Protocols
ELISA
Description
The most suitable primary screening assay for soluble antigen is the enzyme-linked immuno-absorbant assay (ELISA). Soluble antigen is bound to the wells of a 96-well microtitre plate. Tissue culture supernatants are then incubated with this bound antigen and any antibodies present that are antigen specific will bind to the well in the plate. An enzyme linked secondary antibody with specificity for the monoclonal immunoglobulins is used to detect wells where the primary antibody has bound. This binding is then visualised by the addition of an appropriate enzyme substrate that, after reaction with the enzyme, yields a coloured product.
Schematic diagram of a completed ELISA.
The purple wells represent antigen/antibody complexes formed by the binding of specific antibodies to antigen present in the well, C+ marks the position of the positive control well.
Method
Preparation of ELISA plate
- Add 50μl of antigen (diluted 5μg/1ml of distilled water) to a 96 well microtite plate (Nunc Medisorp plates 467320).
- This microtitre plate must be a high protein-binding capacity polystyrene ELISA plate
- Cover the microtitre plate(s) and incubate for 1 hour at room temperature or 30 minutes at 37ºC.
- Tip out the antigen solution and blot the plate(s) on tissues.
- Wash the ELISA plate(s) 3 times with PBS+0.1%Tween 20 (PBS/Tween).
- Blot the plate(s) on tissues.
- Add 200μl of PBS containing 2% dry milk powder to each well of the ELISA plates(s). This blocks any free protein binding sites present in the wells.
- Cover and leave the plates overnight at 4ºC.
- Next day, wash the ELISA plate(s) three times in PBS/Tween.
- Blot the plate(s) on tissues.
- At this stage, the plates are ready for use or can be wrapped in aluminum foil and stored at minus 20ºC for up to one year.
Performing the ELISA
- On the day, remove the ELISA plate(s) from the freezer and allow to warm to room temperature before removing the foil.
- Add 50μl of each supernatant that you wish to test to each well.
- Controls should consist of 50μl of the last test mouse serum diluted 1:100 and 1:200 in PBS as a positive control while the negative controls should be 50μl of PBS or an unrelated antibody.
- Leave the plate(s) for 60 minutes at room temperature or 30 minutes at 37ºC.
- Wash 3 times as before in PBS/Tween.
- Dilute the goat anti-mouse conjugated to horseradish peroxidase (DAKO P 0447) 1:1000 in PBS. Add 50 μl of goat anti mouse to each of your ELISA wells.
- Leave the ELISA plate(s) for 60 minutes at room temperature or 30 minutes at 37oC.
- Wash three times with PBS/Tween.
- Wash further three times using distilled water only. (The presence of Tween will prevent the substrate from working).
- Add 50 μl of the substrate (ABTS Solution Roche Diagnostics 1684 302) to each well.
- Leave the substrate to develop for 15 minutes to 60 minutes and evaluate the results.