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Star Republic: Guide for Biologists

Enzyme Linked Immunosorbent Assay --- ELISA

Virtualab Protocols (Chang Bioscience)
1. Coat a 96-well ELISA plate with 50 l/well of solubilized antigen at maximal coating (1-10 g/ml) in borate buffered saline (100 mM Boric acid, 25 mM Sodium borate, 75 mM Sodium chloride, pH 8.5) or 50 mM sodium carbonate pH 8.5 overnight at 4 C.

2. Wash the plate 5 times with the ELISA wash buffer (10 mM Tris pH8.0, 0.05% Tween-20, 0.03% Sodium azide).

3. Block with 200 l Blocking buffer (0.05% Tween-20, 1.0% BSA, 0.03% Sodium azide in PBS or 5% dried milk/PBS) and incubate for 30 minutes at room temperature or 37 C.

4. Wash the plate 5 times with the ELISA wash buffer (10 mM Tris pH8.0, 0.05% Tween-20, 0.03% Sodium azide).

5. Add 50 l of antibody (hybridoma supernatant, dilute for more concentrated stocks) per well.

6. Wash the plate 5 times with the ELISA wash buffer.

7. Add 50 l of anti-mouse IgG-alkaline phosphatase conjugate (1:4,000 in PBS). and incubate for 30 minutes at room temperature or 37 C.

8. Wash the plate 5 times with the ELISA wash buffer.

9. Add 50 l of substrate (0.06% p-nitrophenyl phosphate 9.6% Diethanolamine (v/v), 1.0 mM Magnesium chloride, pH9.8 or 1 tablet of p-nitrophenyl reagent in 5 ml of 10% diethanolamine, pH 9.8).

10. Incubate for 30 minutes at room temperature or 37 C.

11. Stop the reaction with 12.5 l 3M NaOH and observe the yellow color change or read using a plate reader at 405 nm.