Comparison of different enzyme immunoassays for measuring indole‐3‐acetic acid in vegetative citrus tissues

An enzyme‐linked immunosorbent assay (ELISA) using polyclonal antibodies, which were raised against indole‐3‐acetic acid (IAA) conjugated to bovine serum albumin (BSA) via the indolic nitrogen (IAA‐N₁‐BSA), has been developed. The sensitivity and specificity of these antibodies were compared to those of polyclonal and monoclonal antibodies raised against IAA conjugated to BSA via C₁ of the carboxyl group (IAA‐C₁‐BSA). The sensitivity of the assays improved in the following order: monoclonal antibodies > antibodies to IAA‐C₁‐BSA > antibodies to IAA‐C₁‐BSA. Antibodies against IAA‐C₁‐BSA had less cross‐reactivity to indoles structurally related to IAA, excluding indole‐3‐pyruvic acid. A rapid and effective method for purification of IAA in citrus tissues before analysis by ELISA is described. Values of IAA in citrus (Citrus sinensis [L.] Osbeck cv. Shamouti orange) shoot tips obtained with all three antibodies were similar. However, in leaf tissues which contain lower amounts of IAA compared to shoot tips, monoclonal antibodies gave higher values of IAA than polyclonal antibodies. Estimation of free IAA levels in purified extracts of citrus shoot tips, very young leaves, and mature leaves was ca 380, 248, and 74 ng (g fresh weight)⁻¹ respectively.