ELISA Basic Principle - Berthold Technologies GmbH & Co.KG

Step 1 – coating

ELISA begins with a coating step where the ELISA plate (typically either a 96- or 384-well plate) is treated with a coating solution of either antigen or antibody. The antigen or antibody is absorbed to the well-surface of the ELISA plate, creating the first layer. Then the coating buffer is removed, followed by a series of washes to remove unbound antigen or antibody.

Washes are repeated between each ELISA step to remove unbound materials. Furthermore, it is essential that excess liquid is removed to avoid dilution of the solution added in the next assay step. We provide specialised microplate washers to ensure uniformity.

Step 2 - blocking

In the second step, all free binding sites are blocked using a buffer containing unrelated proteins. This step is important to eliminate a potential source of false positive results. Blocking buffer removal is again followed by a series of washing steps.

Step 3 – detection

Detection is the most complex step of the ELISA procedure as multiple layers of antibodies can be used to amplify the signal. The plate gets incubated with a solution containing enzyme-conjugated detection antibody that binds specifically to the target antigen or antibody. This is followed by buffer removal and another series of washes to remove all unbound antibodies.

Step 4 – Substrate addition & analysis

In the final step a colorimetric substrate is added to the wells that forms a coloured solution when catalysed by the enzyme. The result is read using an ELISA reader or microplate reader.