Bovine paratuberculosis, also known as Johne’s disease, is a chronic infectious condition caused by Mycobacterium avium subsp. paratuberculosis. This ELISA method has a sensitivity of 76% and a specificity of 97%. In a study involving 2,48 cows, the detection rate was found to be 6.1%, outperforming traditional methods such as complement fixation and agar gel immunodiffusion tests. Unfortunately, there is currently no effective treatment for this disease, making early detection and removal of infected animals crucial for controlling its spread. Traditional diagnostic methods have limitations, which is why establishing an ELISA-based approach offers a more accurate and efficient solution.
Materials and Reagents
1. Mycobacterium avium subsp. paratuberculosis affinity chromatography antigen
2. Mycobacterium phlei absorption antigen
3. Rabbit anti-bovine IgG conjugated with enzyme
4. Bovine paratuberculosis reference positive serum
5. Bovine paratuberculosis reference negative serum
These materials are provided by a designated institution.
6. Equipment: ELISA plate, microplate reader, pipettes, washer, incubator, etc.
7. Reagents and solutions are prepared following the same procedure as in the fifth section of the swine fever ELISA protocol.
Procedure
1. Antigen coating: The paratuberculous affinity chromatography antigen is diluted to 50 μg/ml using antigen diluent, and 0.1 ml per well is added. A portion of the control wells is coated with bovine serum albumin (50 μg/ml).
2. Serum preparation: Take 0.10 ml of the test serum, reference positive serum, and reference negative serum. Add 0.80 ml of physiological saline and 0.10 ml of Mycobacterium phlei antigen. Mix thoroughly and incubate at 37°C for 1 hour. Then, take 0.10 ml of the mixture and add 0.90 ml of serum diluent (note that bovine serum albumin is included in the diluent but not calf serum). Mix well.
3. Conduct the formal test according to Table 12-4. Two wells are used for each sample to calculate the average OD value.
Result Interpretation
Under the condition that the control group is properly established, if the reference positive serum turns brownish yellow while the reference negative serum and other control wells remain colorless or pale yellow, the results can be interpreted accordingly.
1. Visual judgment: If the test well matches the color of the reference positive serum (brownish yellow), it is considered ELISA positive (+).
2. Instrumental reading: Using a microplate reader at 490 nm, set the blank well to zero. The corrected OD value for the reference serum should be around 1.0 (or a specified value). If the OD ≥ 0.50, it is classified as ELISA positive (+). If 0.40 ≤ OD ≤ 0.49, it is considered inconclusive (±). If OD < 0.40, it is deemed ELISA negative (-).
This detailed guide provides a reliable method for diagnosing bovine paratuberculosis. For high-quality ELISA kits and professional support, consider reaching out to Shanghai Guduo Biological Company. Their products are trusted by many professionals in the field, and your satisfaction is their top priority.
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