Development of a class-specific competitive enzyme-linked immunosorbent assay for the detection of pyrrolizidine alkaloids in vitro

A two-step enzyme-linked immunosorbent assay (ELISA) was developed for the determination of the presence of retronecine in the parts per billion range. By modification of retronecine two haptens were synthesized. An I₅₀ value of 11 ± 3 ppb (Figure 2a) was obtained, with detection limits of 1.0-100 ppb. Indirect detection of monocrotaline (325 g/mol), retrorsine (351 g/mol), and senecionine (352 g/mol) was achieved by hydrolytic digestion of these naturally occurring pyrrolizidine alkaloids (PAs), demonstrating the utility of the assay as a class-specific detection method. PAs were detected in Senecio vulgaris and Crotalaria retusa plant sample extracts. No cross-reactivity was seen with either swainsonine or lupinine, both of which are structurally similar to retronecine in that they are also fused bicyclic alkaloids.