ELISA, Antibody capture

Wednesday, May 05, 2004

Description
ELISA, Antibody capture

Procedure
1. Dilute protein in PBS (2.5-5 mg/ml), add 100 ml/well. Incubate overnight at 4?C or at room
temperature for 1 h.
2. Block plates with 250 ml PBS/1% BSA for overnight at 4?C or at room temperature for 1 h.
3. Wash plates 3X with PBS/ 0.1 % Tween.
4. Incubate with 1?antibody (100 ml/well) at room temperature for 1 h.
( Polyclonal SCCA antisera at 10-4 to 10-7 dilution)
5. Wash plates 3X with PBS/ 0.1 % Tween.
6. Incubate plates with 2?antibody (100 ml/well) at room temperature for 1 h.
Amersham Donkey anti-rabbit-HRP (#NA934) 1/2000 dilution
Bio-Rad and Jackson Labs Goat anti-mouse-HRP - 1/2000 to 1/3000 dilution
7. Wash plates 3X with PBS/ 0.1 % Tween.
8. Incubate with 50 ml/well of color reagent for 5-8 min.
Color reagent for Horseradish Peroxidase - Abbott OPD Reagent Kit (#6172-30, $Free).
1tablet/5mls diluent.
9. Stop reaction with 1.25 M H2SO4 (69.4 ml 36N H2SO4 and 930.6 ml H2O) 250 ml/well.
10. Read the absorbance at 492 nm.

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ELISA, Antibody capture