Jacalin coated - Binding Capacity 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. 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 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.
  3. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween 20® four times
  4. Add 100 µl /well of TMB substrate solution and incubate 5 minutes at room temperature.
  5. 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 data show that a plateau has got starting with a biotinylated human IgA concentration of 6 µg/ml.
This concentration means the well binding capacity we can express as:
µg/well = 0.6 ( 600 ng/well )
pmol/well= 4.0 ( this result is calculated considering the IgA1 M.W. = 150.000 )

Figure 1.