Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
Anal Biochem. 2011 Oct 15;417(2):188-94. doi: 10.1016/j.ab.2011.06.012. Epub 2011 Jun 16.
Naive libraries of single domain antibodies (sdAbs) enable rapid isolation of binders to nearly any target. These binders, however, lack the benefits bestowed by in vivo affinity maturation and typically have low affinity toward their targets. We expressed five low-affinity toxin binding sdAbs, previously selected from a naive library derived from variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive mutant Escherichia coli alkaline phosphatase (AP) and examined the impact on apparent affinity and utility. AP spontaneously dimerizes in solution, effectively dimerizing the fused sdAbs, imparting avidity in place of the lower affinity monomeric interactions. The sdAb-AP fusion also combines the target recognition domain with a signal transduction domain, commonly used in enzyme-linked immunosorbent assays (ELISAs). The functional affinity of the sdAb-AP fusions, often increased by a factor of 10 over unfused sdAbs, and their utility as tracer reagents in ELISAs was dramatically improved, giving limits of detection of 300 ng/ml or less, whereas parental sdAbs gave no discernible signal at the toxin concentrations tested. The fusion of sdAbs to AP presents a valuable route to facilitate the implementation of sdAb-based immunoreagents rapidly selected from existing naive libraries toward new or emerging threats.
天然的单域抗体（sdAb）文库能够快速分离到几乎任何目标的结合物。然而，这些结合物缺乏体内亲和力成熟带来的好处，通常对其靶标亲和力较低。我们将先前从骆驼重链抗体可变区衍生的天然文库中筛选出的五种低亲和力毒素结合 sdAb 表达为与超活性突变大肠杆菌碱性磷酸酶（AP）的融合体，并研究了其对表观亲和力和实用性的影响。AP 在溶液中自发二聚化，有效地将融合的 sdAb 二聚化，赋予了亲合力，而不是较低亲和力的单体相互作用。sdAb-AP 融合体还将靶标识别结构域与信号转导结构域结合在一起，该结构域常用于酶联免疫吸附测定（ELISA）中。sdAb-AP 融合体的功能亲和力通常比未融合的 sdAb 提高了 10 倍以上，其作为 ELISA 示踪试剂的实用性也得到了显著提高，检测限达到 300ng/ml 或更低，而亲本 sdAb 在测试的毒素浓度下没有可检测到的信号。将 sdAb 与 AP 融合为从现有的天然文库中快速选择针对新出现或新兴威胁的 sdAb 基免疫试剂提供了一条有价值的途径。
