Protocol for ELISA
screening of antibodies:
The 96 well plate was prepared for Elisa by suspending lyophilized Helicosporidia in coating solution (pH 9.6) and letting the cells settle in the plate over night at 4oC. Then the cells were fixed in 2.5% paraformaldehyde for 1 h. The plate was washed 3 times in PBS with 0.02% TWEEN 20. Next, the plate was blocked in 5% nonfat dry milk for 15 min. The primary antibody was added to blocking solution and added to each well (titration from 1/400 to 1/600), then incubated for 1 h at room temperature. The plate was washed again, 3 times, in the washing solution. The secondary antibody, Anti-Mouse IgG (Whole Molecule), was suspened with Alkaline Phosphatase conjugate, 1:30,000 concentration in blocking solution. This solution was added to each well and incubated for 1 h. The plate was washed 5 times in PBS, making sure there was no residual liquid before adding the Nitrophenyl phosphate substrate. Once the substrate was added, the plate was read after a significant color change was evident (about 1 h).
Alkaline Phosphatase Substrate:
9.7 ml Diethanol amine in 100 ml of water, with an adjusted pH of 9.8. This buffer is combined with a commercially bought Nitrophenyl phosphate pill.