Bumbaca Daniela, Tong Raymond K, Koch Alexander W, Xiang Hong, DeForge Laura E, Shen Ben-Quan, Lu Yanmei
Department of Pharmacokinetic & Pharmacodynamic Sciences, Genentech, Inc., CA, USA.
Bioanalysis. 2012 Mar;4(6):703-11. doi: 10.4155/bio.12.30.
In evaluating the serum concentrations in mice of a Sema3E IgG1 Fc fusion protein, a possible antitumor agent, two ELISAs were developed: a generic assay detecting only the Fc portion of the therapeutic and a specific receptor-binding assay detecting intact protein.
An unexpected discrepancy was observed in the measured in vivo serum concentrations, with the generic ELISA yielding higher concentrations than the specific ELISA. Size-exclusion HPLC and SDS-PAGE analysis of in vitro serum stability samples revealed extensive aggregation of Sema3E-Fc. The generic assay recovered more Sema3E-Fc in the presence of aggregates than the specific assay.
Biophysical characterization combined with immunochemical analysis was key to elucidating not only the nature of the protein instability, but also the cause for the assay discrepancy.
在评估一种可能的抗肿瘤药物Sema3E IgG1 Fc融合蛋白在小鼠体内的血清浓度时，开发了两种酶联免疫吸附测定法（ELISA）：一种通用测定法仅检测治疗性蛋白的Fc部分，另一种特异性受体结合测定法检测完整蛋白。
在体内血清浓度测量中观察到意外差异，通用ELISA测得的浓度高于特异性ELISA。对体外血清稳定性样本进行的尺寸排阻高效液相色谱法（HPLC）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示，Sema3E-Fc发生了广泛聚集。在存在聚集物的情况下，通用测定法比特异性测定法回收的Sema3E-Fc更多。
生物物理表征与免疫化学分析相结合不仅是阐明蛋白质不稳定性本质的关键，也是解释测定差异原因的关键。
