High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay
High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay
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微囊藻毒素-LR免疫小鼠噬菌体抗体库的高灵敏单链可变片段筛选及其在免疫分析中的应用
DOI：
10.1016/j.talanta.2019.01.064
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发表时间：
2019
期刊：
影响因子：
6.1
通讯作者：
Liu Xianjin
中科院分区：
文献类型：
--
作者：
Xu Chongxin;Liu Xiaoqin;Liu Yuan;Zhang Xiao;Zhang Cunzheng;Li Jianhong;Liu Xianjin
微囊藻毒素-LR（Microcystin-LR，MC-LR）是水体中常见的由蓝藻产生的高毒性生物毒素之一。因此，需要一种灵敏度高、操作简便的检测方法对MC-LR进行监测。为了建立一种高灵敏度的基于单链抗体的间接竞争酶联免疫吸附试验（IC-ELISA）检测MC-LR，从MC-LR免疫小鼠的噬菌体单链抗体库中筛选出16个阳性的抗MC-LR噬菌体单链抗体，成功构建了容量为8.67 × 107CFU/mL的噬菌体单链抗体库。在大肠杆菌HB2151中成功表达了抗MC-LR的单链抗体MscFv7。分子量（M.W.）SDS-PAGE和蛋白定量分析表明，表达蛋白的分子量约为30 kDa，纯化蛋白的浓度为512.6 μ g/mL。基于MscFv7-scFv的IC-ELISA对MC-LR的半数抑制（IC50）为0.471 μ g/L，检出限（LOD）为0.044 μ g/L，低于饮用水中MC-LR的最大残留限量（MRL）1.0 μ g/L。MscFv7-scFv对MC-RR和MC-YR具有较强的交叉识别能力，交叉反应率分别为93.1%和85.9%，而对MC-LW的交叉识别能力较弱，仅为9.7%，对MC-WR、MC-LF和MC-LY甚至不识别。IC-ELISA检测不同清洁度水样中MC-LR的回收率为81.2-106.3%，批内、批间变异系数为2.62-10.22%。结果表明，获得了高灵敏度的抗MC-LR单链抗体，建立的基于MscFv7-scFv的IC-ELISA有望用于水样中MC-LR、MC-RR和MC-YR的超灵敏检测。
Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 107CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed inEscherichia coli(E.coli) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 μg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC50) of 0.471 μg/L and a limit of detection (LOD) of 0.044 μg/L, which is below the maximum residue limit standard (MRLs) of 1.0 μg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2–106.3% with CVs of 2.62–10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.