Preparation and Characterization of Two Immunogens and Production of Polyclonal Antibody with High Affinity and Specificity for Darunavir.

Darunavir (DRV) is a potent antiviral drug used for treatment of infections with human immunodeficiency virus (HIV). Effective and safe treatment with DRV requires its therapeutic drug monitoring (TDM) in patient's plasma during therapy. To support TDM of DRV, a specific antibody with high affinity is required in order to develop a sensitive immunoassay for the accurate determination of DRV in plasma. In this study, two new and different immunogens were prepared and characterized. These immunogens were the DRV conjugates with keyhole limpet hemocyanin (KLH) protein. The first immunogen (DRV-KLH) was prepared by zero-length direct linking of DRV via its aromatic amino group with the tyrosine amino acid residues of KLH by diazotization/coupling reaction. The second immunogen (G-DRV-KLH) was prepared by conjugation of the N-glutaryl derivative of DRV (G-DRV) with KLH. The 5-carbon atoms-spacing G-DRV hapten was synthesized by reaction of DRV via its aromatic amino group with glutaric anhydride. The reaction was monitored by HPLC and the chemical structure of G-DRV was confirmed by mass,