Hatamifar Mona, Mosavari Nader, Kazemi Javad
Department of Basic Sciences, Ashkezar Branch, Islamic Azad University, Yazd, Iran.
Bovine Tuberculosis Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Tehran, Iran.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S188. doi: 10.1016/j.ijmyco.2016.10.021. Epub 2016 Oct 27.
BACKGROUND/OBJECTIVE: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), also called as Johne's disease, and is considered as the cause of irreparable economic losses in livestock industry. This bacterium is excreted in large amounts through the milk and feces of infected animal. For the detection of the PTB, indirect ELISA has been highly considered as a simple method with high sensitivity and specificity. Accordingly, this study aims at designing a system of indirect ELISA for the detection of PTB.
In this study, 100 serum samples from 10 herds of dairy farms in Tehran and Alborz provinces, where PTB has been proven by culture, were selected and surveyed using the IDXX commercial kit, and the in-house ELISA system was designed. To design ELISA system, surface antigens and real positive and negative serum samples were used, and checkerboard titration was performed. To determine the cutoff point, the results of the commercial kit were used as gold standard and mean+2SD formula was also used in this regard.
According to the commercial ELISA kit results (15 positive samples and 85 negative samples), the best concentration of antigen and antibody dilution were evaluated as 1.2μg and 1/100, respectively. Furthermore, the cutoff point was determined as 0.44. The sensitivity and specificity were evaluated as 67% and 100%, respectively.
Surface antigens in MAP are sensitive to detect the infected animals, and the indirect ELISA system can be used to detect antibody in the early stages of the disease.
背景/目的：副结核分枝杆菌（MAP）是副结核病（PTB，又称约翰氏病）的病原体，被认为是畜牧业不可挽回经济损失的原因。这种细菌通过受感染动物的乳汁和粪便大量排出。对于PTB的检测，间接酶联免疫吸附测定（ELISA）被高度认为是一种具有高灵敏度和特异性的简单方法。因此，本研究旨在设计一种用于检测PTB的间接ELISA系统。
在本研究中，从德黑兰和阿尔伯兹省的10个奶牛场的牛群中选取了100份血清样本，这些牛场的PTB已通过培养得到证实，使用IDXX商用试剂盒进行检测，并设计了内部ELISA系统。为设计ELISA系统，使用了表面抗原以及真正的阳性和阴性血清样本，并进行了棋盘滴定。为确定临界值，将商用试剂盒的结果用作金标准，在这方面还使用了均值+2标准差公式。
根据商用ELISA试剂盒的结果（15份阳性样本和85份阴性样本），抗原的最佳浓度和抗体稀释度分别评估为1.2μg和1/100。此外，临界值确定为0.44。灵敏度和特异性分别评估为67%和100%。
MAP中的表面抗原对检测受感染动物很敏感，间接ELISA系统可用于在疾病早期检测抗体。
