Solution Preparation
Coating Solution:
Capture antibody is diluted in coating solution for immobilization
onto the microplate. Commonly used coating solutions are: 50
mM sodium carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; or 10
mM PBS, pH 7.2. A protein concentration of 1-10 µg/ml is usually
sufficient.
Blocking Solution:
Commonly used blocking agents are: BSA, nonfat dry milk,
casein, gelatin, etc. Different assay systems may require different
blocking agents.
Detection Antibody Solution:
Biotinalyted detection antibody should be diluted in 1x Blocking
solution to help prevent non-specific binding.
AKP Conjugated Streptavidin Solution:
Enzyme conjugate should be diluted in 1x Blocking solution to
help prevent non-specific binding.
Wash Solution:
Typically 0.1 M Phosphate-buffered saline or Tris-buffered saline
(pH 7.4) with a detergent such as Tween 20 (0.02%-0.05% v/v).

Protocol
1. Dilute the capture antibody to appropriate dilution in Coating
solution.
2. Add 100 µl diluted antibody to appropriate wells. Incubate 2
hours at room temperature or 4ºC overnight.
3. Empty plate and tap out residual liquid.
4. Wash twice with 300 µl Wash solution.
5. Add 300 µl Blocking solution to each well. Incubate 1 hour.
6. Empty plate and tap out residual liquid.
7. Wash twice with 300 µl Wash solution..
8. Add 100 µl diluted biotinylated detection antibody to each
well. Incubate 1 hour at 37ºC or 3 hours room temperature.
9. Empty plate, tap out residual liquid.
10.Fill each well with Wash solution. Invert plate to empty, tap
out residual liquid. Repeat 3 times.
11.Add 100 µl diluted AKP conjugated streptavidin to each well.
Incubate 1 hour at room temperature.
12.Empty plate, tap out residual liquid and wash as described
in step 10.
13.Give final 5 minutes soak with Wash solution. Tap residual
liquid from plate. This washing step is critical to reduce
signal background.
14.Fill each well with Wash solution. Invert plate to empty, tap
out residual liquid. Repeat 5 times.
15.Dispense 100 µl substrate (pNPP) into each well. Develop
the color for 30 minutes, and immediately read plate with
plate reader at 405-410 nm.

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