Bovine paratuberculosis, also known as Johne's disease, is a chronic infectious disease caused by *Mycobacterium avium* subspecies *paratuberculosis*. This condition affects cattle and can lead to severe economic losses due to reduced milk production, weight loss, and eventual death. The ELISA diagnostic method described here has a sensitivity of 76% and a specificity of 97%, with a detection rate of 6.1% when tested on 2,48 cows. Compared to traditional methods like complement fixation and agar gel immunodiffusion, this ELISA technique offers improved accuracy and efficiency.
There is currently no effective treatment for bovine paratuberculosis, making early detection and culling of infected animals the primary strategy for controlling the disease. Traditional diagnostic methods are often time-consuming and less sensitive, which is why the development of an ELISA-based approach is highly valuable in modern veterinary practice.
**Materials and Reagents**
1. *M. avium* subspecies *paratuberculosis* affinity chromatography antigen
2. *M. phlei* absorption antigen
3. Rabbit anti-bovine IgG enzyme-labeled antibody
4. Bovine paratuberculosis reference positive serum
5. Bovine paratuberculosis reference negative serum
6. ELISA plate, microplate reader, pipettes, washer, incubator, etc.
7. Reagents and solutions are prepared following the same protocol as in the fifth section of the swine fever ELISA test.
**Procedure**
1. **Antigen Coating**: Dilute the *M. avium* subspecies *paratuberculosis* affinity chromatography antigen to 50 μg/ml using antigen diluent. Add 0.1 ml per well. A few wells are coated with bovine serum albumin (50 μg/ml) as a control.
2. **Serum Treatment**: Take 0.10 ml of the serum sample, reference positive serum, and reference negative serum. Add 0.80 ml of physiological saline and 0.10 ml of *M. phlei* absorption antigen. Mix thoroughly and incubate at 37°C for 1 hour. Then, add 0.90 ml of serum diluent (without calf serum, but containing bovine serum albumin), and mix again.
3. **ELISA Testing**: Follow the procedure outlined in Table 12-4. Each sample is tested in duplicate to calculate the average OD value.
**Result Interpretation**
Under proper control conditions, the reference positive serum should show a brownish yellow color, while the reference negative serum and other control wells remain colorless or pale yellow.
1. **Visual Judgment**: If the test well matches the color of the reference positive serum (brownish yellow), it is considered ELISA positive (+).
2. **Instrumental Measurement**: Using a microplate reader at 490 nm, set the blank well to zero. If the corrected OD value of the reference serum is 1.0 (or the specified value), then:
- OD ≥ 0.50: ELISA positive (+)
- 0.40 ≤ OD ≤ 0.49: ELISA inconclusive (±)
- OD < 0.40: ELISA negative (-)
This ELISA method provides a reliable and efficient way to detect bovine paratuberculosis, supporting early diagnosis and disease management. For high-quality ELISA kits and professional support, consider products from Shanghai Guduo Biological Company. Your satisfaction is our top priority.
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