Protocol for ELISA screening of antibodies:

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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 4°C. 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 suspended with Alkaline Phosphatase conjugate, 1:30,000 concentration in blocking solution. This solution was added to each well and incubated for 1 h. Then 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.