Editing Elisa protocol (section)

=== Coating antigen to microplate ===

Dilute the antigen to a final concentration of 20 µg/ml in PBS or other carbonate buffer. Coat the wells of a PVC microtiter plate with the antigen by pipetting 50 µl of the antigen dilution in the top wells of the plate. Dilute down the plate as required.

<br/>Test samples containing pure antigen are usually pipetted onto the plate at less than 2 µg/ml. Pure solutions are not essential, but as a guideline, over 3% of the protein in the test sample should be the target protein (antigen). Antigen protein concentration should not be over 20 µg/ml as this will saturate most of the available sites on the microtiter plate.

Ensure the samples contain the antigen at a concentration that is within the detection range of the antibody.

Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature, or 4°C overnight. The coating incubation time may require some optimization.<br/>Remove the coating solution and wash the plate three times by filling the wells with 200 µl PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.