Kikuchi Yasufumi, Uno Shinsuke, Nanami Masahiko, Yoshimura Yasushi, Iida Shin-ichiro, Fukushima Naoshi, Tsuchiya Masayuki
Chugai Pharmaceutical Co. Ltd., Fuji-Gotemba Research Laboratories, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.
J Biosci Bioeng. 2005 Sep;100(3):311-7. doi: 10.1263/jbb.100.311.
An assay method using a surface plasmon resonance (SPR) biosensor has been developed that allows quantitative measurement of the specific antibody concentration in crude materials. By injecting non-labeled antibody samples onto a biosensor surface on which antigen was immobilized at high densities, the concentration of active antibodies can be accurately measured. To clarify applicability of this method to pharmacokinetic studies, the concentration of active antibodies in mouse plasma was measured for 4 h after injection of antibodies in mice. Although this period of measurement might be insufficient for determining the pharmacokinetics of blood pool clearance, this method has some advantages over conventional methods in measurement of single-chain antibody fragment (scFv) concentrations. Using the SPR biosensor, scFv and antibodies without epitope tag peptides were easily detected in real time, requiring as little as 20 mul of blood sample. Moreover, from the apparent dissociation rate in the dissociation phase of the sensorgrams, we could identify whether the antibody fragments existed as bivalent or monovalent in animal blood. We also evaluated the antigen binding activity of the scFvs against human CD47 and found scFvs had slightly weak affinity to their antigen (K(D), about 10 nM) compared with F(ab')2 and Fab' fragments (K(D), about 3-4 nM). This assay method promises to be a convenient tool for quality control, screening, and simple pharmacokinetic analysis of antibody fragments and other recombinant proteins not having epitope tags.
已经开发出一种使用表面等离子体共振（SPR）生物传感器的检测方法，该方法能够对粗制品中的特异性抗体浓度进行定量测量。通过将未标记的抗体样品注射到高密度固定有抗原的生物传感器表面上，可以准确测量活性抗体的浓度。为了阐明该方法在药代动力学研究中的适用性，在给小鼠注射抗体后4小时测量了小鼠血浆中活性抗体的浓度。尽管这段测量时间可能不足以确定血池清除的药代动力学，但在测量单链抗体片段（scFv）浓度方面，该方法比传统方法具有一些优势。使用SPR生物传感器，可以轻松实时检测无表位标签肽的scFv和抗体，所需血样量低至20微升。此外，根据传感器图解离阶段的表观解离速率，我们可以确定抗体片段在动物血液中是以二价还是单价形式存在。我们还评估了scFv对人CD47的抗原结合活性，发现与F(ab')2和Fab'片段（K(D)约为3-4 nM）相比，scFv与它们的抗原的亲和力略弱（K(D)约为10 nM）。这种检测方法有望成为一种方便工具，用于抗体片段和其他无表位标签的重组蛋白的质量控制、筛选以及简单的药代动力学分析。
