Leonard Paul, Säfsten Pär, Hearty Stephen, McDonnell Barry, Finlay William, O'Kennedy Richard
Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
J Immunol Methods. 2007 Jun 30;323(2):172-9. doi: 10.1016/j.jim.2007.04.010. Epub 2007 May 15.
Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day.
分子进化策略的进展使得识别具有高度特异性的抗体成为可能，并且还能针对几乎任何特定应用对其生物物理特性进行微调。根据所需功能，抗体/抗原相互作用可以是长寿命的或短寿命的，因此，在寻求识别具有特定反应速率和亲和力特性的抗体时需要特别注意。表面等离子体共振（SPR）生物传感器通常能从抗体/抗原相互作用中生成用于治疗和诊断应用的灵敏且可靠的动力学数据。然而，许多基于动力学的筛选测定在分析之前需要进行严格的样品制备和纯化。为改善这个问题，我们开发了一种直接从粗细菌裂解物中表征重组单链抗体片段（scFv）的快速且可靠的测定方法。通过噬菌体展示从用C反应蛋白（CRP）免疫的鸡中筛选出96种scFv抗体，并使用Biacore A100蛋白质相互作用阵列系统进行评估。抗体从传感器芯片表面的粗细菌提取物中捕获，并根据在缓冲液中解离后剩余复合物的百分比（% left）进行排序。获得了六个在广泛亲和力范围（2.54×10⁻⁸至3.53×10⁻¹⁰ M）内结合单体CRP的克隆的动力学速率常数（k(a)和k(d)）以及亲和力（K(D)）数据。使用这种测定形式，A100生物传感器产生了高质量的动力学数据，每天可筛选近400个抗体克隆。
