ELISA for the Detection of the Prohibited Doping Agent Higenamine

Higenamine is a natural benzyltetrahydroisoquinoline alkaloid produced by various plants. In the World Anti-Doping Agency report of 2020, higenamine is classified as a class S3 (selective and nonselective β2-agonist) prohibited substance. To minimize the problems resulting from the misuse of higenamine-containing products as well as from the abuse of doping agents in sport, numerous higenamine-detection methods have been investigated. In the present study, a monoclonal antibody against the (S)-enantiomer of higenamine was successfully produced and applied in the indirect competitive ELISA to detect the content of (S)-higenamine in plant samples and related products. By immunizing BALB/c mice with higenamine-BSA, the aforementioned monoclonal antibody was produced even when the hapten number, which was the higenamine molecules conjugated to the BSA molecule, was relatively low (approximately 4). The MAb was characterized and utilized in the established icELISA assay with a detectable range of 7.81 - 125 ng/mL. The assay limit of detection (LOD) was 4.41 ng/mL, indicating higher sensitivity than the conventional HPLC-UV methods. Various validation processes demonstrated that icELISA was precise, with the maximum CV (%) of the intra- and inter-assays of 11.58% and 10.18%, respectively. Moreover, the assay was accurate, with the recovery rates of spiked (S)-higenamine ranging from 82% to 113%, and sufficiently reliable for the detection of (S)-higenamine in various kinds of samples. Notably, the present study describes the first immunoassay for (S)-higenamine.