Development of Simple Immunochemical Screening Methods for Psychotropic Drugs and Its Application to Forensic Toxicology

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16590542
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Legal medicine
• Research Institution: Kumamoto University
• Principal Investigator: TSUNENARI Shigeyuki Kumamoto University, Graduate School of Medical Sciences, Professor, 大学院・医学薬学研究部, 教授 (80040202)
• Co-Investigator(Kenkyū-buntansha): YONEMITSU Kosei Kumamoto University, Graduate School of Medical Sciences, Associate Professor, 大学院・医学薬学研究部, 助教授 (10128332) ; KOREEDA Ako Kumamoto University, Graduate School of Medical Sciences, Assistant Professor, 大学院・医学薬学研究部, 助手 (80284751)
• Project Period (FY): 2004 – 2005
• Keywords: psychotropic drug / drug screening / fluvoxamine / forensic toxicology / dot-ELISA

Research Abstract

We have tried to develop a simple immunochemical screening method to detect three drugs, which are important in the field of forensic and clinical toxicology. Those drugs are, 1) fluvoxamine (FLV), a selective serotonin reuptake inhibitor ; SSRI, 2) milnacipran (MLP), a serotonin noradrenaline reuptake inhibitor ; SNRI and 3) phenothiazines (PTZ, an antipsychotic drug).
1. Preparation of antigens for immunization
As the three drugs themselves have little antigenicity, each drug was coupled with BSA. Mice were immunized with the BSA-drug conjugates as an antigen. The antisera obtained from the mice immunized with BSA-FLV reacted specifically to FLV, which revealed the production of anti-FLV antibody. On the other hand, BSA conjugates of MLP or PTZ did not induce their antibodies. Optimal method to make antigen for MLP or PTZ is now under reconsideration.
2. Preparation of anti-FLV monoclonal antibody (MF124)
Hybridoma was created by using myeloma and splenic cells from BSA-FLV immunized mouse. The cell strain producing anti-FLV monoclonal antibody (MF124) was selected by ELISA and cultured in a serum free medium. MF124 was purified from the medium chromatographically.
3. A simple FLV screening method
A new dot-ELISA system for FLV screening was established by using MF124 and DAKO EnVision+^【○!R】 (EV). MF124 and EV were mixed in a tube to prepare MF124-EV complex solution (MF124-EV). Sample solution was applied to MF124-EV and standed at room temperature for 30 min. Next, a detection membrane coated with BSA-FLV was immersed in the mixture and then transferred to substrate solution. If the sample is FLV negative, the detection membrane will be stained reddish brown. On the other hand, the membrane will not be stained when the sample is FLV positive. The detection limit of FLU was 1μg/ml.