基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定

doi:10.3864/j.issn.0578-1752.2012.02.015

中国农业科学 ›› 2012, Vol. 45 ›› Issue (2): 330-337.doi: 10.3864/j.issn.0578-1752.2012.02.015

基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定

刘媛, Huovinen Tuomas, 刘贤金, 梁颖, 张存政, 谢雅晶, 贺江, 王耘, 张霄

1.江苏省农业科学院食品质量安全与检测研究所，中国南京 200014
2.芬兰图尔库大学生物系，芬兰图尔库 20520

收稿日期:2011-05-19 出版日期:2012-01-15 发布日期:2011-11-09
通讯作者: 通信作者刘贤金，Tel： 025-84390396；E-mail：jaasliu@jaas.ac.cn
作者简介:刘媛，E-mail: liuyuan@jaas.ac.cn
基金资助:
国家自然科学基金项目（30871658）、江苏省农业自主创新项目（CX（10）236）、农业部“948”项目（2011-Z46）

Screening and Identification of Single-Chain Antibodies Against Microcystin-LR by Magnetic Beads and Time Resolved Fluorescence Immunoassay

LIU Yuan, Huovinen Tuomas, LIU Xian-Jin, LIANG Ying, ZHANG Cun-Zheng, XIE Ya-Jing, HE Jiang, WANG Yun, ZHANG Xiao

1.江苏省农业科学院食品质量安全与检测研究所，中国南京 200014
2.芬兰图尔库大学生物系，芬兰图尔库 20520

Received:2011-05-19 Online:2012-01-15 Published:2011-11-09

摘要/Abstract

摘要： 【目的】从鼠源合成噬菌体抗体库中，快速分离获得微囊藻毒素LR单链抗体。【方法】采用磁珠筛选和负筛选方法，设计两轮微囊藻毒素LR单链抗体的筛选方案。用噬菌体产出投入比和多克隆噬菌体免疫分析，对每轮筛选后微囊藻毒素LR特异性噬菌体的富集效果进行鉴定。再将第2轮筛选获得的次级库提取单链抗体基因，转入可溶性表达载体；诱导表达后，采用时间分辨荧光免疫法，对单个噬菌体克隆鉴定并测序。【结果】两轮筛选的噬菌体产出投入比分别为4.8×10-8和2.88×10-6。第2轮筛选后的次级库，经多克隆噬菌体免疫分析得到的荧光信噪比，比原始抗体库提高了22.8倍。最终鉴定得到5株不同的阳性噬菌体克隆，其中最佳的克隆对微囊藻毒素LR的检测灵敏度（IC10）为13 ng•mL-1，抑制中浓度（IC50）为435 ng•mL-1，线性范围在31—5 952 ng•mL-1。【结论】本研究筛选到的单链抗体，有潜力应用于微囊藻毒素LR的免疫学检测或免疫亲和柱的制备，同时也探索了一种高效的微囊藻毒素LR单链抗体的库筛选、鉴定方法。

关键词: 微囊藻毒素LR, 单链抗体, 时间分辨免疫分析, 磁珠

Abstract: 【Objective】 Single chain antibodies (scFv) against microcystin-LR were rapidly isolated from a synthetic mouse phage display library. 【Method】 Two rounds of binding were designed by using magnetic beads and subtractive panning. After each round of binding, the ratio of out to input of phage and polyclonal phage immunoassay were employed to investigate the enrichment of specific phage. The scFv genes after the second round of binding were extracted and cloned into the soluble expression vector. The soluble format of scFv was induced by IPTG and determined by time resolved fluorescence immunoassay, and then the positive clones were sequenced. 【Result】 The ratio of out to input of phage after two rounds of binding were 4.8 × 10-8 and 2.88 × 10-6, respectively. In polyclonal phage immunoassay, the signal to background ratio for the library after the second round of binding increased by 22.8 fold to the primary library. Finally, 5 unique positive clones were isolated from the libraries, and the detection limit (IC10) and IC50 for the best clone was 13 ng•mL-1and 435 ng•mL-1, respectively. The linear range for microcystins-LR detection was between 31-5 952 ng•mL-1. 【Conclusion】 In this study, these isolated single-chain antibodies were potentially used in the immunological detection for microcystin LR or preparation of immunoaffinity columns. Meanwhile, an efficient screening and identification methods of scFv against microcystin-LR was also explored.

Key words: microcystin-LR, single chain antibodies, time resolved fluorescence immunoassay (TRFIA), magnetic beads

刘媛, Huovinen Tuomas, 刘贤金, 梁颖, 张存政, 谢雅晶, 贺江, 王耘, 张霄. 基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定[J]. 中国农业科学, 2012, 45(2): 330-337.

LIU Yuan, Huovinen Tuomas, LIU Xian-Jin, LIANG Ying, ZHANG Cun-Zheng, XIE Ya-Jing, HE Jiang, WANG Yun, ZHANG Xiao. Screening and Identification of Single-Chain Antibodies Against Microcystin-LR by Magnetic Beads and Time Resolved Fluorescence Immunoassay[J]. Scientia Agricultura Sinica, 2012, 45(2): 330-337.

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基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定

Screening and Identification of Single-Chain Antibodies Against Microcystin-LR by Magnetic Beads and Time Resolved Fluorescence Immunoassay

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