中国农业科学 ›› 2020, Vol. 53 ›› Issue (5): 1071-1080.doi: 10.3864/j.issn.0578-1752.2020.05.017
• 畜牧·兽医·资源昆虫 • 上一篇
胡骁飞1,李青梅1,姚静静1,胡思宇2,孙亚宁1,邢云瑞1,邓瑞广1,张改平1,3()
收稿日期:
2019-01-08
接受日期:
2019-10-08
出版日期:
2020-03-01
发布日期:
2020-03-14
通讯作者:
张改平
作者简介:
胡骁飞,E-mail:13598412657@163.com。
基金资助:
HU XiaoFei1,LI QingMei1,YAO JingJing1,HU SiYu2,SUN YaNing1,XING YunRui1,DENG RuiGuang1,ZHANG GaiPing1,3()
Received:
2019-01-08
Accepted:
2019-10-08
Online:
2020-03-01
Published:
2020-03-14
Contact:
GaiPing ZHANG
摘要:
【目的】获取高灵敏度的玉米赤霉醇(zearalanol,ZAL)单克隆抗体,为提高玉米赤霉醇免疫学检测方法灵敏度研究奠定基础。【方法】利用ZAL的结构类似物玉米赤霉酮(zearalanone, ZAN)制备人工完全抗原。肟化改造ZAN得到ZAN-O;碳二亚胺(EDC)法把ZAN-O分别连接到牛血清白蛋白(BSA)和卵清蛋白(OVA)上制备出ZAN-O-BSA和ZAN-O-OVA。ZAN-O-BSA免疫小鼠,免疫剂量为50 μg蛋白/只鼠。选取血清效价高、灵敏度好的小鼠进行细胞融合。阳性杂交瘤筛选过程中,利用ZAL替代ZAN作为阻断剂,筛选能分泌抗ZAL单克隆抗体的杂交瘤细胞。体内诱生腹水法来批量制备ZAL单抗,并对单抗的免疫学性能进行了鉴定。【结果】通过细胞融合,阳性杂交瘤筛选得到了1株能分泌抗ZAL单克隆抗体的杂交瘤细胞,命名为12B10A7,其分泌的ZAL单抗灵敏度(半数抑制浓度, IC50)为577 pg?mL -1,亲和力常数Ka=6.21×10 7L?mol -1,与结构类似物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)分别有43.06%、15.51%、15.22%、77.65%及9.79%的交叉反应率,而与其他霉菌毒素及载体蛋白交叉反应率均<0.06%。【结论】基于抗体交叉反应特性,利用ZAN制备人工抗原,得到了ZAL的单克隆抗体,所制备的单克隆抗体亲和力高,灵敏度好,特异性强。
胡骁飞,李青梅,姚静静,胡思宇,孙亚宁,邢云瑞,邓瑞广,张改平. 基于结构类似物的交叉反应性制备高灵敏度 玉米赤霉醇单克隆抗体[J]. 中国农业科学, 2020, 53(5): 1071-1080.
HU XiaoFei,LI QingMei,YAO JingJing,HU SiYu,SUN YaNing,XING YunRui,DENG RuiGuang,ZHANG GaiPing. Development of High Sensitive Zeranol Monoclonal Antibody Based on the Cross Reactivity of Structural Analogs[J]. Scientia Agricultura Sinica, 2020, 53(5): 1071-1080.
表2
ZAN-O-BSA免疫小鼠后血清抗体效价"
小鼠编号 Number of mice | 血清稀释倍数 Dilution ratio of serum | |||||||
---|---|---|---|---|---|---|---|---|
200 | 400 | 800 | 1600 | 3200 | 6400 | 12800 | 空白Blank | |
1 | 3.471 | 2.438 | 1.462 | 0.718 | 0.418 | 0.329 | 0.240 | 0.105 |
2 | 2.656 | 1.898 | 1.047 | 0.611 | 0.345 | 0.244 | 0.205 | 0.083 |
3 | >3.50 | 2.937 | 2.084 | 1.120 | 0.656 | 0.384 | 0.309 | 0.107 |
4 | 2.179 | 2.012 | 1.828 | 1.432 | 1.043 | 0.822 | 0.670 | 0.089 |
5 | 1.879 | 1.855 | 1.392 | 0.938 | 0.692 | 0.539 | 0.373 | 0.105 |
6 | 2.977 | 2.728 | 2.122 | 1.560 | 1.049 | 0.692 | 0.502 | 0.089 |
表3
ZAN-O-BSA免疫小鼠后血清灵敏度"
小鼠编号 Number of mice | ZAL浓度 ZAL concentration (ng·mL-1) | 阳性 Positive | 空白 Blank | |||||
---|---|---|---|---|---|---|---|---|
500 | 250 | 125 | 62.5 | 31.25 | 15.62 | |||
1 | 0.681 | 0.836 | 1.099 | 1.148 | 1.286 | 1.802 | 1.910 | 0.104 |
2 | 0.736 | 0.844 | 0.915 | 0.986 | 1.085 | 1.128 | 1.550 | 0.109 |
3 | 1.078 | 1.043 | 1.101 | 1.176 | 0.993 | 1.168 | 1.544 | 0.092 |
4 | 0.569 | 0.608 | 0.731 | 0.820 | 0.987 | 1.069 | 1.591 | 0.107 |
5 | 0.865 | 0.879 | 0.927 | 0.972 | 0.994 | 1.077 | 1.346 | 0.103 |
6 | 0.288 | 0.402 | 0.510 | 0.621 | 0.653 | 0.741 | 1.276 | 0.106 |
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