1. Prepare target plate :
a) Design the experiment to leave at least one well, row or column blank for the appropriate controls , including standards.
b) Add target antigen ( e. g purified soluble CD 4 ) diluted in buffer
( e. g. PBS / NaN3 ) at 50 µg/ml to an ELISA plate, 50 µl/well. Leave 60 ' at room temperature.
2. While the target plate is being prepared, prepare the inhibitor plate by pre incubate supernatants with Antibody :
a) In a round bottomed plate add 80/100 µl supernatant to each well.
b) Make a dilution series of a standard (e.g. purified CD 4 ) and place it in one column. (A suitable initial concentration is 2 µg/ml for 2 fold dilution's and 10µg/ml for 5 fold dilution's)
c) Add 40 µl /50 µl Antibody diluted in 0.1-0.5% BSA in PBS / NaN3. (Determining the antibody concentration to use.) Seal round bottomed (inhibitor ) plate and incubate for 60' on shaker in cold room.
After 60 minutes
3. Remove Antigen from the target plate for re-use. Wash plate 1 x with PBS/NaN3.
4. Block remaining sites on the target plate with 0.5% BSA in PBS/ NaN3 for 30 minutes at room temperature ( or 10 minutes at 37 oC).
5. Wash the target plate twice with PBS / NaN3 .
When the target plate is ready
6. Transfer 50 µl from each well of the inhibitor plate to duplicate wells on the target plate. Seal target plate and incubate for 60' on a shaker in cold room.
7. Wash target plate 3 x PBS / NaN3 and add 50µl /well of Alkaline - Phosphatase coupled anti-mouse IgG or appropriate species diluted 1 : 1000 (Sigma ). Seal target plate and incubate for 60' on shaker in cold room. Whilst waiting for this take the substrate (Sigma 104 phosphatase substrate) from the freezer and warm to room temperaturebefore opening.
8. Make up enough substrate for 100 µl to be added to all the wells of the target plate by dissolving 50 mg of substrate in 10 ml of Diethanolamine buffer pH 9.8 .
9. Wash the plate 3 x PBS / NaN3 and add 100µl of substrate to each well. Put the plate at 37 oC. In a standard CD 4 assay , colour should develop in 30 - 60 min.
10. Read at 405 nm. The maximum absorbence for linear
readings is 2. In a standard CD 4 assay 50% inhibition is achieved with 50 ng CD 4 / ml.
M. H. B. MRC C. I.R.U. Dec. 1992
Update Jan. 1996. RVH