Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
Kayyali Chair for Pharmaceutical Industries, Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia.
Molecules. 2020 Sep 7;25(18):4075. doi: 10.3390/molecules25184075.
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, H-NMR, and C-NMR spectroscopic techniques. The hapten (G-DRV) was linked to the KLH protein by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling procedure. The pertinence of the coupling reactions of haptens to protein was confirmed, and the immunogens were characterized by ultraviolet (UV) spectrophotometry. Both DRV-KLH and G-DRV-KLH were used for the immunization of animals and the animal's antiserum that showed the highest affinity was selected. The collected antiserum (polyclonal antibody) had very high affinity to DRV (IC value = 0.2 ng mL; defining IC as the DRV concentration that can inhibit antibody binding by 50% of its maximum binding) and high specificity to DRV among other drugs used in the combination therapy with DRV. Cumulative results from direct and competitive enzyme-linked immunosorbent assay (ELISA) using this polyclonal antibody proved that the immunogens were highly antigenic and elicited a specific polyclonal antibody. The produced polyclonal antibody is valuable for the development of highly sensitive and selective immunoassays for TDM of DRV.
达芦那韦 (DRV) 是一种强效的抗病毒药物，用于治疗人类免疫缺陷病毒 (HIV) 感染。DRV 的有效和安全治疗需要在治疗期间对患者的血浆进行治疗药物监测 (TDM)。为了支持 DRV 的 TDM，需要具有高亲和力的特异性抗体，以便开发用于准确测定血浆中 DRV 的灵敏免疫分析。在这项研究中，制备并表征了两种新型和不同的免疫原。这些免疫原是与血蓝蛋白 (KLH) 蛋白偶联的 DRV 衍生物。第一个免疫原 (DRV-KLH) 通过 DRV 芳香族氨基与 KLH 中酪氨酸残基的零长度直接连接，通过重氮化/偶联反应制备。第二个免疫原 (G-DRV-KLH) 通过 DRV 的 N-谷氨酰衍生物 (G-DRV) 与 KLH 偶联制备。通过 DRV 芳香族氨基与戊二酰酐的反应合成了具有 5 个碳原子间距的 G-DRV 半抗原。反应通过 HPLC 进行监测，并通过质量、H-NMR 和 C-NMR 光谱技术确认 G-DRV 的化学结构。半抗原 (G-DRV) 通过水溶性 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺 (EDC) 偶联程序与 KLH 蛋白连接。确认了半抗原与蛋白质的偶联反应的相关性，并通过紫外 (UV) 分光光度法对免疫原进行了表征。DRV-KLH 和 G-DRV-KLH 均用于动物免疫，选择亲和力最高的动物抗血清。收集的抗血清（多克隆抗体）对 DRV 具有非常高的亲和力（IC 值=0.2ng/mL；将 IC 定义为可抑制 50%最大结合的 DRV 浓度），并且在与 DRV 联合治疗中使用的其他药物中具有很高的特异性。使用该多克隆抗体的直接和竞争性酶联免疫吸附测定 (ELISA) 的累积结果证明免疫原具有高度抗原性，并引起特异性多克隆抗体。所产生的多克隆抗体对于开发用于 DRV TDM 的高度敏感和选择性免疫分析具有重要价值。
Zotero 插件
