1. Coating
Dilute capture antibody with DW and add to each ELISA plate
well. Incubate for at least 1 hr at room temperature. (or
overnight at 4°C). After incubation, remove the solution from
each well and wash the plate 3-5 times. Make sure that
residual buffer in each well was removed.
2. Blocking
Add 200ul Blocking Solution to each well. Incubate for at least
1 hr at room temperature.After incubation, remove the solution
from each well and wash the plate 3-5 times.
3. Reaction
Dilute standard or sample in Diluent and add to each well.
Incubate for at least 1 hr at room temperature. After
incubation, remove the standard or sample from each well and
wash the plate 3-5 times.
4. Detection
Dilute detection antibody in diluent and add 100ul per well.
Incubate for at least 1 hr at room temperature. After
incubation, remove the solution from each well and wash the
plate 3-5 times.
5. Enzyme Conjugate
Dilute Avidin-HRP at 1:2000 in diluent and add 100ul per well.
Incubate for at least 1 hr at room temperature. After
incubation, remove the solution from each well and wash the
plate 3-5 times.
6. Color Reaction and Reading
Mix 50ul TMB solution and 100ul Substrate solution prior to use.
Add this mixture to each well. After sufficient color development
(5-10minutes at room temperature or at 37°C), add 50 ul Stop
Solution (2M H2SO4) to each well. Using a microtiter plate
reader, read the plate at the wavelength that is appropriate
for the substrate used (450 nm for TMB).
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