A biotin-avidin-based enzyme immunoassay to quantify three phytohormones: auxin, abscisic acid and zeatin-riboside

Avidin-biotin complexes have been used in an ELISA assay of three phytohormones: auxin (IAA), abscisic acid (ABA) and zeatin-riboside (ZR). Anti-hormone antibodies were elicited in rabbits immunized with hormone-bovine serum albumin conjugates. Microtitration plates coated with antigen protein were used in the ELISA method. The competition between plate-bound and free (standards or samples) antigen for a limiting amount of specific antibody results in a variable level of antibody adsorbed to the wells after washes. Such antibodies were labelled in two steps: first, incubation with biotinylated goat anti-rabbit IgG and, second, incubation with avidin-alkaline phosphatase conjugate. Bound ezyme activity was measured spectrophotometrically with p-nitrophenylphosphate and standard curves and ZR. The detection limit was 3–5 pg of plant hormone and the working range was between 10–1000 pg. This compares favourably with the best systems of analysis described in the literature. To avoid unwanted cross-reactions, a rapid and efficient HPLC purification step was included prior to measuring IAA, ABA and ZR in the same plant extract. About 100 mg fresh weight of a tomato sample could be analyzed by this technique.