Jacalin coated - Sensitivity test

  1. Add 100 µl of different concentrations of biotinylated human IgA ( from 0.05 to 10 µg/ml ) to the wells of Jacalin coated plate diluted in pure distilled water containing 1 mM CaCl2.2 H2O + 1 mM MnCl2.4 H2O and incubate for 60 minutes at room temperature.
  2. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween 20® four times
  3. Add 100 µl /well of Streptavidin-HRP ( BioSpa product code SB01-61 , diluted 1:20.000 in pure distilled water containing 1 mM CaCl2.2 H2O + 1 mM MnCl2.4 H2O) and incubate for 30 minutes at room temperature
  4. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween 20® four times
  5. Add 100 µl /well of TMB substrate solution and incubate 5 minutes at room temperature.
  6. Stop the substrate reaction by adding 100 µl /well of sulphuric acid 0,3 N and read the optical density values at 450 nm

The microplate sensitivity was calculated as the lowest biotinylated IgA concentration higher than the mean optical density plus 5 S.D. of 0 µg/ml biotinylated IgA concentration.

Our experiment gave the following results:

Figure 2.