Sandwich ELISA Procedure
 |
Required Reagents
A: Coating Buffer: 10 mM phosphate buffer, 145 mM NaCl, pH 7.2.
B: Washing and dilution buffer: 10 mM phosphate buffer, 500 mM NaCl, 0.1% TWEEN® 20, pH 7.2.
C: 100 mM Citric acid-phosphate buffer, pH 5.0
(These buffers, if stored at +4°C, are stable for up to 2 months).
D. Chromogenic substrate: 2 mg OPD tablet + 12 ml of buffer A. Allow 15-20 minutes for complete dissolution and add 5 ml of 30% hydrogen peroxide. Prepare this reagent just before use. Allow it to warm to room temperature. After addition of hydrogen peroxide, make sure that the reagent maintains a slight yellow tinge. This reagent is stable for about 2 hours, if stored protected from light. Avoid contact with skin.
E. 1 M Sulfuric acid: Always add acid to water and not vice versa. To prepare, add 56 ml of sulfuric acid to 944 ml of water.
ELISA Procedure
a) Coating of wells with antibody: Add 100 ml of diluted (with buffer A) antibody to each well. The antibody should be directed against the antigen to be determined. Cover the plate with plastic film or aluminium foil. Incubate overnight at +4°C.
b) Washing: Empty the plate by inversion and tap the plate against a few layers of soft tissue paper to remove any residual liquid. Wash the plate by filling the wells by immersion in buffer B. Drain to empty the plate. Repeat washing two more times.
c) Incubation with test samples: Add 100 ml of sample and/or standard, diluted in buffer B, to each well. Cover the plate and allow to stand at room temperature for about two hours.
d) Incubation with peroxidase-conjugated antibody: Add 100 ml of peroxide conjugated antibody to each well. Cover the plate and allow to stand at room temperature for 1 hour. The peroxidase-conjugated antibody should be directed against the antigen to be determined.
e) Wash as described in step (b).
f) Color development: Add 100 ml of chromogenic substrate to each well. Cover the plate and allow to stand at room temperature for 15 minutes or until color has developed. Protect the plate from light during this period.
g) Stopping the reaction: Stop the reaction by adding 150 ml of 1M sulfuric acid to each well. For quantitation purposes, it is important to make sure that each well has been incubated with color reagent for exactly the same length of time.
h) Measuring the absorption: Read the absorption in a suitable photometer or ELISA plate reader (set at 492 nm) within 3 hours of color development. Plot the standard curve on semilogarithmic paper with A492 as ordinate and log10 concentration as abscissa.
|
