Protein G coated - Sensitivity test

  1. Add 100 μl of different concentrations of human biotinylated IgG ( from 1.56 to 100 ng/ml ) to the wells of Protein G 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 Streptavidin-HRP ( BioSpa product code SB01-61 , diluted 1:20.000 ) 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 microplate sensitivity was calculated as the lowest biotinylated IgG concentration higherthan the mean optical density plus 5 S.D. of 0 ng/ml biotinylated IgG concentration.

Our experiment gave the following results:

  1. 0 ng/ml biotinylated IgG optical density mean ( coming from 8 replicates ) = 0.133
  2. standard deviation = 0.012
  3. mean + 5 S.D. = 0.193
  4. sensitivity = 0.056 ng/well of human IgG

Figure 2.