Topic 19.3

Auxin Measurement by Radioimmunoassay

A radioimmunoassay (RIA) allows the measurement of physiological levels (10^–9 g = 1 ng) of IAA in plant tissues. An RIA (Web Figure 19.3.A), requires two chemicals: (1) specific antibodies that recognize IAA; and (2) a radioactively labeled IAA in which several hydrogen atoms have been replaced by radioactive tritium. Because of the sensitivity and selectivity of the antibodies used in this technique, RIA is as sensitive as some of the best physicochemical methods (Caruso et al. 1995). Radioimmunoassays also allow one to quantify a specific auxin. In a modified RIA, the radiolabeled IAA is replaced by an auxin conjugated to an enzyme, generally an alkaline phosphatase. This enzyme-linked immunosorbent assay (ELISA) affords sensitivities that are similar or better than the RIA.

Web Figure 19.3.A Radioimmunoassay for the amount of auxin in a plant tissue extract. In the preparation of auxin antibodies, IAA is first conjugated to a large protein and injected into a mouse or rabbit. The animal produces antibodies to all of the antigenic determinants on the protein, including the IAA. The anti-IAA antibodies can then be purified for use in the RIA. Unlabeled antigen (IAA) competes with a known amount of radioactively labeled antigen—e.g., [¹⁴C]IAA—for binding to antibodies. The more unlabeled antigen present, the less radioactivity from the radioactive antigen will be found in the antibody precipitate. (Click image to enlarge.)