Identifying Rhizobia by the Indirect Enzyme-Linked Immunosorbent Assay

Abstract

The enzyme-linked immunosorbent assay (ELISA) is a colorimetric enzyme immunoassay used to detect antigens. Several other variations of the assay have been developed. An indirect method is described here that does not require the conjugation of the primary (rabbit) antibody with an enzyme. In this procedure, the antigen is immobilized in the wells of polystyrene microtiter plates and is reacted with specific (primary) antibodies. After a washing step, the primary antibodies bound to the antigens in the wells are reacted with alkaline phosphatase conjugated to secondary antibodies that bind specifically to the primary antibodies. Reaction with an enzyme substrate causes a color change, the intensity of which corresponds to the amount of primary antibodies present.