Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan.
J Immunol Methods. 2011 Mar 7;366(1-2):60-8. doi: 10.1016/j.jim.2011.01.006. Epub 2011 Jan 28.
Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.
烟曲霉引起高致死性侵袭性曲霉病（IA）。在本研究中，我们通过针对烟曲霉膜部分（AMF）或 HM-1 杀伤毒素（HM-1）中和单克隆抗体（nmAb-KT）作为抗原进行淘选，鉴定了一种新型抗真菌药物来预防侵袭性疾病。关键步骤是用 pH 值为 7.0 的磷酸盐缓冲盐水（PBS）洗脱结合的噬菌体。可溶性 scFv AF1 抗体对抗原的特异性通过 ELISA 进行了验证，该抗体特异性结合 AMF 和 nmAb-KT。核苷酸测序后，通过 HisTrap HP 亲和柱进行克隆表达和纯化，scFv AF1 显示出对烟曲霉的体外抗真菌活性。通过 SPR 分析，它显示出与 nmAb-KT 的高结合亲和力（K(d)=5.22×10(-11) M）。分离 scFv AF1 的方法是一种新方法，我们相信它将适用于分离针对任何酵母或真菌的膜蛋白的特异性 scFv。
