Virtualab Protocols (Chang Bioscience)
1. Coat a 96-well ELISA plate with 50 µl/well of solubilized antigen at maximal coating (1-10 µg/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 µl 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 µl 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 µl 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 µl 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 µl 3M NaOH and observe the yellow color change or read using a plate reader at 405 nm.
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