These facts highlight the need to develop new diagnostic techniques in order to collaborate for the control and eradication of brucellosis. However, there are some difficulties concerning these tests, such as the need for highly trained staff, the use of labile reagents that need to be constantly prepared, and titrated, toxic reagents. 1,2ĭue to the social and economic importance of this disease, the Ministry of Agriculture, Livestock and Supply of Brazil (MAPA) has set up a control and eradication program, which defined as official tests for the diagnosis of bovine and buffalo brucellosis: Milk ring test (MRT) used for monitoring the health condition and as a diagnostic tool in epidemiological surveillance systems, Rose Bengal test (RB) as a screening test and 2-mercaptoethanol (2-ME) and complement fixation (CF) as confirmatory tests in addition to the fluorescence polarization assay (FP). It is also a public health problem, for being a zoonosis of occupational character, transmitted by contact with contaminated fetal membranes with the causative agent Brucella abortus, and foodborne by unpasteurized milk intake, fresh cheese and undercooked meat from animals with brucellosis. The evaluated test proved to be effective and reliable, besides being easy to handle and interpret the results.īrucellosis is an infectious disease of chronic character that affects animals, causing great losses to livestock. Using the combination of the test results 2-ME and CF to establish the true condition of the animal, the dot-blot showed relative sensitivity of 100%, and relative specificity of 99.91%. The comparison of the dot-blot results with 2-ME showed Kappa index of 0.9939, sensitivity of 99.48%, and specificity 99.91%, with CF, Kappa index of 0.8226, sensitivity 100% and specificity 95.32%. At the end of standardization, it was established as optimal for the antigen obtained from Brucella abortus B19 after passing through a microorganism rupture process, the blood serum samples diluted at 1:100, and the conjugate at 1:30,000. Fifty bovine blood serum samples were used for the test standardization, and 1315 samples were used for evaluation and comparison between the tests the results were compared using the Kappa indicator. Proceed with immunodetection (see Immunodetection using a chemiluminescent detection method or Immunodetection using a chromogenic detection method).The objective of this study was to standardize and validate the dot-blot test for the serological diagnosis of bovine brucellosis, compare the results with those found in the 2-mercaptoethanol (2-ME) and complement fixation test (CF), and estimate the relative sensitivity and specificity of the dot-blot compared to these tests.Tip: For larger sample volumes, suitable equipment is available from several suppliers. After applying the samples, the membrane should be dried for a short time at room temperature before proceeding with the detection process.Tip: To differentiate between nonspecific and positive signals, an extra sample containing 1 µl of a cell extract of the host strain without plasmid (or other suitable control) should also be applied to the membrane and treated together with the protein of interest. In most cases diluting the protein with buffer containing denaturing reagents will increase epitope exposure and give better results. Note: Under native conditions especially, the antibody epitope must be at least partially exposed to allow antibody binding. It is also possible to use crude cell lysate and apply 1 µl samples with an estimated concentration of 1–100 ng/µl protein. Apply 1 µl samples of diluted protein directly onto membrane.Tip: The protein of interest is diluted in dilution buffer for denaturing conditions, dilution buffer for native conditions, or another preferred buffer. Dilute protein samples in buffer to final protein concentrations of 1–100 ng/µl.
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