1. 50mM carbonate/bicarbonate buffer, pH 9.6:

Na2CO3 1.59g

NaHCO3 2.93g

Dissolve in 1 liter deionized water

Thimerosal 0.10g/liter (can be added as preservative if necessary).

2. Phosphate buffered saline, pH 7.4, containing 0.05% Tween 20 (PBS-T):

NaCl 8.00g

KH2PO4 0.20g

Na2HPO4 1.15g

KCl 0.20g

Thimerosal 0.10g (optional)

Tween 20 0.5ml

All made up to 1 liter in deionized water. Add 1g bovine serum albumin (BSA, Sigma A-7030) to 100ml PBS-T to make 1% BSA PBS-T.

3. Streptavidin-Peroxidase:

Reconstitute 0.25mg Streptavidin-Peroxidase (Sigma S5512) in 1ml distilled water and store at -20°C in 50µl aliquots. Dilute 1/1000 in 1% BSA PBS-T for use in the assay.

4. Substrate solution, 1mM ABTS in 70mM citrate-phospate buffer, pH4.2:

70mM citrate-phosphate buffer, pH 4.2

Solution A = 0.1M anhydrous citric acid, 19.21g/L

Solution B = 0.2M Dibasic Na Phosphate.7H2O, 53.65g/L

For 500ml buffer, mix 147ml A + 103ml B and make up to 500ml with deionized H2O. Add 274mg ABTS to 500ml buffer to make the substrate solution (contains 1mM ABTS).

ABTS = 2,2'-azino-di-(3 ethylbenzthiazoline sulfonic acid), Sigma A1888. The substrate solution is stable at 4°C in the dark. Immediately prior to adding to assay plates, add 1µl 30% H2O2 solution/ml ABTS. The assay will not work if you do not add the H2O2.