRM) Brucella abortus 19. The choice of this antigen is based on the following provisions. Firstly, gram-negative microorganisms, although they have great similarity in LPS, differ significantly in BVM antigens. Secondly, in the immunized organism, the vaccine strains are delayed, as a rule, for a short time (1-3 months). Therefore, for the immune system of vaccinated animals, LPS located on the surface of the cell is more accessible than the protein antigens "disguised" by a complex complex of the surface structure. In this regard, the vaccinated organism has time to develop antibodies mainly on LPS. In the organism of the infected animal, there is a long persistence of brucellas, accompanied by the reaction of the immune system to the causative agent of the disease, which tries to localize in the organs most favored by it. This leads to the accumulation in the tissues of a large number of decay products of brucellae, among which there are also BVM, which were previously not available for the immune system.
The ELISA test system for the detection of anti-brucellosis antibodies in bovine serum, developed by the employees of KazSU named after S.Seifullin, is based on the use of BVM brucella. As the results of the studies showed, the test was more specific than the well-known serological reactions. The ELISA was performed in two versions. In indirect ELISA, BVMs were immobilized to the solid phase nonspecifically, and in the "sandwich" variant - by means of monoclonal antibodies (MCA) having a specificity for the protein epitope with a molecular weight of 50 kD. Comparing the diagnostic significance of the two methods of staging ELISA, researchers prefer a "sandwich" option. First, in the latter case, the protein antigen, in which the epitope of monoclonal antibodies is present, is fixed to the solid phase specifically. At the same time, other protein components of BRUC brucella are removed during the washing of the well of the plate. Thus, during the formulation of the "sandwich" ELISA, the first antibodies select antigens specific for Brucella from the Brucella BMB. This certainly increases the specificity of this test. The developer has scientific and technical documentation and prototypes of immunoenzyme diagnosticum (registered in the State Register of Veterinary Preparations for No. RK-VP-2-0126-04 dated January 27, 2004). Copyrights are protected by patent No. 14230 of the Republic of Kazakhstan "Method for the determination of antibodies against the causative agent of brucellosis" (Bul. No. 4, published on April 15, 2008). High specificity of protein antigens Leptospira, serogroup Hebdomadis was noted by us in serological ELISA diagnostics of cattle for leptospirosis in comparison with other immunological methods. (AK Bulashev, 2005). Diagnostics significantly exceeded the sensitivity and specificity of the reaction of microagglutination and lysis (RMAL) and 6% more revealed seropositive animals than its foreign counterpart "IFA-leptospirosis-BPH" (MA Kuibagarov, 2006). In the literature there are reports of the prospects of using BVM in the diagnosis of other infectious diseases. For example, a pseudotuberculosis species-specific antigenic polymeric diagnosticum was designed for the volumetric agglutination reaction on the basis of the YVBM Yersinia pseudotuberculosis. At the same time, the sensitivity of the diagnosticum was 100%, the specificity was 85.7-89.5% (DI Simakova et al., 2011).
The methods of detection of antigens of pathogens of infectious diseases, proposed by scientists of KazAU named after S.Seifullin, are of great practical importance. Thus, the diagnostic kit, which is based on the MKA strains of the hybridomas BAMA and BIMA, makes it possible to detect Brucella abortus in the patch material, namely in the parenchymal organs and lymph nodes, and in the stomach contents of abortion fetuses (AK Bulashev, 1992). The MCA of the above strains were used by NA Buzyzova and co-authors. (2011) for the development of rapid immunochromatographic tests (ICT) for the detection of Brucella abortus antigens and antibodies against this pathogen. Tests to determine the antigen Brucella abortus allow for 10-15 minutes to identify in the biomaterial LPS in concentrations up to 5 ng / ml, a single standard brucellosis antigen up to 1 mln.kl / ml. The results of serological studies with the help of ICT completely coincided with the data of ELISA.
The express test on the basis of MCA was tested by us and for the detection of the causative agent of tuberculosis in biological material. It was established that the "dot" ELISA using antibodies to Mycobacterium bovis both in the "sandwich" and in the direct version for sensitivity and specificity, and, naturally, in the time of conducting (3.5-4 hours) significantly exceeds the classical methods of research ( microscopic, bacteriological) and comparable to PCR diagnostics. This test, unlike other used ones, made it possible to differentiate Mycobacterium bovis from other species, including representatives of atypical mycobacteria (AK Bulashev et al., 2010; 2011). An immunoenzymatic diagnosticum intended for serological examination of cattle for tuberculosis deserves attention (A.K.Bulashev, 2003). Based on the results of approbation, all lesions that showed positive results at the same time on the ELISA and allergic test were found in the lungs and lymph nodes, characteristic of tuberculosis, whereas in cows that reacted only to PPD tuberculin, no visible changes in the internal organs were detected.
Due to the unfortunate epizootic situation in the Siberian anthrax, foot and mouth disease and rabies, the veterinary service of Kazakhstan needs effective rapid tests for monitoring the sanitary condition of environmental objects. Employees of KazAU named after S.Seifullin and the National Center of Biotechnology of the Republic of Kazakhstan (NCB RK) developed a highly specific method of indicating the causative agent B.anthracis in soil, based on the use of two types of MCA in "dot" ELISA on nitrocellulose paper (AK Bulashev et al. , 2005). The sensitivity of the test was 4,500 m.p./ml. In addition, it can be used to detect anthrax bacilli and other objects of the external environment (water, feed), as well as in raw materials of animal origin.
Scientists of the two mentioned scientific institutions created 3 strains producing the MCA for foot-and-mouth disease virus and 4 hybridomas synthesizing antibodies to the rabies virus and suggested ELISA methods for laboratory diagnostics of the mentioned viral diseases (AK Bulashev et al., 2005). A test system for the identification of rabies virus antigens by the ELISA method was developed by the Research Institute for Biological Safety Problems of the Republic of Kazakhstan (E.S. Zhilin et al., 2011). As a result of studies, 13 of the 15 field materials (brain and saliva) collected from cows, sheep, foxes and corsacs with clinical signs of rabies have been identified as having antigen of the rabies virus. The results were confirmed by testing a similar panel of field materials using the commercial ELISA kit for rabies diagnostics (All-Russian Scientific Research Veterinary Institute, Kazan). NCB RK and the Institute of Physical and Chemical Medicine (Moscow) received a E.coli producer strain of recombinant non-structural protein 3A of foot and mouth disease virus. ELISA based on the above-mentioned antigen, having high specificity, detected antibodies in the blood serum of sick cows at a dilution of 1: 320 (KN Mukantayev et al., 2011).
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