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Serologiczna diagnostyka boreliozy mimo opracowania i wprowadzenia różnych metod wciąż wymaga weryfikacji. Metody przesiewowe najczęściej stosowane do oceny przeciwciał anty - Borrelia to metoda jakościowa ELFA (Enzyme Linked Fluorescent Assay, analizator Vidas), metoda immuno - fluorescencji pośredniej (IIFT) oraz metoda immunoenzymatyczna ELISA. Wszystkie wymienione metody charakteryzują się wysoką czułością. Ze względu na możliwość wystąpienia nieswoistych reakcji krzyżowych i wyników fałszywie dodatnich wskazana jest weryfikacja wyników wątpliwych i dodatnich uzyskanych metodami przesiewowymi. Weryfikacji dokonuje się testem Western biot (Wb) o wysokiej swoistości. Technika Wb pozwala na identyfikację przeciwciał dla białek wysoce specyficznych dla Borrelia burgdorferi: VIsE, p83, p39, p31 (OspA), p30, p25 (OspC), p21, p19, p17, wśród których białko VIsE jest najbardziej swoiste dla odpowiedzi w klasie IgG oraz białko OspC, którego obecność związana jest z produkcją przeciwciał IgM. Celem niniejszej pracy była analiza wyników uzyskanych metodą ELFA (z analizatorem Vidas) i metodą IIFT jako testów przesiewowych stosowanych w diagnostyce zakażeń krętkiem B. burgdorferi.
Monoclonal antibodies (MAbs) for diagnosis of classical swine fever have been produced. Hog cholera virus, the Alfort strain, was used as an antigen for the immunization of mice; it was propagated in the culture of primary swine kidney cells. Mice of the BALB-c strain were immunized in four cycles and the spleen cells of the mice characterized by the highest titers of antibodies were taken for hybridization. The spleen cells were fused with S p2/o myeloma cells in the presence of PEG 4000. The hybrydoma cells were cultivated in a 20 per cent DMEM HY-HT medium at 37°C with a 5 per cent presence of CO2. The supernatant fluids were tested for the presence of specific antibodies by ELISA and NPLA assays. The selected cultures were subcloned twice in a semi-solid agar, then they were isotyped using the Immuno-Select TM set. Intraperitoneal inoculation of hybri- doma cells male BALB/c mice resulted in the production of ascites fluids containing MAbs. The ascites fluids were purified by means of an Econo-Pac Protein A Kit and conjugated with horse-radish peroxidase.
The seroneutralization test based on the inhibition of syncytial effect (SN-ST) turned out to be very useful in the serological diagnostics of enzootic bovine leukaemia. The mean coefficient value of the SN-ST for positive sera (ELISA titer from 200 to 3200) was 0.1, for doubtful (ELISA = 100) - 0.52 and for negative sera 0.94. In case of negative serum the multinuclear cells (syncytium) were very large and numerous and with 85 to 100 nuclei; for doubtful sera they were small, scarce and with 25 to 30 nuclei. No syncytia were observed with the presence of negative sera. The time of incubation of BLV with positive sera played a very important role in the process of seroneutralization. The best results of SN-ST, which corresponded to the ELISA, were found after two hours of neutralization.
The aim of the study was to perform a preliminary evaluation of the ELISA test for the diagnosis of bovine tuberculosis in Polish conditions. The Idexx kit of immunoassays for detecting the presence of antibodies specific for Mycobacterium bovis in samples of cattle’s blood serum and plasma was used to examine a total of 50 serum samples from clinically healthy animals, 44 sera from cows with previous positive result of tuberculin tests, but negative in microbiological examination, 25 sera from animals from whose tissues strains of Mycobacterium bovis had been isolated (including 10 sera from cows with characteristic tuberculous lesions and 15 sera from animals without these lesions), and 16 serum samples from animals reacting positively for Johne’s disease. On the basis of the results of these examinations, the specificity of the test was determined at 100% and sensitivity at 90%. There were no cross-reactions, and the results indicate that the ELISA Idexx kit may be used as an important diagnostic tool in the diagnosis of bovine tuberculosis.
Sera taken from 449 cows which aborted in years 1991-1994 have been assessed by the complement fixation test for the presence of Q-fever antibodies. It was found that 10.7% of the animals had antibodies against Coxiella burnetii II antigen. Moreover, in the examinations repeated three times on 150 cows coming from a farm in which several cases of abortions were observed, about 15% of seroreagents were noted. At the same time 14 persons working in that farm and additionally 22 humans drinking milk of the cows were tested by the fluorescent antibody test. Specific antibodies against C. burnetii I and II were detected; however lower concentrations of antibodies were found against C. burnetii phase I.
The porcine respiratory and reproductive syndrome is a relatively new swine disease first described in the USA in 1987, and in Europe in 1990. Serodiagnosis of the disease can be performed by using an immunoperoxidase mono- layer assay (IPMA), To evaluate this test, the following materials were used to perform the assay: Hesse strain of PRRSV, MARC-145 cells, 42 sera obtained from PRRS suspected herd, 60 sera from herds free from PRRS, and positive reference anti-PRRSV serum. In order to test the sera for the presence of antibodies to PRRSV, 50 pi of successive, 2-fold serum dilutions (from undiluted to diluted 1:256) were distributed in triplicate on the prepared 96-well microplate. The serum samples were considered as positive if brownish-red precipitates were found in the cell cytoplasm of at least one of the tested serum dilutions. The presence of antibodies to PRRSV was found in 23 out 42 examined sera from a suspected farm. In 10 out of 23 positive serum samples the titre was >1:256. No antibodies to PRRSV were detected in the sera that came from swine herds free from PRRS. Taking into account our results, the IPMA can be applied for routine serodiagnosis of PRRS in Poland.
Отмечено положительное влияние абсорбции неспецифических рецепторов в сыворотках скота при применении 30% суспензии кровяных телец гуся. Абсорбция проводилась в темп. 37°С в течение 30 мин. с использованием 3,1X10⁷—9,2Х10⁷ эритроцитов/0,7 мл сыворотки. Добавка телец к усследуемой сыворотке в отношении 1:10, не влияет отрицательно на титр противотел для виртса РI₃ в микротесте HI. Результаты теста, проведенного на 3 плитках, указывают на повторяемость полученных результатов. Неабcoрбирoванные сыворотки, дающие отрицательный результат тесте, после абсорбции показывали титр 1:10—1:40±. Проведенные исследования указывают на полную пригодность кровяных телец гуся и плиток фирмы PLASTOMED к кутиновой серологической диагностике параинфлюэнцы скота.
The aim of the study was to evaluate the application of SDS-PAGE electrophoresis and western-blotting for serological diagnostics of glanders. Six commercial Burkholderia mallei antigens, eight anti-B. mallei sera and two negative sera were used in the study. In the first part of the study electrophoretic profiles of antigens in SDS-PAGE were determined at a gradient of 4% to 20% of polyacrylamide gel. Optimal conditions of western-blotting reaction were determined. In the case of all examined antigens and anti-B. mallei sera, the presence of protein fractions in the range from 42 to 33 kDa and one band of the molecular weight of 16 kDa were demonstrated by immunoblotting reaction. Additional bands of the different molecular weight were also observed. The antigens examined did not show reaction with negative sera. In the next part of the study, a sensitivity of the western-blotting method and the CFT were compared. The western-blotting method was two - four times more sensitive than the CFT.
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