Preparation of a monoclonal antibody against obacunone and its application in obacunone-specific enzyme-linked immunosorbent assay (ELISA)

Abstract

Obacunone, a limonoid compound structurally related to limonin, is commonly found in plants of the Rutaceae family, including Phellodendron and Citrus species. Obacunone serves as a marker for assessing the quality of herbal medicines and traditional Chinese medicine (TCM) formulations. However, the current detection method for obacunone requires expensive equipment, such as high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), and lacks convenient and rapid detection alternatives. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for obacunone detection. The half-maximal inhibitory concentration (IC₅₀) for the icELISA was established at 0.44 ng mL⁻¹, with a detection range extending from 0.09 ng mL⁻¹ to 3.52 ng mL⁻¹. The spike-recovery experiments conducted demonstrated excellent method stability and reliability, with recovery rates consistently ranging from 94.6% to 105.5%, well within the acceptable criteria for analytical method validation in pharmaceutical analysis. The obacunone content in seven TCM formulations and two herbal medicine samples was analyzed using both icELISA and UPLC-MS, demonstrating consistent results between the two analytical methods. The results demonstrated that the developed immunoassay method exhibited excellent applicability for obacunone detection in both herbal medicines and TCM formulations, with significant advantages in terms of analytical efficiency and stability.