Sandwich - ELISA

 

 

1.      Microtiter plates 16 h with 100 µl per well

         Primary antibody(10 µg/ml) in Carbonate / Bicarbonate buffer, pH = 9.6 coaten

2.      Dry plates

3.      Block with 150 µl PBS / 1% BSA per Well, 1h, RT

4.      Wash mit PBS + 0.05% Tween 20 (= PBST)

5.      Dilute samples with PBST + 1% BSA

6.      Use triplikates of each sample, add background and positive control

7.      Incubate plate for 2-5 h

8.      Wash with PBST

9.      + 100 µl / Well of biotinylated secondary antibody (1-10 µg/ml) in PBST-BSA, 1-2 h

10.    Wash with PBST, dry

11.    + 100 µl / Well Streptavidine-Peroxidase-conjugate (dil.: 10^-3), 30 min

12.    Wash with PBST, dry

13.    + 100 ml / Well ABTS-Peroxidase substrate, incubate for 10-15 min

14.     Measure Absorbance with Microtiterplate reader