| Brucellosis is a widespread and economically crucial zoonosis with important public health significance. Immunization and accurate diagnosis are the most important measures to control and prevent this disease. At present, most of the diagnostic methods for brucellosis was based on LPS antigen, and a LPS-iELISA was developed in this study for antibody detection against brucella in goats and sheep.In order to improve the specificity, LPS which was the dominant antigen of brucella must be purified. A heat pheno soluble bacteria crude extract LPS was obtained according to OIE manual, and Cold Methanol Precipitattion, Ammonium Sulfate Precipitation, Enzyme Digestion were compared for purification of crude LPS. At last, Enzyme Digestion was selected as purification protocol of crude LPS from brucella。An indirect ELISA for detection of antibody against brucella in sheep and goats was developed based on LPS antigen, and the reaction conditions of the iELISA were optimized. In this study, block titration was used to search the optimum coating concentration of LPS antigen and dilution of serum samlpes. The coating concentration of LPS and dilution of sera tested were determined as0.17ug/ml and1:100, according to the test results. The other conditions such as blocking solution, blocking time, incubating time of serum to detect, working dilution and incubating time of peroxidase-conjugatted anti-sheep immunoglobulin, the time of showing color was determined by a single variable method. The ultimate reaction conditions determined was that plate was coated at4℃for8hours with0.17μg/ml LPS, blocked at37℃with0.4%gelatin for30minutes, serum to detect was incubated at37℃for10min, HRP-IgG was diluted by1:40000and incubated at37℃for lOmin, showing color was at room temperature (25℃) for lOmin. A385of sheep sera known background were detected with the iELISA and the test results were analyzed by ROC curve. According to test data and analytical results, the criterion for diagnosis was determined as S/P≥0.2for positive and S/P<0.2for negative., and the O.D.450mm value of strong positive serum,weak positive serum and negative serum was determined as>0.83,>0.35and<0.15respectively to guarantee the test effective.Evaluation of the iELISA developed was done from its sensitivity, specificity, repeatability(in and among batches), stability, and coincidence rate. Sensitivity test results shows that the iELISA constructed was60-150times more sensitive than that of SAT and RBPT. Specificity test showed that the iELISA can differentiate IgG induced by brucella and Escherichia coli0:157, Salmonella Dublin effectively. Repeatability test in and among batches showed that coefficient of variability were both in10%, indicating good repeatability. Stability test showed that plates coated with LPS was effective in11months, showing good stability. A385of sheep sera samples from different areas were detected with iELISA and the test results were compared with that of RBPT and SAT, showing that the coincidence of the iELISA developed and RBPT&SAT was98.41%and96.65respectively. The results of1932clinical samples tested by iELISA and RBPT were compared, showing that the whole coincidence was 91.7%.Some documents and research results showed that serological diagnostic methods based on brucella LPS antigen cannot effectively differentiate IgG induced by brucella and Yersinia enterocolitica0:9. Outer membrane protein28(OMP28) was an important antigen of brucella and does not exist in Yersinia enterocolitica O:9. In this study, omp28gene of different brucella species were cloned and sequenced. The result showed that omp28gene has a high similarity among different brucella species. Omp28gene of Brucella melitensis16M were cloned into pET32a plasmid and expressed in vitro, OMP28expressed was purified and identified with SDS-PAGE and western-blot, and the result showed that recombined protein obtained has a good reactivity with brucella serum and being a good potential antigen for brucellosis diagnosis. |
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