School of Environmental Sciences, University of Guelph, Guelph, Ontario, Canada.
J Immunol Methods. 2011 Oct 28;373(1-2):8-18. doi: 10.1016/j.jim.2011.07.006. Epub 2011 Jul 18.
Three V(H)Hs against the model hapten, azoxystrobin (MW 403), were isolated from a hyper-immunized phage-displayed V(H)H library. This library was constructed by isolating the V(H)H-coding genes from the lymphocytes collected from a Llama glama that was immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones containing V(H)Hs specific to the hapten. After screening 95 clones, three V(H)Hs (A27, A72, and A85) with different amino acid sequences were identified, expressed in soluble format in Escherichia coli HB2151, and purified using nickel-immobilized metal affinity chromatography. Competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) showed that A27 and A85 were specific to azoxystrobin while A72 was not. The IC(50) values of A27 and A85 V(H)Hs were 7.2 and 2.0μM, respectively. To our knowledge A85 is one of the highest affinity V(H)Hs that has yet been isolated against a hydrophobic hapten such as azoxystrobin.
从针对阿唑菌酮（MW 403）的超免疫噬菌体展示 VH 文库中分离出了 3 种针对模型半抗原的 VH。该文库是通过从用阿唑菌酮偶联牛血清白蛋白（BSA）免疫的羊驼淋巴细胞中分离 VH 编码基因而构建的。针对阿唑菌酮与卵清蛋白（OVA）或兔血清白蛋白（RSA）偶联物进行了 6 轮淘选，以富集含有针对半抗原的 VH 的克隆。筛选了 95 个克隆后，鉴定出了 3 种具有不同氨基酸序列的 VH（A27、A72 和 A85），它们在大肠杆菌 HB2151 中以可溶性形式表达，并通过镍固定金属亲和层析进行纯化。竞争性抑制酶联免疫吸附测定（CI-ELISA）表明，A27 和 A85 特异性针对阿唑菌酮，而 A72 则不是。A27 和 A85 VH 的 IC50 值分别为 7.2 和 2.0μM。据我们所知，A85 是针对疏水性半抗原（如阿唑菌酮）分离出的亲和力最高的 VH 之一。
