中國農業科學 ?? 2020, Vol. 53 ?? Issue (5): 1071-1080.doi: 10.3864/j.issn.0578-1752.2020.05.017

? 畜牧·獸醫·資源昆蟲 ? 上一篇    

基于結構類似物的交叉反應性制備高靈敏度 玉米赤霉醇單克隆抗體

胡驍飛1,李青梅1,姚靜靜1,胡思宇2,孫亞寧1,邢云瑞1,鄧瑞廣1,張改平1,3()   

  1. 1 河南省農業科學院動物免疫學重點實驗室,鄭州 450002
    2 河南農業大學生命科學學院,鄭州 450002
    3 河南農業大學,鄭州 450002
  • 收稿日期:2019-01-08 接受日期:2019-10-08 出版日期:2020-03-01 發布日期:2020-03-14
  • 通訊作者: 張改平 E-mail:[email protected]
  • 作者簡介:胡驍飛,E-mail:[email protected]
  • 基金資助:
    河南省科技攻關項目(192102310513);國家“十二五”科技支撐計劃課題(2014BAD13B05)

Development of High Sensitive Zeranol Monoclonal Antibody Based on the Cross Reactivity of Structural Analogs

HU XiaoFei1,LI QingMei1,YAO JingJing1,HU SiYu2,SUN YaNing1,XING YunRui1,DENG RuiGuang1,ZHANG GaiPing1,3()   

  1. 1 Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002
    2 College of Life Sciences, Henan Agricultural University, Zhengzhou 450002
    3 Henan Agricultural University, Zhengzhou 450002
  • Received:2019-01-08 Accepted:2019-10-08 Online:2020-03-01 Published:2020-03-14
  • Contact: GaiPing ZHANG E-mail:[email protected]

摘要:

【目的】獲取高靈敏度的玉米赤霉醇(zearalanol,ZAL)單克隆抗體,為提高玉米赤霉醇免疫學檢測方法靈敏度研究奠定基礎。【方法】利用ZAL的結構類似物玉米赤霉酮(zearalanone, ZAN)制備人工完全抗原。肟化改造ZAN得到ZAN-O;碳二亞胺(EDC)法把ZAN-O分別連接到牛血清白蛋白(BSA)和卵清蛋白(OVA)上制備出ZAN-O-BSA和ZAN-O-OVA。ZAN-O-BSA免疫小鼠,免疫劑量為50 μg蛋白/只鼠。選取血清效價高、靈敏度好的小鼠進行細胞融合。陽性雜交瘤篩選過程中,利用ZAL替代ZAN作為阻斷劑,篩選能分泌抗ZAL單克隆抗體的雜交瘤細胞。體內誘生腹水法來批量制備ZAL單抗,并對單抗的免疫學性能進行了鑒定。【結果】通過細胞融合,陽性雜交瘤篩選得到了1株能分泌抗ZAL單克隆抗體的雜交瘤細胞,命名為12B10A7,其分泌的ZAL單抗靈敏度(半數抑制濃度, IC50)為577 pg?mL -1,親和力常數Ka=6.21×10 7L?mol -1,與結構類似物β-玉米赤霉醇(β-zearalanol, β-ZAL)、α-玉米赤霉烯醇(α-zearalenol, α-ZEL)、β-玉米赤霉烯醇(β-zearalenol, β-ZEL)、玉米赤霉酮、玉米赤霉烯酮(zearalenone, ZEN)分別有43.06%、15.51%、15.22%、77.65%及9.79%的交叉反應率,而與其他霉菌毒素及載體蛋白交叉反應率均<0.06%。【結論】基于抗體交叉反應特性,利用ZAN制備人工抗原,得到了ZAL的單克隆抗體,所制備的單克隆抗體親和力高,靈敏度好,特異性強。

關鍵詞: 玉米赤霉醇, 玉米赤霉酮, 結構類似物, 交叉反應, 單克隆抗體

Abstract:

【Objective】The aim of this study was to obtain highly sensitive monoclonal antibody of Zearalanol (ZAL). 【Method】Zearalanone (ZAN), as the structural analogs of ZAL, was employed to synthesize the artificial complete antigen. ZAN structure was modified by oximation reaction to obtain ZNA-O. ZAN-O was coupled with bovine serum albumin and ovalbumin by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) respectively to prepare ZAN-O-BSA and ZAN-O-OVA. ZAN-O-BSA was used to immunize the mice with the immune dose of 50 μg protein/mouse. The mouse with higher serum titer and sensitivity was selected for cell fusion. During the process of cell fusion to screen the positive hybridoma cells, ZAL was used as a blocker instead of ZAN to screen the hybridoma cell lines that could secrete monoclonal antibody against ZAL. ZAL monoclonal antibody was prepared in batches by ascites induction in vivo, and the immunological properties of the monoclonal antibodies were identified. 【Result】By cell fusion and screening for positive hybridoma cell lines, one hybridoma cell line named 12B10A7 that could secrete monoclonal antibody against ZAL was obtained. The sensitivity (half inhibitory concentration, IC50) of the prepared monoclonal antibody was 577 pg?mL -1, the affinity constant Ka was 6.21×10 7 L?mol -1, the cross-reactivity rate with structural analogues such as β-zearalanol, α-zearalenol, β-zearalenol, zearalanone and zearalenone were 43.06%, 15.51%, 15.22%, 77.65% and 9.79%, respectively, and the cross reaction rates with other toxins and carrier proteins were all less than 0.06%. 【Conclusion】In conclusion, based on the cross reaction characteristics of antibody, in this study, we prepared the artificial antigen of ZAN, and got the monoclonal antibody against ZAL, which had high affinity, sensitivity and specificity.

Key words: Zearalanol, Zearalanone, structural analogs, cross reaction, monoclonal antibody

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