QC PROTOCOL FOR STREPTAVIDIN CONJUGATES

ASSAY

1. 12-well immulon-4 Elisa strips (Cat. No. 011-010-6404, Dynatech Laboratories) are prepared as follows:
 

a) Triplicate wells are coated with

100 µl 0.1 M Sodium Carbonate buffer pH 9.5

100 µl 0.1 M Sodium Carbonate buffer pH 9.5 with 0.1 ng Biotinylated BSA (Sigma A-8549)

100 µl 0.1 M Sodium Carbonate buffer pH 9.5 with 0.2 ng Biotinylated BSA (Sigma A-8549)

100 µl 0.1 M Sodium Carbonate buffer pH 9.5 with 0.4 ng Biotinylated BSA (Sigma A-8549)
 

b) Incubate the wells overnight at 4°C
 

c) Wash and block the wells for 1-2 hours at room temperature with TBSBT (25 mM Tris, 150 mM NaCl, 0.1% Tween-80, 1% BSA, pH 7.5)

2. Dilute the standard conjugate and any test conjugates 1:1000 (final 1 µg/ml conjugate) in TBSBT. Using a different test strip for each conjugate to be assayed, add 100 µl of diluted conjugate to each well of the test strip and incubate for 2 hours at room temperature.
 

3. Wash the wells 3 times with TBST (25 mM tris, 150 mM NaCl, 0.1% tween-80, pH 7.5). Then wash the wells once briefly with DI water.
 

4. Add 100 µl of substrate (we use Moss ABTS Peroxidase Substrate for HRP conjugates or Moss pNPP Single Component AP Substrate (ELISA) for AP conjugates). Develop 15-20 minutes at room temperature and read results at 405 nm.
 

5. Average OD vs [biotin-bsa] is plotted and the slope determined by linear regression for each conjugate. A passing conjugate must have greater than 80% of the slope of the standard material. New standards must have 100% or greater slope compared to the standard lot they are replacing.