Homogeneous FRET immunoassays for use in personalized medicine

Abstract

IntroductionDrug replacement and maintenance therapy of methadone has been used all over the world in maintenance treatment programmes of opioid dependence. The effectiveness of drug maintenance therapy in a patient is highly depended on the drug levels in the patient's blood. A popular technique in drug monitoring is competitive immunoassay for example ELISA which has proven to be simpler and less expensive. However, multiple processing steps are still required to achieve the required sensitivity. In order to increase the efficiency for detection of drugs, homogeneous immunoassays that do not require any time-consuming washing and separation steps have an advantage. ObjectiveTo develop a one-step homogeneous FRET immunoassay against methadone for use in personalized medicine. MethodsFRET based homogeneous immunoassay against methadone was designed by using fluorescein isothiocyanide – Lissamine™ Rhodamine B Ethylenediamine as donor fluorophore and acceptor fluorophore. These organic dye molecules were then conjugated with the monoclonal antibodies which against methadone and the antigen (methadone). The measurement of the signal of this FRET assay was performed after the mixing of this donor-acceptor pair. Then, the percentage of energy transfer was calculated and compared. The optimum conditions and reproducibility for this assay was determined in this study. Results & DiscussionFRET based immunoassay that was developed from this study is highly reproducible. This method was proven to be a very rapid method for quantitative detection of methadone. ConclusionFRET based immunoassay can be used as suitable alternative for the monitoring of MMT serum levels.