Required Reagents:
Antigen (preferably purified)
HRP-Conjugated Primary Antibody
Coating Buffer, 0.05 M Carbonate-Bicarbonate, pH 9.6
Wash Solution, 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0
Blocking (Postcoat) Solution, 50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0
Conjugate Diluent, 50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0
Enzyme Substrate, TMB
Stop Solution, 0.18 M H2SO4 or other appropriate solution
High Protein Binding Microtiter Plate (i.e. Nunc C bottom Immunoplate 96 well, Cat. No. 446612)
Preparation of Reagents:
Bethyl Laboratories ELISA Accessory Kit may be used or prepare the following reagents as specified below.
Coating Buffer
3.7 g Sodium Bicarbonate (NaHCO3)
0.64 g Sodium Carbonate (Na2CO3)
1 L of distilled water
Tris Buffered Saline (TBS)
6.06 g Tris Base
8.2 g NaCl
6.0 ml 6 M HCl
1 L of distilled water
pH should be 7.2 to 7.8, conductivity should be 14,000 to 16,000
Wash Solution
1 L of TBS
5 ml of 10% Tween 20
Blocking (Postcoat) Solution
1 L of TBS
10 g BSA
Conjugate Diluent
1 L of TBS
10 g of BSA
5 ml of 10% Tween 20
0.18 M H2SO4
Stock is 18 M
10 ml of Stock solution
1L distilled water
Procedure:
(Perform all steps at room temperature.)
Coat with Antigen
Blocking (Postcoat)
HRP Conjugated Primary Antibody
Enzyme Substrate Reaction
Notes: This procedure is suitable for alkaline phosphatase conjugated antibody. Use an appropriate substrate for alkaline phosphatase