animal model for shigella
Posted 19 May 2002 - 07:10 PM
Posted 19 July 2017 - 10:47 PM
Cynomolgus monkeys (Macaca fascicularis) or Rhesus monkeys (Macaca mulatta) are today used for shigella vaccines study. To evaluate humoral immune response to inoculation shigella bacteria, a good ELISA method on microplate (MTP) is that following described.
Shigella antibody measurements : a) Plate coated : 100 μl/well of S. dysenteriae 1 LPS at 5 μg/ml in carbonate buffer 0.05 M at pH 9.6, are dispensed in MTP (BioHit) and, after sealing, incubate for 3 h at 37 °C. Plate are blocked overnight at +4 °C by using 10% dried milk (Nestle or Sigma) in PBS. After each incubation, plates were washed 6 times with PBS containing 0.05% Tween 20; Plasma/serum procedure :100 μl of sample, diluted 1:2 in 10% dried milk in PBS containing 0.05% Tween 20, are dispensed in well and incubate for 1 h at 37 °C. After wash such as above described, 100 μl of anti-human IgG or anti-human IgA both peroxidase labeled from Dako, diluted 1:1000 and 1:500 respectively in sample diluent are dispensed in separate wells and incubate for 1 h at 37 °C. After further wash, dispense 100 μl of TMB substrate chromogen (see you - http://www.protocol-...posts/6375.html) and incubate for 15 min. The reaction is stopped by the addition of 100 μl of 1 mol/L H3PO4, and the optical density at 450 nm measured in any ELISA microplate reader