| Paratuberculosis or Johne's disease is a chronic intestinal inflammation in ruminants caused by infection with an acid-fast micro-organism, Mycobacterium avium subsp. paratuberculosis(Map). It has a global distribution and affects many species of farm livestock as well as free-living species and is considered by many to be the most serious infectious disease currently plaguing the world's cattle, sheep, and goat industries.The key to effective control of paratuberculosis is the rapid and accurate detection of infected animals. Enzyme-linked immunosorbent assay—An indirect ELISA was considered as the most sensitive and specific method to detect antibodies to M. paratuberculosis.The purpose of this study was to develop an indirect ELISA to detect the paratuberculosis serum antibody in deer serum. An affinity-purified antigen derived from PT strain 18 was used as the coated antigen and serum samples were absorbed by antigen of M. phlei before examination. The threshold value for a positive ELISA response was set at OD 0.56. Test protocol as follow: wells of a microtitration enzyme immunoassay plate were coated with 100ul of 40ug/ml of the antigen in carbonate butter and incubated 3h at 37 ℃.Plate were rinsed by flooding them 3 times (3 minutes each) with a wash solution containing 0.15NaCl and 0.05% Tween-20.Test sera were diluted 1:100 in ELISA buffer(0.15M NaCl,0.01M PB,0.1%bovine serum albumin, and 0.05% Tween-20) and 100ul applied to each well. Plates were incubated for 90 minutes at 37℃,and rinsed 3 times. Horseradish peroxidase-conjugated rabbit anti-deer IgG was diluted 1:4 000 in ELISA buffer, and 100ul was added to each well. The plates were incubated for 90 minutes and rinsed as before, 100ul substrate containing OPD-H2O2 system was added to each well. Plates were read after 20 minute when positive control sera reached an optical density (OD) of 1.0.760 blood samples were collected and examined by this indirect ELISA, 61 samples were ELISA-positive. |
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