Evaluation of binding specificity towards monoclonal anti-calmodulin

  1. Dilute anti-calmodulin monoclonal antibody from 1: 100 to 1: 3200 with 0,1 M PBS pH 7,2 containing 0,2 % BSA.
  2. Add 100µl of each dilution to the wells of Calmodulin coated plate and incubate 60' RT. Add the same solutions to Albumin coated plate as comparison for evaluate the specificity of binding.
  3. Leave blank wells as control
  4. Empty the wells and wash with 0,1M PBS pH 7,2 + 0,05% Tween® 20 four times
  5. Add 100 µl / well of conjugate goat anti-mouse-POD and incubate 30' RT.
  6. Add 100 µl /well of TMB substrate solution and incubate 10 minutes at room temperature
  7. Stop the substrate reaction by adding 100 µl of sulphuric acid 1 N and read the optical density values at 450 nm.