Production of Anti-Opioid Drug Antibodies in HEK293 via All-In-One Vector system

Abstract

Fentanyl and morphine are powerful opioids widely used to alleviate severe pain; however, overdose can induce a paradoxical state characterized by maintained consciousness alongside analgesia, anxiolysis, euphoria and sedation. This effect is a key reason why certain opioids are classified as controlled substances. Current drug detection methods involve sample pretreatment, such as with urine or hair, followed by sophisticated analytical techniques. Among these, antibody-based methods are particularly favored in diagnostic applications due to their high sensitivity and specificity, enabling the detection of very low drug concentrations. Antibody expression is typically achieved using mammalian or microbial expression systems. Mammalian cell systems, while producing antibodies with correct folding and post-translational modifications, are associated with higher costs and slower production rates. Conversely, microbial systems offer a faster and cost-effective alternative but are often hindered by protein folding issues. In this study, we chose a mammalian cell-based expression system using Human embryonic kidney (HEK293) cells to ensure stable production. To overcome the issue of low yield in mammalian systems, we employed Kozak sequences and an all-in-one vector system for antibody expression. Our approach resulted in successful antibody production, as confirmed by SDS-PAGE, yieding approximately 8.0 mg/L. Further analysis using Indirect ELISA and Competitive ELISA demonstrated antigen-specific antibody activity within the range of 0.1 nM to 1 ？M, with a limit of detection (LOD) approximately 1 nM. These findings provide the future development of a highly sensitive and selectivity antibody-based sensor for opiod detection.