Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys
Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys
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DOI：
10.1016/j.jpba.2010.07.040
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发表时间：
2011-01-05
影响因子：
3.4
通讯作者：
Cheng, Yuanguo
中科院分区：
文献类型：
--
作者：
Chen, Zhihang;Liu, Ruorui;Cheng, Yuanguo
建立了一种灵敏、准确、精确的酶免疫分析法（EIA），用于恒河猴血清中完整人B7.1-Fc的定量，并利用表面等离子体共振（SPR）和流式细胞术分别鉴定了融合蛋白的87.1和Fc片段的特征。B7.1-Fc分别与CD28和CTLA-4结合，K-d值分别为45.1和9.58 nM，与文献报道非常接近，Fc受体结合实验和IL-8释放实验也证实了融合蛋白Fc部分的功能。为了监测完整蛋白，EIA方法采用夹心结构，以多克隆抗人IgG （Fc特异性）抗体和单克隆抗人B7.1抗体分别作为捕获抗体和检测抗体。该EIA具有0.5-32 ng/ml的可靠响应范围。定量限为0.5 ng/ml。精密度和准确度分别为6.1 ~ 8.8%和（3.0 ~ 9.0）%，精密度和准确度分别为5.7 ~ 11.5%和（10.7 ~ 9.1）%。在一定条件下建立了稳定性，没有发现显著差异。该方法成功地应用于B7.1-Fc在恒河猴体内静脉滴注后的药代动力学行为评估，并在研究剂量范围（32 ~ 320 μ g/kg）内建立了非线性特征。(C) 2010 Elsevier B.V.版权所有
A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of 87.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K-d values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal antihuman B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 mu g/kg). (C) 2010 Elsevier B.V. All rights reserved.