School of Life Sciences, Liaoning Normal University, Dalian, Liaoning Province, People's Republic of China.
Department of Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, Liaoning Province, People's Republic of China.
PLoS Negl Trop Dis. 2023 Aug 17;17(8):e0011568. doi: 10.1371/journal.pntd.0011568. eCollection 2023 Aug.
rLj-RGD3, a new member of the RGD (Arginine-Glycine-Aspartate)-motif toxin protein family obtained from Lampetra japonica by means of recombinant DNA techniques, has been demonstrated to be a platelet fibrinogen receptor antagonist and holds potential as a drug candidate for a specific indication. The present article reports an innovative validated highly sensitive and specific biotin-avidin enzyme linked immunosorbent assay (BA-ELISA) to provide a bio-analytical method for pharmacokinetic (PK) studies of rLj-RGD3. The concentration of picogram level rLj-RGD3 in rat plasma was measured using the developed double sandwich BA-ELISA assay, which used two mouse anti-rLj-RGD3 monoclonal antibodies that recognize different epitopes for capture and detection. This method was verified to be highly specific (blank plasma did not interfere with detection), precise (RSD <15%), and accurate (86%-113%). Absolute recovery was in the 94%-119% range. The calibration curve showed good linearity within the 50 to 1600 pg/mL range. The LOQ was as low as 50 pg/mL. The above validated assay was successfully employed to assess PK of rLj-RGD3 in rats. After i.v. and s.c. dosing with 30 μg/kg, the rLj-RGD3 plasma concentration declined bi-exponentially with time. This decay was best fitted to a two-compartment model. In conclusion, the BA-ELISA method described here meets all requirements for PK studies of rLj-RGD3 with an effective pharmacological dose in the μg/kg BW range.
rLj-RGD3 是一种新的 RGD（精氨酸-甘氨酸-天冬氨酸）基 motif 毒素蛋白家族成员，通过重组 DNA 技术从日本七鳃鳗中获得，已被证明是血小板纤维蛋白原受体拮抗剂，并有可能成为特定适应症的药物候选物。本文报道了一种创新的、经过验证的高度敏感和特异性生物素-亲和素酶联免疫吸附测定法（BA-ELISA），为 rLj-RGD3 的药代动力学（PK）研究提供了一种生物分析方法。使用开发的双夹心 BA-ELISA 测定法，以识别不同表位用于捕获和检测的两种抗 rLj-RGD3 单克隆抗体，可测量大鼠血浆中皮克级水平的 rLj-RGD3 浓度。该方法被证明具有高度特异性（空白血浆不干扰检测）、精确性（RSD<15%）和准确性（86%-113%）。绝对回收率在 94%-119%范围内。校准曲线在 50 至 1600 pg/mL 范围内显示出良好的线性。LOQ 低至 50 pg/mL。经验证的测定法成功用于评估 rLj-RGD3 在大鼠中的 PK。静脉注射和皮下注射 30 μg/kg 后，rLj-RGD3 血浆浓度随时间呈双指数下降。这种衰减最适合二房室模型。总之，这里描述的 BA-ELISA 方法符合 rLj-RGD3 在μg/kg BW 范围内有效治疗剂量的 PK 研究的所有要求。
