Development of methyl ester antibody-based competitive indirect ELISA for quantitative detection of Mitragynine in human urine

• Main Authors: Mustafa, Radhiahtul Raehan, Sukor, Rashidah, Mohd. Nor, Siti Mariam, Saari, Nazamid, Mohsin, Aliah Zannierah
• Format: Article
• Language: English
• Published: American Chemical Society 2023
• Subjects: H Social Sciences (General)
• Online Access: http://eprints.utm.my/104866/1/RadhiahtulRaehanMustafa2023_DevelopmentofMethylEsterAntibodyBased.pdf; http://eprints.utm.my/104866/; http://dx.doi.org/10.1021/acsomega.3c02734
• Institution: Universiti Teknologi Malaysia

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH3 IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 µg/mL, respectively. The measurement range was between 0.01 and 100.0 µg/mL, with a minimal inhibition (IC50) value of 0.152 µg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.