Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (2): 330-337.doi: 10.3864/j.issn.0578-1752.2012.02.015

• STORAGE·FRESH-KEEPING·PROCESSING • Previous Articles     Next Articles

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. 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

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

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[1] ZHANG Liu-Quan-1, 2 , ZHANG Xiao-1, LIU Yuan-1, XU Zhong-Xin-1, ZHANG Cun-Zheng-1, XIE Ya-Jing-1, KOU Li-Ping-2, LIU Xian-Jin-1. Selection and Identification of Anti-Cry1Ac scFv from a Phage Display Antibody Library by Magnetic Beads and Sandwich ELISA Immunoassay [J]. Scientia Agricultura Sinica, 2014, 47(9): 1802-1810.
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