Bever Candace R S, Majkova Zuzana, Radhakrishnan Rajeswaran, Suni Ian, McCoy Mark, Wang Yanru, Dechant Julie, Gee Shirley, Hammock Bruce D
Department of Entomology and Nematology, University of California-Davis , Davis, California 95616, United States.
Anal Chem. 2014 Aug 5;86(15):7875-82. doi: 10.1021/ac501807j. Epub 2014 Jul 9.
An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies.
已开发出一种基于抗体的分析方法，用于检测一种化学阻燃剂，该方法使用从羊驼分离的抗体片段。一种特定的化学阻燃剂同系物，2,2',4,4'-四溴二苯醚（BDE-47），通常是人类和环境样品中主要的多溴二苯醚（PBDE）同系物，也是最常检测到的同系物。用共价连接到载体蛋白上的BDE-47替代物免疫羊驼。分离出编码重链抗体可变区（VHH）的mRNA，转录成cDNA，并克隆到噬菌粒载体中用于构建噬菌体展示文库。通过在精心改良的条件下淘选实现对识别BDE-47的VHH的筛选。检测BDE-47的分析灵敏度低至十亿分之一（微克/升）水平。交叉反应分析证实该方法对BDE-47和选定的羟基化代谢物具有高度选择性。当暴露于高温时，骆驼科动物VHH抗体比针对相同目标分析物产生的多克隆抗体保留更多的反应活性。将这种固定在金电极上用于阻抗生物传感的VHH抗体试剂的使用证明了VHH抗体具有更高的通用性。
