Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Anal Chem. 2011 May 15;83(10):3717-24. doi: 10.1021/ac200159x. Epub 2011 Apr 20.
Recombinant single-chain variable fragment antibodies (scFv) were specifically generated and selected for the measurement of environmental uranium with an antibody-based sensor. These sFvs, which recognized UO(2)(2+) complexed to 2,9-dicarboxyl-1,10-phenanthroline-acid (DCP), were produced using genetic material obtained from the spleen cells of rabbits immunized with UO(2)(2+)-DCP conjugated to keyhole limpet hemocyanin. Immunoglobulin light chain and heavy chain genes were amplified and cloned into the phagemid pSD3 for generation of a recombinant antibody library and phage-displayed antibodies. The screening process was designed to isolate antibodies that bound to a "loaded" noncovalent complex with high affinity, while selecting against binding to an "unloaded" complex. After five rounds of panning, individual positive scFv clones were used to infect E. coli TG1 and soluble scFv antibodies were purified and characterized. Binding studies showed that the best scFv bound tightly to the UO(2)(2+)-DCP complex (K(d), 19.6 nM). However, because of the depletion experiments performed on this library during the panning process, this scFv bound 1200-fold less tightly (K(d), 23.5 μM) to metal-free DCP. This scFv (clone 3A) was subsequently used to accurately determine the UO(2)(2+) concentrations in environmental water samples using a sensor based on kinetic exclusion analysis. The present studies demonstrate that recombinant scFvs with properties engineered for specific applications (i.e., biosensor-based measurement of metals in groundwater) can be prepared if the correct genetic material and techniques are employed. The phage display system permitted the generation of proteins with very specific binding properties (in this case, high affinity for a metal-chelate complex and low affinity for metal-free chelator). The recombinant scFvs isolated in these studies will be the basis for rapid and affordable assays for the detection of residual uranium in environmental water samples.
针对基于抗体的传感器对环境铀进行测量，专门生成和选择了重组单链可变片段抗体 (scFv)。这些 scFv 识别与 2,9-二羧酸-1,10-菲啰啉酸（DCP）络合的 UO2(2+)，是使用从小兔脾脏细胞中获得的遗传物质产生的，这些细胞经 UO2(2+)-DCP 与血蓝蛋白缀合后进行了免疫。免疫球蛋白轻链和重链基因被扩增并克隆到噬菌粒 pSD3 中，以生成重组抗体文库和噬菌体展示抗体。筛选过程旨在分离与高亲和力结合的“负载”非共价复合物的抗体，同时选择不与“空载”复合物结合的抗体。经过五轮淘选，单个阳性 scFv 克隆被用于感染 E. coli TG1，可溶性 scFv 抗体被纯化并进行了表征。结合研究表明，最佳 scFv 与 UO2(2+)-DCP 复合物紧密结合（Kd，19.6 nM）。然而，由于在淘选过程中对该文库进行了耗尽实验，该 scFv 与无金属的 DCP 的结合力弱 1200 倍（Kd，23.5 μM）。随后，使用基于动力学排除分析的传感器，该 scFv（克隆 3A）被用于准确测定环境水样中的 UO2(2+)浓度。本研究表明，如果使用正确的遗传物质和技术，具有针对特定应用（即基于传感器的地下水金属测量）设计的特性的重组 scFv 可以制备。噬菌体展示系统允许生成具有非常特定结合特性的蛋白质（在这种情况下，对金属螯合物复合物具有高亲和力，对无金属螯合剂具有低亲和力）。本研究中分离的重组 scFv 将成为快速、经济的环境水样中残留铀检测方法的基础。
