19-去甲睾酮异源性ciELISA试剂盒的研制及应用

doi:Q511

中国生物工程杂志

2010, Vol. 30

Issue (09): 68-74 DOI: Q511

技术与方法

19-去甲睾酮异源性ciELISA试剂盒的研制及应用

姜金庆^1,2,张海棠¹,王自良¹,王建华²,范国英¹

1.河南科技学院动物科学学院新乡 453003
2.西北农林科技大学动物医学院杨凌 712100

Development and Application of Heterologous ciELISA Kit for Rapid Detection of 19-Nortestosterone Residues

JIANG Jin-qing^1,2,ZHANG Hai-tang¹,WANG Zi-liang¹,WANG Jian-hua²,FAN Guo-ying¹

1.College of Animal Science, Henan Institute of Science and Technology, Xinxiang 453003, China
2.College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

全文: PDF(689 KB) HTML

摘要：

筛选抗19-去甲睾酮（NT）单克隆抗体，建立异源性ciELISA试剂盒。用人工抗原NT-17-BSA免疫Balb/C小鼠，细胞融合技术建立1株抗NT单克隆杂交留细胞NT1-B7，同种型为IgG1 /k，腹水效价达0.64×105，亲和常数（Ka）为4.31×109 L/mol，半数抑制浓度（IC50）为0.55 ng/mL，与群勃龙和雌二醇的交叉反应率（CR）分别为2.2%和1.6%，与其它化合物无CR。异源性ciELISA试剂盒在猪肉、猪尿、牛肉和牛尿四种基质中的检测限分别为0.24、0.23、0.22和0.17 ng/mL，批内和批间相对标准差（RSD）在4.4％～21.8％，添加回收率在85％～110％，4 ℃下保存期至少6个月。NT-Kit的基质pH值适用范围为6.5～7.5，温育过程优化后可节约检测时间30～90 min。试剂盒灵敏度高、特异性强，可应用于样品基质中NT残留检测。

关键词： 19-去甲睾酮; 单克隆抗体; 间接竞争ELISA; 异源性; 快速检测试剂盒

Abstract:

To screen anti-Nortestosterone monoclonal antibody (NT mAb) and develop heterologous ciELISA kit. Balb/C mice were immunized with artificial antigen of NT-17-BSA and a strain of hybridoma cell named NT1-B7 was produced, which can stably secret NT mAb. The isotype of mAb was IgG1 /k, ascites titer was 0.64×105, affinity constant (Ka) was 4.31×109 L/mol and IC50 value determined by ciELISA was 0.55 ng/mL. Of all the analogous competitors, 2.2% and 1.6% cross-reactivity (CR) were observed for trenbolone and estradiol, respectively, while no CR was obtained for other compounds. With coating antigen of NT-3-OVA, heterologous ciELISA kit was developed, which limits of detection in pork, swine urine, beef and cattle urine were 0.24, 0.23, 0.22 and 0.17 ng/mL, respectively. The recoveries of NT spiked in the four matrix were in the range of 85％-110％, while intra- and inter-relative standard deviation (RSD) were in the range of 4.4％-21.8％. when stored at 4 ℃, the NT-Kit could preserve 6 months or more. The pH value of validation detection in matrix ranged from 6.5 to 7.5, and the program could save 30-90 min after the incubation process was optimized. With high sensitivity and specificity, this ciELISA kit could be advantageously utilized for the primary screening of sample matrix for presence of 19-NT residue.

Key words: 19-nortestosterone Monoclonal antibodyci ELISAHeterology Rapid test kit

收稿日期: 2010-05-10 出版日期: 2010-08-25

基金资助:

“十一五” 国家科技支撑计划重点项目(2006BAK02A21/1)、河南省高校科技创新人才支持计划(2010HASTIT026)资助项目

通讯作者: 王自良 E-mail: wangzl_2008@yahoo.com.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	姜金庆
	张海棠
	王自良
	王建华
	范国英

引用本文:

姜金庆张海棠王自良王建华范国英. 19-去甲睾酮异源性ciELISA试剂盒的研制及应用[J]. 中国生物工程杂志, 2010, 30(09): 68-74.

JIANG Jin-Qiang, ZHANG Hai-Tang, WANG Zi-Liang, WANG Jian-Hua, FAN Guo-Yang. Development and Application of Heterologous ciELISA Kit for Rapid Detection of 19-Nortestosterone Residues. China Biotechnology, 2010, 30(09): 68-74.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q511 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I09/68

［1］ Yamada M, Aramaki S, Kcrosawa M, et al. Simultaneous doping analysis of main urinary metabolites of anabolic steroids in horse by iontra Pgas chromatographytandem mass spectrometry. Anal Sci, 2008, 24(9):11991204.
［2］ Aman C S, Pastor A, Cighetti G, et al. Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotopedilution GC–MS/MS. Anal Bioanal Chem, 2006, 386(6):18691879.
［3］ Council of the European Communities.Council Directive 86/469/ EEC. Off.J.Eur. Commun.1986, Document No.L 275:36.
［4］周艳飞. 高效液相色谱法测定黄体酮、丙酸睾酮及苯甲酸雌二醇含量. 中国兽药杂志, 2003, 12:1921. Zhou Y F. Chinese Journal of Veterinary Drug, 2003, 12:1921.
［5］ Bagnati R, Fanelli R. Determination of 19nortestosterone, testosterone and trenbolone by gas chromatographynegativeion mass spectrometry after formation of the pentafluorobenzyl carboxymethoximetrimethylsilyl derivatives. J Chromatogr, 1991, 547(12):325334.
［6］ Seo J, Kim H Y, Chung B C, et al. Simultaneous determination of anabolic steroids and synthetic hormones in meat by freezinglipid filtration, solid phase extraction and gas chromatographymass spectrometry. J Chromatogr A, 2005, 1067(12):303309.
［7］ Dehennin L, Bonnaire Y, Plou P. Urinary excretion of 19norandrosterone of endogenous origin in man: quantitative analysis by gas chromatographymass spectrometry. J Chromatogr B Biomed Sci Appl, 1999, 721(2):301307.
［8］ Kim J Y, Choi M H, Kim S J, et al. Measurement of 19nortestosterone and its esters in equine plasma by highperformance liquid chromatography with tandem mass spectrometry. Rapid Commun Mass Spectrom, 2000, 14(19):18351840.
［9］ Pozo O J, Van Eenoo P, Deventer K, et al. Development and validation of a qualitative screening method for the detection of exogenous anabolic steroids in urine by liquid chromatographytandem mass spectrometry. Anal Bioanal Chem, 2007, 389(4):12091224.
［10］ Grace P B, Drake E C, Teale P, et al. Quantification of 19nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom, 2008, 22(19):29993007.
［11］ Macdougall D F, Jondorf W R. 19nortestosterone detection in canine plasma after intramuscular administration of 19nortestosterone phenylpropionate. Res Vet Sci, 1989, 47(3):399401.
［12］ Roda A, Manetta A C, Portanti O, et al. A rapid and sensitive 384well microtitre format chemiluminescent enzyme immunoassay for 19nortestosterone. Luminescence, 2003, 18(2):7278.
［13］ Degand G, Schmitz P, Maghuinrogister G. Enzyme immunoassay screening procedure for the synthetic anabolic estrogens and androgens diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone in bovine urine. J Chromatogr, 1989, 489(1):235243.
［14］ Liu L, Peng C, Jin Z, et al. Development and evaluation of a rapid lateral flow immunochromatographic stri Passay for screening 19nortestosterone. Biomed Chromatogr, 2007, 21(8):861866.
［15］ Conneely G, Aherne M, Lu H H, et al. Electrochemical immunosensors for the detection of 19nortestosterone and methyltestosterone in bovine urine. Sensors and Actuators B, 2007, 121:103112.
［16］ Galarini R, Piersanti A, Falasca S, et al. A confirmatory method for detection of a banned substance: the validation experience of a routine EU laboratory. Anal Chim Acta, 2007, 586(12):130136.
［17］ Erlanger B F, Borek F, Beiser S M, et al. Steroidprotein conjugates. I. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J Biol Chem, 1957, 228(2):713727.
［18］余德河，胥传来，彭池方，等. 食品中残留19去甲睾酮免疫原的合成与鉴定. 食品科学, 2006, 27(5):195198. Yu D H, Xu C L, Peng C F, et al. Food Science, 2006, 27(5):195198.
［19］ Beatty J D, Beatty B G, Vlahos W G, et al. Measurement of monoclonal antibody affinity by noncompetitive enzyme immunoassay. J Immunol Methods, 1987, 100(12):173179.
［20］张海棠，王自良，邓瑞广，等. 高亲和力莱克多巴胺单克隆抗体的研制及ciELISA检测方法的建立. 中国生物工程杂志, 2009, 29(1):5055. Zhang H T, Wang Z L, Deng R G, et al. China Biotechnology, 2009, 29(1):5055.
［21］ Hao X L, Kuang H, Li Y L, et al. Development of an enzymelinked immunosorbent assay for the alphacyano pyrethroids multiresidue in Tai lake water. J Agric Food Chem, 2009, 57(8):30333039.
［22］ Mercader J V, Suarezpantaleon C, Agullo C, et al. Hapten synthesis and monoclonal antibodybased immunoassay development for the detection of the fungicide kresoximmethyl. J Agric Food Chem, 2008, 56(5):15451552.
［23］ Liang Y, Liu X J, Liu Y, et al. Synthesis of three haptens for the classspecific immunoassay of O,Odimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity. Anal Chim Acta, 2008, 615(2):174183.
［24］ Holthues H, Pfeiferfukumura U, Sound I, et al. Evaluation of the concept of heterology in a monoclonal antibodybased ELISA utilizing direct hapten linkage to polystyrene microtiter plates. J Immunol Methods, 2005, 304(12):6877.

[1]	赵妍淑,张金华,宋浩. 工程原核生物和酵母菌中生产单克隆抗体和抗体片段研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(8): 74-83.
[2]	王猛,宋慧茹,程雨洁,王毅,杨波,胡征. 以核糖体蛋白L7/L12为分子标志物精准检测肺炎链球菌的研究 ^*[J]. 中国生物工程杂志, 2020, 40(4): 34-41.
[3]	孔建涛,庄英萍,郭美锦. 基于DOE设计和氨基酸补加策略提高CHO细胞表达抗CD20单克隆抗体^*[J]. 中国生物工程杂志, 2020, 40(12): 41-48.
[4]	吝建华,韩君,徐寒梅. PD-1/PD-L1免疫检查点抗体药物制剂稳定性开发[J]. 中国生物工程杂志, 2020, 40(10): 35-42.
[5]	江一帆,贾宇,王龙,王志明. 细胞培养过程对单克隆抗体糖基化修饰的影响和调控[J]. 中国生物工程杂志, 2019, 39(8): 95-103.
[6]	高倩,江洪,叶茂,郭文娟. 全球单克隆抗体药物研发现状及发展趋势 ^*[J]. 中国生物工程杂志, 2019, 39(3): 111-119.
[7]	刘国芳,刘晓志,高健,王志明. 宿主细胞残留蛋白质对单克隆抗体药物质量影响及其质量控制 ^*[J]. 中国生物工程杂志, 2019, 39(10): 105-110.
[8]	徐婧雯,张雪梅,吴忠香,朱文兵,蒋曦,巩蔚,严丽蔚,宋杰,李慧,董少忠. 抗树鼩CD3ε单克隆抗体的制备及生物学特性鉴定[J]. 中国生物工程杂志, 2018, 38(4): 54-62.
[9]	任建委,李军,李尚泽. 人源CT55蛋白原核表达及单克隆抗体的制备 ^*[J]. 中国生物工程杂志, 2018, 38(11): 1-8.
[10]	毛开云,范月蕾,王恒哲,王跃,陈大明. 全球PD-1/PD-L1单克隆抗体市场竞争格局 ^*[J]. 中国生物工程杂志, 2018, 38(11): 103-115.
[11]	孙静静,周伟伟,周雷鸣,赵巧辉,李桂林. 杂交瘤细胞体外大规模培养研究进展[J]. 中国生物工程杂志, 2018, 38(10): 82-89.
[12]	王云龙, 赵二霞, 李玉林. Thymidine Kinase 1(TK1)重组蛋白的表达、纯化及鉴定[J]. 中国生物工程杂志, 2017, 37(9): 15-22.
[13]	李敏, 吴日伟. 国内外单抗药物市场概述[J]. 中国生物工程杂志, 2017, 37(3): 106-114.
[14]	邓义熹, 李继东, 李乐, 蒙国基, 于玉根. 添加SNS能显著提高CHT填料使用寿命的研究[J]. 中国生物工程杂志, 2017, 37(1): 81-88.
[15]	任华景, 刘晓志, 王志明, 高健. 中枢神经系统疾病治疗性抗体药物应用进展[J]. 中国生物工程杂志, 2016, 36(9): 54-58.

Viewed

Full text

Abstract

Cited

Shared

Discussed