An ELISA for the determination of salicylic acid in plants using a monoclonal antibody

Abstract A mouse monoclonal antibody (McAb) against salicylic acid (SA) was produced and characterized for the first time. The McAb, based on the study of immuno-response of rabbit to immunogens conjugated SA to carrier proteins through different methods, and effect of changing conjugation bridge between hapten and carrier proteins on the specialty of salicylate polyclonal antibodies (PAbs), was derived from an immunogen in which 5-aminosalicylic acid (5-ASA) was conjugated to KLH (hemocyanin from keyhole limpet) through its (C 5 )-NH 2 group. The antibody shows high specificity to SA and even more high specificity to 5-ASA (the cross-reaction is 144.8%), but show very little cross-reactivity with 5-sulfosalicylic acid (0.9%) and 4-aminosalicylic acid (0.3%). Other compounds structurally similar to SA, such as 4-hydroxybenzoic acid, 4-aminobenzoic acid and salicylaldoxime, were not found to exhibit any cross-reactivity. Based on this antibody, a direct enzyme-linked immunosorbent assay (ELISA) with high sensitivity and precision was developed which displays a linear range from 0.0195 to 20 nmol of SA. Using this ELISA, the fluctuation of endogenous SA levels of cucumber after infected with Pseudomonas syringae pv. syringae were examined.