CP4-EPSPS蛋白抗原表位鉴定及其快速DAS-ELISA检测方法的建立

doi:10.3969/j.issn.1674-7968.2021.01.015

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摘要中国每年进口大量转基因大豆(Glycine max),其中主要为cp4 epsps转基因大豆,需建立一种针对cp4 epsps转基因大豆的高效的快速ELISA鉴定方法。本研究利用合成多肽法将CP4-EPSPS蛋白分段表达,初步定位了5株CP4-EPSPS单克隆抗体识别的抗原表位,采用方阵法对识别不同抗原表位的单抗进行配对,以P/N最大值确定工作抗体及其浓度,通过控制变量法,确定最佳检测条件,建立cp4 epsps转基因大豆快速双抗夹心ELISA (double antibody sandwich ELISA, DAS-ELISA)检测方法。并通过特异性试验、重复性试验、阳性判定值试验,对建立的检测方法进行性能评估,利用建立的检测方法和商品化试剂盒同时对130份样品进行检测,比较其符合率。通过方阵法确定单抗2D3为捕获抗体,单抗1D10为检测抗体时检测信号最强,2D3工作浓度为20 μg/mL,1D10工作浓度为10 μg/mL。捕获抗体最佳包被条件为37 ℃封闭2 h,4 ℃过夜,样品与检测抗体先后加入酶标板37 ℃共同孵育10 min。该方法灵敏度为叶片或籽粒稀释160倍(g/mL),待检叶片最佳稀释范围是10~80 (g/mL)、籽粒最佳稀释范围是10~40 (g/mL)。板内、板间变异系数小于25%,阳性判定值为1.41。对70份大豆叶片、40份大豆籽粒和20份豆浆进行检测,结果与CP4-EPSPS蛋白ELISA试剂盒检测结果比较,符合率为100%,且与其他蛋白不发生交叉反应。本研究建立的快速ELISA检测方法具有良好的准确性、重复性和特异性,该检测方法仅需30 min完成检测,适用于大豆植株、大豆籽粒及豆浆的快速鉴定。

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	候吉超
	李忠鹏
	梁雨欣
	张春雨
	于寒松
	李小宇
	王永志

关键词 ： CP4-EPSPS, 双抗夹心ELISA (DAS-ELISA), 大豆, 检测

Abstract：China imports a large number of transformed soybeans (Glycine max) from abroad every year, mainly cp4 epsps transformed soybean. It is necessary to establish a rapid-effective ELISA method for identification of cp4 epsps transformed soybean. This study, CP4-EPSPS protein was expressed in segments by synthetic peptides method, the antigenic epitopes recognized by 5 strains of CP4-EPSPS monoclonal antibodies were mapped. The monoclonal antibodies that recognized different epitope were paired by matrix approach, the working antibody and working concentration of antibody were determined by the maximum value of P/N. The optimal detection conditions were determined by controlling variable method, and the cp4 epsps transformed soybean rapid double antibody sandwich ELISA detection method was established. The performance of the established detection method was evaluated by specific test, repeatability test and positive determination value test, at the same time, 130 samples were detected by the established detection method and commercial kits, and compared their coincidence rates. The monoclonal antibodies 1D10 and 2D3 were paired and had the best matching detection effect by matrix approach. The monoclonal antibody 2D3 and 1D10 were determined as capture antibody and detection antibody, working concentration of 2D3 was 20 μg/mL, 1D10 was 10 μg/mL. The capture antibody coating condition was 37 ℃ 2 h, 4 ℃ overnight. The sample and detection antibody were added in ELISA plate at the same time, then co-incubated at 37 ℃ for 10 min. The sensitivity of this method was 160 (g/mL) times dilution for leaf and seed, the optimal dilution rate for soybean leaves and seeds were 10~80 and 10~40 (g/mL), respectively. The intra-plate and inter-plate variation coefficient was less than 25%, and the positive determination value was 1.41. 70 soybean leaves, 40 soybean seeds and 20 soybean milk samples were detected, the coincidence rate was 100%, and no cross react with other proteins. This detection method has good accuracy, repeatability and specificity. This testing program can be completed within 30 min, and is suitable for rapid qualitative detection for plant, seed and soybean milk.

Key words： CP4-EPSPS Double-antibody sandwich ELISA (DAS-ELISA) Soybean Detection

收稿日期: 2020-05-28

ZTFLH:	S412
	TS214.2

基金资助:吉林省农业科技创新工程自由创新项目(CXGC2018DC003); 吉林省省级产业创新专项资金项目(2017C057-1); 吉林省科技厅重点研发项目(20180201084SF)

通讯作者: *lxyzsx@163.com; yzwang@126.com

引用本文:

候吉超, 李忠鹏, 梁雨欣, 张春雨, 于寒松, 李小宇, 王永志. CP4-EPSPS蛋白抗原表位鉴定及其快速DAS-ELISA检测方法的建立[J]. 农业生物技术学报, 2021, 29(1): 159-168.
HOU Ji-Chao, LI Zhong-Peng, LIANG Yu-Xin, ZHANG Chun-Yu, YU Han-Song, LI Xiao-Yu, WANG Yong-Zhi. Mapping of Antigenic Epitopes on CP4-EPSPS Protein and Detection Method Establishment of Rapid DAS-ELISA. 农业生物技术学报, 2021, 29(1): 159-168.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.01.015 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I1/159

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