Jacalin coated - Binding specificity

In order to evaluate the Jacalin coated plate specificity an assay to capture human lgA was carried out.

lgA assay

  1. normal human serum containing: 1170 mg/dl IgG, 280 mg/dl lgA, 90 mg/dl lgM was diluted 1:200 in PBS 0,05M pH 7,2
  2. 100µl diluted human serum and 100 µl PBS 0,05M pH 7,2 as 0 mg/dl of reaction were incubated for 30 min.at room temperature in Jacalin coated wells and in wells coated with bovin serum albumine (BSA), used to verify the test specificity.
  3. a washing step with 0,1M PBS pH 7,2 + 0,05% Tween® 20 was performed
  4. 100µl/well of purified goat anti-human Fc lgA Peroxidase was added to each well and incubated for 30 min. at room temperature
  5. a further washing step was performed
  6. 100µl/well of TMB was added to each well and incubated for 15 min. at room temperature
  7. the reaction was stopped by adding 100 µl/well of sulphuric acid 1N
  8. reading at 450 nm was then performed

The following results expressed in optical density (O.D.), were obtained

Sample Jacalin coated well
O.D.
BSA coated well
O.D.
100 µl PBS 0.05M pH 7.2 0.126 0.076
100 µl 1:200 diluted serum 1.189 0.134