Bovine paratuberculosis is a chronic infectious disease caused by åˆ0 Mycobacterium tuberculosis, also known as johne disease. The method has a sensitivity of 76% and a specificity of 97%. The detection rate is 6.1% by testing 2,48 cows and compared with conventional complement and anomalous reactions. "There is no effective treatment for this disease. The only way to eradicate the disease is to detect, isolate or eliminate sick animals. At present, the quarantine of this disease mainly relies on replenishment and abnormal reactions. This paper establishes ELISA for the diagnosis of bovine paratuberculosis. method.
First, materials and reagents
1. Mycobacterium tuberculosis affinity chromatography antigen
2, Mycobacterium phlei absorption antigen
3, rabbit anti-bovine IgG enzyme labeled antibody
4, bovine paratuberculosis reference positive serum
5, bovine paratuberculosis reference negative serum
1 to 5 items are supplied by the designated unit.
6, equipment ELISA board, enzyme-linked immunosorbent detector, sampler, bottle washing, incubator and so on.
7. The reagents and solutions are prepared in the same way as the fifth section of the swine fever ELISA.
Second, the operation method
1. Antigen coating The paratuberculous affinity chromatography antigen was diluted to 50 μg/ml with an antigen dilution, and 0.1 ml per well was coated. Part of the control well was coated with bovine serum albumin (50 μg/ml).
2, serum treatment to be tested serum, reference positive serum, reference negative serum each 0.10ml, plus physiological saline 0.80ml, add Mycobacterium phlei antigen absorption 0.10ml, mix, 37 °C feeling for 1h. Take 0.10ml, plus Serum dilution 0.90 ml (note that bovine serum albumin is added to the serum dilution without calf serum). Mix well
3. The formal test is carried out according to Table 12-4. Two wells were added to each sample to obtain an OD average.
Third, the result is judged
On the premise of the establishment of the control group, the reference positive serum was brownish yellow, and the reference negative serum and other control wells were colorless or pale yellow, and the test results were determined.
1. The visual judgment is equivalent to the color of the reference positive serum hole, that is, it is brownish yellow, and it is judged as ELISA positive (+).
2. The microplate reader judges (490 nm) to zero the substrate solution control well, and the corrected reference serum OD value is 1.0 (or the corresponding specified value), and the hole to be inspected is determined. When OD ≥ 0.50, ELISA positive (+); 0.40 ≤ OD ≤ 0.49 ELISA coagulation (±); OD < 0.40, ELISA negative (-).
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