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Desorption methods developed for the antigens of infectious pathogens from the surface of magnetodriving microparticules and their detection in serological and genetic tests


Y.Y. Garkusha, E.N. Afanasiev, I.S. Tyumentseva, I.V. Zharnikova, S.A. Kurcheva, E.V. Zhdanova, O.L. Startseva

One of the main condition for increasing the sensitivity and specificity of the diagnostic methods is the selection and concentration infectious agent or their antigens, release samples from the pollutions of the biotic and nonbiotic nature with the subsequently worked up the express tests. For some of the tests (for example, enzyme immunoassay (EIA) no need to elute seeking antigen from the surface of immobilized magnetic matrix which serve as solid phase when working up the laboratory investigation. For the other methods such as passive hemagglutination test (PHAT), passive agglutination of latex (PAL), polymerase chain reaction (PCR) and so on, badly necessary to put out the magnetic particles from the investigated sample. The purpose of investigation was the development desorption method of cells antigens from the surface of tularemia or brucella magnetic immunosorbent (MIS) particles and subsequent detection of antigens using the PHAT, PAL, PCR. The organic silica microgranulated magnetic sorbents, with the covalently joint the specific polyclonal tularemia and brucellaimmunoglobulins of G group (clasis) was used in this work. For the desorption of antigen the different solutions were used after working up the preliminary immunomagnatic separation. To detect the pathogens the kits of reagents for PHAT and PAL were used and the test-system for the DNA detection using PCR with the elecrophoretic results appreciation. The results of investigation are approving that after working up the preliminary immunomagnatic separation the cells of tularemia or brucella for serological tests when using MIS, it is reasonably to take the pathogens desorption from the surface of magnetic immune matrix with the help one of follow solutions: 1 5 pepsin solution, pH 6,2; twim-20 solution, pH 5,6; 0,03 M potassium hydroxide solution, pH 11,4; 60 % acetonytrile solution, pH 8,5; Tris EDTA buffer, pH 8,9. With the help of above mentioned eluents the most effective desorption of the pathogens from surface of the magnetodrivingmicrosize particles, in the PHAT and PAL false positive results were absented. The most active reagent on account of desorption was 0,03 M potassium hydroxide solution: when using this solution the sensitivity of serological tests were 2,5×105 m.c./ml at the same time all the other solutions show four time less sensitivity – 1×106 m.c./ml. In samples desorpted by using 60 % acetonitrile solution or 0,3Мtrisodium phosphate solution the pathogens were detected in concentration 1×102 m.c./ml with the help of PCR. When working up traditional PCR the antigens of pathogens were not detected, because of their low concentration. Finally, the usage of MIS allow to get selected concentration pathogens in samples with their low concentration and PCR detection.


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