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

doi:10.3864/j.issn.0578-1752.2020.05.017

摘要/Abstract

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。

关键词: 玉米赤霉醇, 玉米赤霉酮, 结构类似物, 交叉反应, 单克隆抗体

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

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.

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参考文献 39

[1]	MILES C O, ERASMUSON A F, WILKINS A L, TOWERS N R, SMITH B L, GARTHWAITE I, SCAHILLBRYAN G, HANSEN R P . Ovine metabolism of zearalenone to α-zearalanol (zeranol). Journal of Agriculture and Food Chemistry, 1996, 44(10):3244-3250.
[2]	孟繁静, 阙月美, 韩玉珍, 黎洪霞, 王章存 . 冬小麦越冬茎尖中的玉米赤霉烯酮. 中国科学B辑化学生物学农学医学地学). 1988(12):1261-1266.
	MENG F J, QUE Y M, HAN Y Z, LI H X, WANG Z C . Zearalenone in the stem tip of wheat overwintering. Science in China (B: Chemistry, Biology, agronomy, Medicine, Geoscience), 1988(12):1261-1266. (in Chinese)
[3]	BENNETT G, BEAUMONT W H, BROWN P R . Use of the anabolic agent zearanol (resorcylic acid lactone) as a growth promoter for cattle. Veterinary Record, 1974,94(11):235-239.
[4]	WANG S, WANG X H . Analytical methods for the determination of zeranol residues in animal products: A review. Food Additive and Contaminants Part A Chemistry Analysis Control Exposure and Risk Assessment, 2007,24(6):573-582.
[5]	BO C, ZHAO W, JIA Q, YANG Z, SAI L, ZHANG F, DU Z, YU G, XIE L, ZHANG Z . Effects of alpha-zearalanol on spermatogenesis and sex hormone levels of male mice. International Journal of Clinical and Experimental Medicine, 2015,8(11):20002-20013.
[6]	TROUT W E, HERR C T, RICHERT B T, SINGLETON W L, HAGLOF S A, DIEKMAN M A . Effects of Zeranol upon luteal maintenance and fetal development in peripubertal gilts. Animal Reproduction Science, 2007,99(3/4):408-412.
[7]	WANG Y F, LI L, WANG C C, LEUNG L . Effect of zeranol on expression of apoptotic and cell cycle proteins in murine placentae. Toxicology, 2013,314(1):148-154.
[8]	NAKAMURA U, RUDOLF F O, PANDEY K, KADOKAWA, H. The non-steroidal mycoestrogen zeranol suppresses luteinizing hormone secretion from the anterior pituitary of cattle via the estradiol receptor GPR30 in a rapid, non-genomic manner. Animal Reproduction Science, 2015,156:118-127.
[9]	XU P P, YE W P, LI H, LIN S H, KUO C T, FENG E, LIN Y C . Zeranol enhances leptin-induced proliferation in primary cultured human breast cancer epithelial cells. Molecular Medicine Reports, 2010,3(5):795-800.
[10]	XU P P, YE W P, ZHONG S Y, LI H, FENG E, LIN S H, KUO C T, LIU J Y, LIN Y C . Leptin and zeranol up-regulate cyclin D1 expression in primary cultured normal human breast pre-adipocytes. Molecular Medicine Reports, 2010,3(6):983-990.
[11]	YE W P, XU P P, JEN R, FENG E, ZHONG S Y, LI H, LIN S H, LIU J Y, LIN Y C . Zeranol down-regulates p53 expression in primary cultured human breast cancer epithelial cells through epigenetic modification. International Journal of Molecular Sciences, 2011,12(3):1519-1532.
[12]	XU P P, YE W P, ZHONG S H, JEN R, LI H, FENG E, LIN S H, LIU J Y, LIN Y C . Zeranol may increase the risk of leptin-induced neoplasia in human breast. Oncology Letters, 2011,2(1):101-108.
[13]	BELHASSEN H, JIMENEZ-DIAZ I, GHALI R, GHORBEL H, MOLINA-MOLINA J M, OLEA N, HEDILI A . Validation of a UHPLC-MS/MS method for quantification of zearalenone, alpha- zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone in human urine. Journal of Chromatography B Analytical Technology Biomedical Life Science, 2014,962:68-74.
[14]	LI L P, FAN S, ZHAO R, LI B, LIU W, WU G H . Determination of zearalanol and related mycotoxins in pork by solid-phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry. Se Pu, 2013, 31(7):703-708.
[15]	TAGUCHI S, YOSHIDA S, TANAKA Y, HORI S . Simple and rapid analysis of trenbolone and zeranol residues in cattle muscle and liver by stack-cartridge solid-phase extraction and HPLC using on-line clean-up with EC and UV detection. Shokuhin Eiseigaku Zasshi, 2001,42(4):226-230.
[16]	FANG X, CHEN J, GUO D, WANG G . Detection and identification of zeranol in chicken or rabbit liver by liquid chromatography- electrospray tandem mass spectrometry. Journal of AOAC International, 2002,85(4):841-847.
[17]	MATRASZEK-ZUCHOWSKA I, WOZNIAK B, ZMUDZKI J . Determination of zeranol, taleranol, zearalanone, alpha-zearalenol, beta-zearalenol and zearalenone in urine by LC-MS/MS. Food Additive and Contaminants Part A Chemistry Analysis Control Exposure and Risk Assessment, 2013,30(6):987-994.
[18]	YUNIN M A, METALNIKOV P S, KOMAROV A A, PANIN A N . Development of a rapid method for the analysis of trenbolone, nortestosterone, and zeranol in bovine liver using liquid chromatography tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 2015,407(15):4363-4371.
[19]	HAN H, KIM B, LEE S G, KIM J . An optimised method for the accurate determination of zeranol and diethylstilbestrol in animal tissues using isotope dilution-liquid chromatography/mass spectrometry. Food Chemistry, 2013,140(1-2):44-51.
[20]	DICKSON L C, COSTAIN R, MCKENZIE D, FESSER A C E,, MACNEIL J D, . Quantitative screening of stilbenes and zeranol and its related residues and natural precursors in veal liver by gas chromatography-mass spectrometry. Journal of Agriculture and Food Chemistry. 2009,57(15):6536-6542.
[21]	ZHANG W, WANG H, WANG J, LI X, JIANG H, SHEN J . Multiresidue determination of zeranol and related compounds in bovine muscle by gas chromatography/mass spectrometry with immunoaffinity cleanup. Journal of AOAC International, 2006,89(6):1677-1681.
[22]	SEO J, KIM H Y, CHUNG B C, HONG J . Simultaneous determination of anabolic steroids and synthetic hormones in meat by freezing-lipid filtration, solid-phase extraction and gas chromatography- mass spectrometry. Journal of Chromatography A, 2005,1067(1/2):303-309.
[23]	DICKSON L C, MACNEIL J D, REID J, FESSER A C . Validation of screening method for residues of diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine using immunoaffinity chromatography and gas chromatography/mass spectrometry. Journal of AOAC International, 2003,86(4):631-639.
[24]	MEDINA M B, SCHWARTZ D P . Thin-layer chromatographic detection of zeranol and estradiol in fortified plasma and tissue extracts with Fast Corinth V. Journal of Chromatography, 1992, 581(1):119-128.
[25]	MEDINA M B, NAGDY N . Improved thin-layer chromatographic detection of diethylstilbestrol and zeranol in plasma and tissues isolated with alumina and ion-exchange membrane columns in tandem. Journal of Chromatography, 1993, 614(2):315-323.
[26]	MCCAUGHEY W J . Detection of zeranol in the blood of ram lambs by a radioimmunoassay technique. Veterinary Record, 1988,123(20):511-513.
[27]	刘媛, 文孟棠, 张霄, 徐重新, 杨晶祎, 张留娟, 王恒, 何鑫, 梁颖, 郑勤, 夏兴霞, 刘贤金 . 玉米赤霉醇特异性单克隆抗体及其胶体金检测试纸的研制. 核农学报, 2015,29(6):1101-1107.
	LIU Y, WEN M T, ZHANG X, XU C X, YANG J Y, ZHANG L J, WANG H, HE X, LIANG Y, ZHENG Q, XIA X X, LIU X J . Development of specific monoclonal antibodies and colloidal gold test strips for zeranol. Journal of Nuclear Agricultural Sciences, 2015,29(6):1101-1107. (in Chinese)
[28]	王鹤佳, 曹振, 沈建忠, 史为民 . 玉米赤霉醇单克隆抗体的制备及鉴定. 中国兽医杂志, 2004,40(10):3-5.
	WANG H J, CAO Z, SHEN J Z, SHI W M . Preparation and identification of the monoclonal antibody to Zeranol. Chinese Journal of Veterinary Medicine, 2004,40(10):3-5. (in Chinese)
[29]	孙亚宁 . 赭曲霉毒素A免疫学快速检测方法研究[D]. 洛阳:河南科技大学, 2011.
	SUN Y N . Study of ochratoxin A immunological rapid detection method[D]. Luoyang: Henan University of Science and Technology, 2011. ( in Chinese)
[30]	胡骁飞, 王方雨, 邢广旭, 杨继飞, 张改平, 邓瑞广 . 粮油作物中伏马菌素B1免疫测定方法的建立. 中国粮油学报, 2015,30(7):116-122.
	HU X F, WANG F Y, XING G X, YANG J F, ZHANG G P, DENG R G . Establishment of immunoassay methods to detect fumonisin B1 in grain and oil crops. Journal of the Chinese Cereals and Oils Association, 2015,30(7):116-122. (in Chinese)
[31]	姚静静, 胡骁飞, 韩俊岭, 李青梅, 王方雨, 邓瑞广, 张改平 . 伏马菌素B1单克隆抗体的制备及免疫学检测方法初步应用. 畜牧兽医学报, 2016,47(5):1009-1017.
	YAO J J, HU X F, HAN J L, LI Q M, WANG F Y, DENG R G, ZHANG G P . Development and preliminary application of the monoclonal antibody against fumonisin B1. Acta Veterinaria et Zootechnica Sinica, 2016,47(5):1009-1017. (in Chinese)
[32]	BEATTY J D, BEATTY B G, VLAHOS W G . Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. Journal of Immunological Methods, 1987, 100(1/2):173-179.
[33]	GODING J W. Monoclonal Antibodies: Principles and Practice. 3rd ed. London: Academic Press, 1996: 492.
[34]	王晶, 王耀, 王方雨, 胡骁飞, 姚静静, 邓瑞广, 侯玉泽, 张改平 . 草甘膦人工抗原合成及鼠源多抗血清制备. 核农学报, 2017,31(1):194-200.
	WANG J, WANG Y, WANG F Y, HU X F, YAO J J, DENG R G, HOU Y Z, ZHANG G P . Preparation of glyphosate artificial antigen and mice polyclonal antiserum. Journal of Nuclear Agricultural Sciences, 2017,31(1):194-200. (in Chinese)
[35]	王耀, 胡骁飞, 刘胜男, 裴亚峰, 张改平, 李志西, 邓瑞广 . 伏马菌素B1兔源多克隆抗血清的制备与鉴定. 中国食品学报, 2014,14(4):216-221.
	WANG Y, HU X F, LIU S N, PEI Y F, ZHANG G P, LI Z X, DENG R G . Preparation and identification of fumonisin B1 rabbit polyclonal antiserum. Journal of Chinese Institute of Food Science and Technology, 2014,14(4):216-221. (in Chinese)
[36]	SUN Y, HU X, ZHANG Y, YANG J, WANG F, WANG Y, DENG R, ZHANG G . Development of an immunochromatographic strip test for the rapid detection of zearalenone in corn. Journal of Agriculture and Food Chemistry, 2014,62(46):11116-11121.
[37]	HOLTHUES H, PFEIFER-FUKUMURA U, SOUND I, BAUMANN W . Evaluation of the concept of heterology in a monoclonal antibody- based ELISA utilizing direct hapten linkage to polystyrene microtiter plates. Journal of Immunological Methods, 2005,304(1/2):68-77.
[38]	姜金庆 . 19-去甲睾酮免疫学检测方法的建立及应用[D]. 杨凌:西北农林科技大学, 2011.
	JIANG J Q . Development and application of immunoassays for detection of 19-nortestosterone[D]. Yangling: Northwest A and F University, 2011. ( in Chinese)
[39]	贾万钧 . 抗原抗体反应动力学: 在免疫血清学检测技术中的应用实例. 北京: 军事医学科学出版社, 2004: 18.
	JIA W J. The Reaction Kinetics of Antigen And Antibody: Application Examples in Immunoserological Detection Technology. Beijing: Military Medical Science Press, 2004: 18. (in Chinese)

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ZEN mAb稀释倍数 Dilution ratio of ZEN mAb	1×10⁴	2×10⁴	4×10⁴	8×10⁴	16×10⁴	32×10⁴	64×10⁴	空白 Blank
ZAN-O-BSA	>3.5	3.044	1.847	1.028	0.579	0.343	0.243	0.064
ZAN-O-OVA	1.182	0.869	0.653	0.387	0.289	0.196	0.163	0.042

小鼠编号 Number of mice	血清稀释倍数 Dilution ratio of serum
小鼠编号 Number of mice	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

小鼠编号 Number of mice	ZAL浓度 ZAL concentration (ng·mL^-1)						阳性 Positive	空白 Blank
小鼠编号 Number of mice	500	250	125	62.5	31.25	15.62	阳性 Positive	空白 Blank
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