Molecular Recognition Mechanism of an Anti-Amatoxins mAb and Its Application in Centrifugal Disk-Based Immunoassay

Amatoxins are polypeptides that cause 90% of fatalities from accidental ingestion of poisonous mushrooms. Unfortunately, there are no specific antidotes against amatoxins poisoning, hence preparation of high-affinity antibodies, understanding the receptor (amatoxins) and ligand (antibody) mechanism, and establishing a straightforward screening approach are of great significance for confirming poison agents and clinical diagnosis. Here, anti-amatoxins monoclonal antibody (mAb) 9B2 was prepared and the recognition mechanism was investigated. The approach is useful for designing desirable immunogens, developing new antibodies with improved performance, and constructing effective immunoassays. Based on the mAb, we designed a centrifugal disk-like microfluidics chip and developed a fully automated immunoassay capable of detecting amatoxins poisoning in various samples including serum, urine, and mushrooms. The whole detection process could be automatically accomplished within 30 min, with a limit of detection of 0.08 to 0.12 μg/L for real samples, ∼30-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). Our platform not only provided a practical approach for performing poison agent confirmation and clinical diagnosis but also had important implications for improving the survival of patients with mushroom poisoning.