全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
科学通报  2011 

19-去甲睾酮单克隆抗体的筛选及icELISA方法的建立

, PP. 1622-1628

Keywords: 19-去甲睾酮,杂交瘤细胞株,单克隆抗体,间接竞争ELISA,牛尿

Full-Text   Cite this paper   Add to My Lib

Abstract:

采用细胞融合技术筛选抗19-去甲睾酮(nortestosterone,NT)单克隆抗体(monoclonalantibody,mAb)杂交瘤细胞株,体内诱性腹水法制备NTmAb,建立异源性间接竞争ELISA(indirectcompetitiveELISA,icELISA)标准曲线,并对其特异性、理化因素的影响和基质效应性进行探讨.结果表明,筛选的5株杂交瘤细胞分泌的单抗腹水效价为0.64×105~2.56×105,半数抑制浓度IC50为0.55~1.0ng/mL,抗体为IgG1亚型,具k轻链,亲和常数Ka为2.6×109~4.7×109L/mol.建立的icELISA标准曲线在磷酸盐缓冲液(phosphatebufferedsaline,PBS)中的线性检测范围为0.004~85.8ng/mL,IC50值为0.55ng/mL,检测限(limitofdetection,LOD)为0.002ng/mL;抗体与17a-去甲睾酮的交叉反应率(cross-reactivity,CR)为62%,与其他化合物无CR.标准品稀释液中甲醇的最佳含量为10%,牛尿120倍稀释可消除基质干扰,ELISA方法和液质联用检测(LC-MS/MS)的相关性良好(R2=0.9871).

References

[1]  2 Sauer M J, Samuels T P, Howells L G, et al. Residues and metabolism of 19-nortestosterone laurate in steers. Analyst, 1998, 123:2653-2660??
[2]  3 Conneely G, Aherne M, Lu H, et al. Development of an immunosensor for the detection of testosterone in bovine urine. Anal Chim Acta,2007, 583: 153-160??
[3]  4 Roda A, Manetta A C, Portanti O, et al. A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for19-nortestosterone. Luminescence, 2003, 18: 72-78??
[4]  7 Bahrke M S, Yesalis C E. Abuse of anabolic androgenic steroids and related substances in sport and exercise. Curr Opin Pharmacol, 2004,4: 614-620??
[5]  8 Baume N, Avois L, Schweizer C, et al. Nandrolone excretion in trained athletes: Interindividual variability in metabolism. Clin Chem,2004, 50: 355-364??
[6]  9 Akhter J, Hyder S, Ahmed M. Cerebrovascular accident associated with anabolic steroid use in a young man. Neurology, 1994, 44:2405-2406
[7]  10 Br?nnvall K, Bogdanovic N, Korhonen L, et al. 19-Nortestosterone influences neural stem cell proliferation and neurogenesis in the ratbrain. Eur J Neurosci, 2005, 21: 871-878??
[8]  11 EC (1996) Council Directive (96/22/EC) of 29 April 1996 concerning the prohibition on the use in stock farming of certain substances,having a hormonal or thyrostatic action and of beta-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Off JEur Commun, L125, 3-9
[9]  12 Bagnati R, Fanelli R. Determination of 19-nortestosterone, testosterone and trenbolone by gas chromatography-negative-ion mass spectrometryafter formation of the pentafluorobenzyl carboxymethoxime-trimethylsilyl derivatives. J Chromatogr, 1991, 547: 325-334??
[10]  13 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: 303-309??
[11]  14 Dehennin L, Bonnaire Y, Plou P. Urinary excretion of 19-norandrosterone of endogenous origin in man: Quantitative analysis by gaschromatography-mass spectrometry. J Chromatogr B, 1999, 721: 301-307??
[12]  16 Pozo O J, Van Eenoo P, Deventer K, et al. Development and validation of a qualitative screening method for the detection of exogenousanabolic steroids in urine by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem, 2007, 389: 1209-1224??
[13]  17 Grace P B, Drake E C, Teale P, et al. Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses usingliquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom, 2008, 22: 2999-3007??
[14]  23 Wang L, Zhang Y, Gao X, et al. Determination of chloramphenicol residues in milk by enzyme-linked immunosorbent assay: Improvementby biotin-streptavidin-amplified system. J Agric Food Chem, 2010, 58: 3265-3270??
[15]  25 Zhang Y, He F, Wan Y, et al. Generation of anti-trenbolone monoclonal antibody and establishment of an indirect competitive enzyme-linked immunosorbent assay for detection of trenbolone in animal tissues, feed and urine. Talanta, 2011, 83: 732-737??
[16]  29 Liang Y, Liu X J, Liu Y, et al. Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticidesand effect of hapten heterology on immunoassay sensitivity. Anal Chim Acta, 2008, 615: 174-183??
[17]  1 Sterk S, Herbold H, Blokland M, et al. Nortestosterone: Endogenous in urine of goats, sheep and mares? Analyst, 1998, 123: 2633-2636
[18]  5 Conneely G, Aherne M, Lu H, et al. Electrochemical immunosensors for the detection of 19-nortestosterone and methyltestosterone inbovine urine. Sens Actuators, 2007, 121: 103-112??
[19]  6 Yamada M, Aramaki S, Kurosawa M, et al. Simultaneous doping analysis of main urinary metabolites of anabolic steroids in horse byion-trap gas chromatography-tandem mass spectrometry. Anal Sci, 2008, 24: 1199-1204??
[20]  15 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 chromatographywith tandem mass spectrometry. Rapid Commun Mass Spectrom, 2000, 14: 1835-1840??
[21]  18 K?hler G, Milstein C. Continuous cultures of fused cells secreting antibodies of predefined specificity. Nature, 1975, 256: 495-497??
[22]  19 Chen Y, Wang Z, Wang Z, et al. Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramycinin swine tissues. J Agric Food Chem, 2008, 56: 2944-2952??
[23]  20 Zhao S Q, Sun Y M, Zhang C Y, et al. Studies on purification of methamidophos monoclonal antibodies and comparative immunoactivityof purified antibodies. Biomed Environ Sci, 2003, 16: 119-125
[24]  21 Wang Z, Zhu Y, Ding S, et al. Development of a monoclonal antibody-based broad-specificity ELISA for fluoroquinolone antibiotics infoods and molecular modeling studies of cross-reactive compounds. Anal Chem, 2007, 79: 4471-4483??
[25]  22 Wang Y C, Guo Z Q, Li Y Z, et al. Production and characterization of anti-estrone monoclonal antibody. Biomed Environ Sci, 2002, 15:103-112
[26]  24 Watanabe E, Eun H, Baba K, et al. Synthesis of haptens for development of antibodies to alkylphenols and evaluation and optimization ofa selected antibody for ELISA development. J Agric Food Chem, 2005, 53: 7395-7403??
[27]  26 Strahm E, Saudan C, Sottas P E, et al. Direct detection and quantification of 19-norandrosterone sulfate in human urine by liquid chromatography-linear ion trap mass spectrometry. J Chromatogr B, 2007, 852: 491-496??
[28]  27 James W G. Monoclonal Antibodies: Principles and Practice. New York: Academic Press, 1983. 142-147
[29]  28 Beier R C, Ripley L H, Young C R, et al. Production, characterization, and cross-reactivity studies of monoclonal antibodies against thecoccidiostat nicarbazin. J Agric Food Chem, 2001, 49: 4542-4552??

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133