Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex.
Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex.
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DOI：
10.1021/ac8014774
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发表时间：
2008-11-15
影响因子：
7.4
通讯作者：
Hayhurst, Andrew
中科院分区：
文献类型：
--
作者：
Goldman, Ellen R.;Anderson, George P.;Conway, Jerry;Sherwood, Laura J.;Fech, Melissa;Vo, BaoHan;Liu, Jinny L.;Hayhurst, Andrew
来自驼科动物的免疫球蛋白具有独特的形式，不包含轻链。在这些非常规的重链同源二聚体中，抗原结合是通过一个单一的可变区来调节的。当重组表达时，这些可变区被称为单区抗体(SdAb)，是天然免疫球蛋白衍生的抗原结合单位中最小的一个。SdAb具有良好的溶解性和热稳定性，在加热和化学变性后可以折叠，是一种很有前途的替代识别元件。我们已经从用肉毒神经毒素(BONT)复合毒素(BONT)复合毒素的鸡尾酒免疫的骆驼中构建了一个噬菌体展示的sdAb文库，并在文库中搜寻BONT A复合毒素的结合剂。分离出六种独特的结合剂，发现它们能以类毒素和无毒素的形式与BoNT A复合体特异性结合，当在缓冲液和牛奶中进行最佳组合时，可以检测到100pg/mL的无毒素复合体。与常规多克隆血清相比，所有sdAb在85℃加热1小时后仍保持其与靶标的特异性结合能力。所有sdAb都高度特异于A1亚型，而不是A2亚型，并显示与33 kDa血凝素结合，可能与某种重叠的线性表位结合。这些sdAb的独特性质可以为许多诊断应用提供优势，在这些应用中，长期存储和在线监测需要非常坚固但高度特异的识别元件。
Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb), and are among the smallest naturally IgG derived antigen binding units. SdAb possess good solubility, thermostability, and can refold after heat and chemical denaturation making them promising alternative recogntion elements. We have constucted a library of phage-displayed sdAb from a llama immunized with a cocktail of botulinum neurotoxin (BoNT) complex toxoids and panned the library for binders for BoNT A complex toxoid. Six unique binders were isolated, and found to specifically bind BoNT A complex in toxoid and untoxoided forms and when used in optimal combinations in buffer and milk could detect 100 pg/mL untoxoided complex. All sdAb retained their ability to specifically bind target after heating to 85°C for an hour, in contrast to conventional polyclonal sera. All of the sdAb were highly specific for subtype A1 rather than A2 and demonstrated binding to the 33 kDa hemagglutinin, potentially to a somewhat overlapping linear epitope. The unique properties of these sdAb may provide advantages for many diagnostic applications where long term storage and in line monitoring require very rugged yet highly specific recognition elements.
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影响因子：
5.6
作者：
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