Goldman Ellen R, Anderson George P, Liu Jinny L, Delehanty James B, Sherwood Laura J, Osborn Lisa E, Cummins Larry B, Hayhurst Andrew
Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, DC 20375, USA.
Anal Chem. 2006 Dec 15;78(24):8245-55. doi: 10.1021/ac0610053.
Llamas possess a class of unconventional immunoglobulins that have only heavy chains; unpaired heavy variable domains are responsible for antigen binding. These domains have previously been cloned and expressed as single domain antibodies (sdAbs); they comprise the smallest known antigen binding fragments. SdAbs have been shown to bind antigens at >90 degrees C and to refold after being denatured. To take advantage of the remarkable properties of sdAbs, we constructed a large, semisynthetic llama sdAb library. This library facilitated the rapid selection of binders to an array of biothreat targets. We selected sdAb specific for live vaccinia virus (a smallpox virus surrogate), hen egg lysozyme, cholera toxin, ricin, and staphylococcal enterotoxin B. The selected sdAb possessed high specificity as well as enhanced thermal stability in comparison to conventional IgG and scFv antibodies. We also determined equilibrium dissociation constants as well as demonstrated the use of several antitoxin sdAbs as effective capture and reporter molecules in sandwich assays on the Luminex instrument. The ability to rapidly select such rugged antibodies will enhance the reliability of immunoassays by extending shelf life and the capacity to function in hostile environments.
骆驼拥有一类非常规免疫球蛋白,其只有重链;未配对的重链可变结构域负责抗原结合。这些结构域此前已被克隆并表达为单结构域抗体(sdAbs);它们构成了已知最小的抗原结合片段。已证明sdAbs能在90摄氏度以上结合抗原,并在变性后重新折叠。为利用sdAbs的显著特性,我们构建了一个大型半合成骆驼sdAb文库。该文库有助于快速筛选针对一系列生物威胁靶点的结合物。我们筛选出了对活痘苗病毒(天花病毒替代品)、鸡蛋清溶菌酶、霍乱毒素、蓖麻毒素和葡萄球菌肠毒素B具有特异性的sdAb。与传统IgG和scFv抗体相比,筛选出的sdAb具有高特异性以及更高的热稳定性。我们还测定了平衡解离常数,并证明了几种抗毒素sdAb在Luminex仪器上的夹心测定中作为有效的捕获和报告分子的用途。快速筛选此类坚固抗体的能力将通过延长保质期和在恶劣环境中发挥作用的能力来提高免疫测定的可靠性。