Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375, USA.
Anal Chem. 2010 Sep 1;82(17):7202-7. doi: 10.1021/ac100961x.
Single domain antibodies are the recombinantly expressed binding fragments derived from heavy chain antibodies found in camels and llamas. These unique binding elements offer many desirable properties such as their small size ( approximately 15 kDa) and thermal stability, which makes them attractive alternatives to conventional monoclonal antibodies. We created a phage display library from llamas immunized with ricin toxoid and selected a number of single domain antibodies. Phage selected on ricin were found to bind to either ricin A chain or the intact molecule; no ricin B chain binders were identified. By panning on B chain, we identified binders and have characterized their binding to the ricin B chain. While they have a poorer affinity than the previously described A chain binders, it was found that they performed dramatically better as capture reagents for the detection of ricin, providing a limit of detection in enzyme linked immunosorbent assay (ELISA) below 100 pg/mL and excellent specificity for ricin versus the highly related RCA 120 (1 to 10 000). We also reevaluated the previously isolated antiricin single domain antibody binding kinetics using surface plasmon resonance and found their K(d)s matched closely to those previously obtained under equilibrium binding conditions measured using the Luminex flow cytometer.
单域抗体是从骆驼和羊驼中发现的重链抗体中重组表达的结合片段。这些独特的结合元件具有许多理想的特性，如它们的小尺寸（约 15 kDa）和热稳定性，这使得它们成为传统单克隆抗体的有吸引力的替代品。我们用蓖麻毒素免疫的羊驼创建了一个噬菌体展示文库，并筛选了一些单域抗体。在蓖麻毒素上筛选的噬菌体被发现与蓖麻毒素 A 链或完整分子结合；没有鉴定到蓖麻毒素 B 链结合物。通过对 B 链进行淘选，我们鉴定到了结合物，并对它们与蓖麻毒素 B 链的结合特性进行了表征。虽然它们的亲和力比之前描述的 A 链结合物差，但发现它们作为蓖麻毒素检测的捕获试剂性能要好得多，在酶联免疫吸附测定（ELISA）中的检测限低于 100 pg/mL，并且对蓖麻毒素具有极好的特异性，而对高度相关的 RCA 120（1 比 10 000）则没有交叉反应。我们还使用表面等离子体共振重新评估了之前分离的抗蓖麻毒素单域抗体的结合动力学，发现它们的 K(d)与之前使用 Luminex 流式细胞仪在平衡结合条件下测量的结果非常吻合。
