A highly sensitive and specific enzyme immunoassay for detection of β-amanitin in biological fluids

Polyclonal antisera to beta-amanitin were generated in sheep and used to construct a competitive ELISA for measurement of the toxin in human serum and urine. The assay had a detection limit of about 80 pg mL(-1), a dynamic range of 80-2,000 pg mL(-1), a cross reactivity of 22% with alpha-amanitin, and no cross reactivities with cyclic peptides from algal sources. Assay responses in buffer, serum, and urine were remarkably similar. Coupling of the toxin to carrier proteins was carried out by previously unreported methods. The key step that allowed the construction of the highly sensitive assay was the introduction of a novel heterologous hapten derivative made of beta-amanitin-cyanuric chloride derivative. The new derivative overcame the problems of bridge binding that was, in this case, particularly serious with the homologous hapten derivative. The study demonstrated that the developed antiserum and ELISA procedure can be used to detect beta-amanitin and related toxins from Amanita phalloides in human serum and urine samples from suspected poison cases and enable early treatment to be administered.