Protein A coated - Binding Capacity Test

  1. Add 100 μl of different concentrations of human IgG ( from 0.1 to 20 μg/ml ) to the wells of Protein A coated plate and incubate for 30 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 F(ab)2 goat anti-human IgG-HRP ( Sigma product code A 2290, diluted 1:8000 ) 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 15 minutes at room temperature.
  6. Stop the substrate reaction by adding 100 μl /well of sulphuric acid 1 N and read the optical density values at 450 nm

The data show that a plateau has got starting with an IgG concentration of 7.5μg/ml.

This concentration means the well binding capacity we can express as:

Figure 1