ELISA

A. Antigen Coating
1. Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer (coating buffer), e.g. human IgG (0.025mg/ml) in coating buffer.
2. Pipette 0.2 ml of the above solution to each well of the microtiter plate.
3. Incubate at 37 0C for 30 min.
4. Remove the coating solution. Wash three times with PBS-T (0.2 ml/well).
5. Blocking step: 0.5% BSA-PBS (0.2ml/well) at 370C for 30 min (an additional blocking step may be required to block non-specific binding).
B. Primary Antibody Reaction
1. Dilute the primary (first) antibodies in PBS-T, e.g. Rabbit-anti-human IgG antiserum (different dilutions of antiserum, 1:400~1:51200).
2. Add 0.2 ml of the diluted first antibody to each well. Negative control should be included (No first Ab.).
3. Incubate at 37 0C for 1 hour.
4. Wash three times with PBS-T (0.2 ml/well).
C. Application of Secondary Antibody
1. Dilute the peroxidase conjugated secondary antibody in PBS-T, e.g. Goat-anti-rabbit IgG-HRP (1:20,000). Add 0.2 ml of this solution to each well.
2. Incubate at 37 0C for 30 min.
3. Wash three times with PBS-T (0.2 ml/well).
D. Substrate Preparation
1. During the last incubation and immediately before use, dissolve o-Phenylenediamine in Citric acid-sodium hydrogen phosphate buffer, add 30% H2O2 before use.
E. Development
1. Add 0.2 ml of the freshly prepared substrate to each well.
2. Orange-yellow color should develop in positive wells after 5 minutes.
3. Stop reaction with 50 l per well of preferred stopping reagent and read at 490nm in a microplate reader.

REAGENTS
1. Coating buffer (Carbonate-Bicarbonate buffer, pH 9.6) : Na2CO3 0.15g, NaHCO3 0.293g, add H2O to 100ml (pH 9.6).
2. Phosphate buffered saline (PBS): NaCl 8g, KCl 0.2g, KH2PO4 0.24g,Na2HPO4. 12 H2O 2.9g, add H2O to 1000ml (pH 7.4).
3. Washing buffer (PBS-T): PBS + 0.05% Tween 20.
4. First antibody: Rabbit-ani-human IgG antiserum.
5. Peroxidase conjugated secondary antibody: Goat-anti-rabbit IgG-HRP (1:20,000).
6. Substrate: substrate buffer(fresh):
0.1M Citric acid (2.1g/100ml), 6.1ml;
0.2M Na2HPO4. 12 H2O(7.163g/100ml), 6.4ml;
ddH2O 12.5ml;
dissolve 8mg o-Phenylenediamine;
add 40ml of 30% H2O2 before use.
7. Stopping reagent: 2M H2SO4