恩诺沙星时间分辨荧光免疫和纳米均相荧光免疫技术的建立与考核
|
摘要
人工合成的新型抗菌药物恩诺沙星(enrofloxacin)是第三代喹诺酮类药物(FQNs)之一,它通过抑制细菌DNA螺旋酶而抗菌,具有抗菌谱广、杀菌力强、体内分布广泛及与其它抗生素之间无交叉耐药性等特点,在预防和治疗畜禽的细菌性感染及支原体病方面取得良好的效果,现已广泛地应用于兽医临床。但由于致病菌产生耐药性和长期应用所产生的不良反应以及潜在的致癌性,其残留问题已引起广泛关注。许多国家和组织等都将其列入限制使用的兽药范围。
本研究采用时间分辨荧光免疫分析法(TRFIA),建立了恩诺沙星的高灵敏的检测方法。方法采用稀土离子Eu~(3+)标记技术建立恩诺沙星时间分辨免疫检测法并对其进行方法学的考核,其检测的灵敏度可达5 ng/L,测量范围为0.01μg/L～100μg/L:向鳗鱼、猪肉、鸡肉样品中分别添加0.1μg/kg、10μg/kg、200μg/kg三个水平的恩诺沙星标准品,蜂蜜样品中添加0.34μg/kg、34.3μg/kg、342.8μg/kg,各样品的平均回收率分别为89.1℅、88.4℅、89. 0℅、89.4℅。方法的批内变异系数小于10 %,批间变异小于15 %。特异性实验表明恩诺沙星抗体与其他喹诺酮类药物交叉反应均较低,抗体的特异性很好。
同时,初步建立了恩诺沙星纳米均相发光免疫检测法,并简要的对方法的稳定性、样品添加回收率等方面进行了考核。恩诺沙星纳米均相发光免疫检测法两步共30 min的反应模式大大缩短了检测时间,同时又保证了良好的精密度,批内、批间变异率均小于5 %;方法的灵敏度为2 ng/L,线性范围为0.007~100μg/L。
研究表明,恩诺沙星-时间分辨免疫检测法是目前已报道文献中检测恩诺沙星的最灵敏的方法,具有很好的应用前景。同时,本论文还对新兴的纳米均相技术进行了较全面的探讨,并初步建立了恩诺沙星纳米均相发光免疫检测法,为我国这一领域以及相关领域的研究提供了很有价值的参考资料。
Enrofloxacin, the newly artificially synthesized antimicrobial drug, is one of the third generation quinolone derivatives. It functions effectively on preventing and treating bacterial infection by inhibiting bacterial DNA gyrase. Currently, it is used widely in veterinary clinical attributing to its broad spectrum of activity, strong bactericidal,widely distributed in vivo and no cross-resistance with other antibiotics. However, it is concerned widly of the residues because of its resistance, adverse reaction and potential carcinogenic trait. Many countries and organizations have included it to the scope of restrictions on the use of veterinary drugs.
The main purpose of this thesis is to develop the ultrasensitive detection for enrofloxacin by time-resolved fluoroimmunoassay(TRFIA).The method is to establish the Enrofloxacin -TRFIA by europium (Eu~(3+)) marker and to carry out the evaluation methodology. The sensitivity of detection was 5 ng/L and the practical linear response was from 0.01 to 100μg/L.The average recoveries of detection for enrofloxacin spiked in eel, pork, chicken samples were 89.1℅,88.4℅,89. 0℅ when the added enrofloxacin were 0.1μg/kg,10μg/kg and 200μg/kg respectively,while the honey sample was 89.4℅, with enrofloxacin added 0.34μg/kg,34.3μg/kg and 342.8μg/kg respectively. The intra-assay variation of the method was less than 10℅, and the inter-assay variation was less than15℅.The specificity test indicated that the antibody of Enrofloxacin is low cross reaction with other quinolones and the antibody enables high specificity.
Furthermore, we also initially established a homogenously indirect competitive luminescent oxygen channeling immunoassay(LICLIA) for the detection of enrofloxacin and examined constancy and recovery of the assay briefly. It only took 30min of this immunoassay to complete the detection. The results indicated that the detection limit of the assay was 2ng/L for homogenously indirect competitive LICLIA at a practical working concentration range from 0.007 to 100μg/L.
The research indicated that Enrofloxacin-TRFIA was the most sensitive method of detecting Enrofloxacin reported so far,and it has good prospects of application.Furthermore, the thesis discussed fully on the newly emerged homogeneous nanotechnology,and the enrofloxacin-LICLIA has been initially established, which would provide great valuable reference to this area.
本研究采用时间分辨荧光免疫分析法(TRFIA),建立了恩诺沙星的高灵敏的检测方法。方法采用稀土离子Eu~(3+)标记技术建立恩诺沙星时间分辨免疫检测法并对其进行方法学的考核,其检测的灵敏度可达5 ng/L,测量范围为0.01μg/L～100μg/L:向鳗鱼、猪肉、鸡肉样品中分别添加0.1μg/kg、10μg/kg、200μg/kg三个水平的恩诺沙星标准品,蜂蜜样品中添加0.34μg/kg、34.3μg/kg、342.8μg/kg,各样品的平均回收率分别为89.1℅、88.4℅、89. 0℅、89.4℅。方法的批内变异系数小于10 %,批间变异小于15 %。特异性实验表明恩诺沙星抗体与其他喹诺酮类药物交叉反应均较低,抗体的特异性很好。
同时,初步建立了恩诺沙星纳米均相发光免疫检测法,并简要的对方法的稳定性、样品添加回收率等方面进行了考核。恩诺沙星纳米均相发光免疫检测法两步共30 min的反应模式大大缩短了检测时间,同时又保证了良好的精密度,批内、批间变异率均小于5 %;方法的灵敏度为2 ng/L,线性范围为0.007~100μg/L。
研究表明,恩诺沙星-时间分辨免疫检测法是目前已报道文献中检测恩诺沙星的最灵敏的方法,具有很好的应用前景。同时,本论文还对新兴的纳米均相技术进行了较全面的探讨,并初步建立了恩诺沙星纳米均相发光免疫检测法,为我国这一领域以及相关领域的研究提供了很有价值的参考资料。
Enrofloxacin, the newly artificially synthesized antimicrobial drug, is one of the third generation quinolone derivatives. It functions effectively on preventing and treating bacterial infection by inhibiting bacterial DNA gyrase. Currently, it is used widely in veterinary clinical attributing to its broad spectrum of activity, strong bactericidal,widely distributed in vivo and no cross-resistance with other antibiotics. However, it is concerned widly of the residues because of its resistance, adverse reaction and potential carcinogenic trait. Many countries and organizations have included it to the scope of restrictions on the use of veterinary drugs.
The main purpose of this thesis is to develop the ultrasensitive detection for enrofloxacin by time-resolved fluoroimmunoassay(TRFIA).The method is to establish the Enrofloxacin -TRFIA by europium (Eu~(3+)) marker and to carry out the evaluation methodology. The sensitivity of detection was 5 ng/L and the practical linear response was from 0.01 to 100μg/L.The average recoveries of detection for enrofloxacin spiked in eel, pork, chicken samples were 89.1℅,88.4℅,89. 0℅ when the added enrofloxacin were 0.1μg/kg,10μg/kg and 200μg/kg respectively,while the honey sample was 89.4℅, with enrofloxacin added 0.34μg/kg,34.3μg/kg and 342.8μg/kg respectively. The intra-assay variation of the method was less than 10℅, and the inter-assay variation was less than15℅.The specificity test indicated that the antibody of Enrofloxacin is low cross reaction with other quinolones and the antibody enables high specificity.
Furthermore, we also initially established a homogenously indirect competitive luminescent oxygen channeling immunoassay(LICLIA) for the detection of enrofloxacin and examined constancy and recovery of the assay briefly. It only took 30min of this immunoassay to complete the detection. The results indicated that the detection limit of the assay was 2ng/L for homogenously indirect competitive LICLIA at a practical working concentration range from 0.007 to 100μg/L.
The research indicated that Enrofloxacin-TRFIA was the most sensitive method of detecting Enrofloxacin reported so far,and it has good prospects of application.Furthermore, the thesis discussed fully on the newly emerged homogeneous nanotechnology,and the enrofloxacin-LICLIA has been initially established, which would provide great valuable reference to this area.
引文
[1]郑晶,黄晓蓉,李耀平,等.鳗鱼中恩诺沙星残留的酶联免疫检测方法[J].食品科学,2004,26(10):247～250
[2] Hormazabal V, Yndestad M.Rapid Assay for Monitoring Residues of Enrofloxacin in Milk and Meat Tissues by HPLC [J] .Journal of LiquidChromatography,1994,17 (17):3775～3782
[3]李俊锁,邱月明,王超.兽药残留分析[M].上海:上海科学技术出版社,2002,257~258
[4]蔡勤仁.恩诺沙星单克隆抗体的制备及初步鉴定[D].广州:华南农业大学,2001
[5] WATANABE H,SATAKE A,KIDO Y,et a1.Monoclonal-based enzyme-linked immunosorbent assay and immumochromatographic assay for enrofloxacin in biological matrices[J].Analyst,2002,127(1):98～103
[6] Bassmen JD, Gilmer PR Jr, Gardner FH.Improved classification of anemias by MCV and RDW[J].Am J Clin Pathol ,1983,80(3):322～326
[7]丛玉隆.MCV/ROD贫血分类法临床应用价值初步探讨[J].中华医学检验杂志.1990;11:27
[8] Patel AR. The use of the technical H in the diagnosis of hovediary sphorocytrsis[J]. Blood, 1989;11:27
[9]刘国卿.药理学[M].北京:中国医药科技出版社,2002,385
[10] Krause JR.Automoted differentials in the hematology laboratory[J]. Am J Clin Pathol 1990,93(4 Suppl 1):S11～S16
[11] BROWN SA.Fluoroquinolones in animal health[J].J Vet Pharmacol Ther,1996,19(1):11～14
[12] Smith CR. The adverse effects of fluoroquinolones. J Antimicrob Chemother, 1987,19(6):709～711
[13]邓万俊.喹诺酮类抗菌药物的常见不良反应及救治.临床医药.1994;14(1):37
[14] Ross DW, Watson JS, Davis PH, et al. Evaluation of the Coulter three-part differential screen. Am J Clin Pathol, 1985,84(4):481～484
[15]张婷.氟喹诺酮类抗菌素的光遗传毒性.癌变·畸变·突变,2003,15 ( 2):124～128
[16]邱银生.喹诺酮类药物在动物组织中的残留[J].中国兽药杂志,1996,30 (1):47～49
[17]庄无忌.各国食品和饲料农药兽药残留限量大全[M].北京:中国对外经济贸易出版社,1995:982～1014
[18]蔡勤仁.恩诺沙星单克隆抗体的制备及初步鉴定[D].广州:华南农业大学,2001
[19]农业部公告第235号.动物性食品中兽药最高残留限量[S].2002
[20] Hormazabal V, Yndestad M.Rapid Assay for Monitoring Residues of Enrofloxacin in Milk and Meat Tissues by HPLC [J].Journal of Liquid Chromatography,1994, 17 (17):3775～3782
[21] Andon A,Martinez-Larranage M R.Diaz M J. Pharmacokinetics and Residues of Enrofloxacin in Chickens[J].Journal of American Veterinary Research, 1995,56 (4):50～506
[22] Waggoner T B,Bowman M C. Spectrofluorometric determination of BAY Vp 2674 residues in poultry tissues [J]. J Assoc Off Anal Chem,1987,70 (5)813～818
[23] Okerman L, De Wasch K, Van Hoof J.Analyst,1998,123(11):2 361～2 365
[24]杜黎明,卫洪清,张俊燕.反相高效液相色谱法同时测定6种氟喹诺酮类药物[J].色谱,2003,21(5):503～506
[25]刘嫒,谢孟峡,丁岚.高效液相色谱同时测定鸡蛋中4种氟喹诺酮类药物残留[J].分析化学,2004,32(3):352～355
[26] TYCZKOWSKA K,HEDEEN KM,AUCOIN DP,et a1.High-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue[J]. J Chromatogr, 1989, 493(2):337～346
[27] Turnipseed SB, Walker CC, Roybal JE,et a1.Confirmation of fluoroquinolones in catfish muscle by electrospray liquid chromatography/mass spectrometry[J].J AOAC Int,1998,81(3)554～562
[28]邓国东,杨桂香,陈杖榴.氟喹诺酮类药物在食品动物中的残留检测研究进展[J].中国兽药杂志,2000,34(3):53～58
[29] Horstk?tter C, Jiménez-Lozano E, Barrón D,et a1.Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection[J].Electrophoresis,2002,23(17):3078～3083
[30] Hassapoglidou S, Diamandis E P, Sutherland D J A.Quantification of p53 protein in tumor cell lines, breast tissue extracts and serum with time-resolved immunofluorometry[J]. Oncogene,1993,8:1501～1509
[31] Yalow R S,BersonS A.Assay of Plasma Insulin in Human Subjects by Immunological Methods[J].Nature,1959,184:1648～1649
[32]蔡勤仁,曾振灵,杨桂香,等.恩诺沙星单克隆抗体的制备及鉴定[J].中国农业科学,2004,37(7): 1060～1064
[33]郑晶,黄晓蓉,李耀平,等.鳗鱼中恩诺沙星残留量的酶联免疫检测方法[J].食品科学,2004,25(10):247～250
[34] Hiroo W,Atsuko S,Yasumasa K, et.Monoclonal based enzyme- lined immunosorbent assay and immunoc hromatographic assay for enrofloxacin in biological matrices[J].AriaI,2002,12(1):98～103
[35] Ambrose WP,Goodwin PM,Jett JH,et al. Single molecule fluorescence spectroscopy at ambient temperature [J].Chem Rev,1999,99(10):2929～2956
[36] Ekins R.More sensitive immunoassays[J].Nature,l980,284 (5751):14～15
[37] Ito K,Oda M,Tsuji A,et a1. Simultaneous determination of alpha-fetoprotein, human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay[J].J Pharm Biomed Anal,1999,20(1-2):169～178
[38]田振,郭周义,贾雅丽.时间分辨荧光免疫分析及其在临床检测中的应用[J].激光生物学报,2002,11(4):290～293
[39]李振甲,陈泮藻,高平.时间分辨荧光分析技术与应用[M].北京:科学出版社,1996
[40] Alfthan H.Comparison of immunoradiometric and immunofluorometric assays for serum hCG[J].J Immunol Methods,1986,88(2):239～244.
[41] Mitrunen K,Pettersson K,Piironen T,et al.Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum[J].Clin Chem,1995,41(8 Pt 1):1115～1120
[42] Ito K,Oda M,Tsuji A,et a1. Simultaneous determination of alpha-fetoprotein, human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay[J].J Pharm Biomed Anal,1999,20:169～175
[43] Szekeres P, Leong K, Day T, Kingston A, and Karran E Development of homogeneous 384-well high-throughput screening assays for Ab 1-40 and Ab 1-42 using AlphaScreen technology[J].J Biomol Screen, 2008, 13(2):101～111
[44] Beasley JR, Swanson R, Yang J, and Dunn D. Conversion of ELISA to no-wash technologies.Poster: SBS 2007 Conference, Montreal
[45] Sehr P, Pawlita M, and Lewis J. Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen[J].J Biomol Screen, 2007, 12(4):560～567
[46] Peppard J, Glickman F, He Y, Hu SI, Doughty J, and Goldberg R. Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen)[J].J Biomol Screen, 2003,8(2):149～156
[47] Poulsen F and Jensen KB A. luminescent oxygen channeling immunoassay for thedetermination of insulin in human plasma[J].J Biomol Screen, 2007,12(2):240～247
[48] HUANG B, XIAO H L, ZHANG J, et a1.Dual-label time-resolved fluoroimmunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A[J]. Arch Toxicol, 2009,83(6):619～624
[49]刘红,曾振灵.恩诺沙星ELIS快速检测方法的建立[J].中国兽药,2006,40(11):13～15
[50] Hemmila I,Holttinen S,Pettersson K,et al.Double-label time-resolved immunofluorometry of lutropin and follitropin in serum[J].Clin Chem,1987,33(12):2281- 2283
[51] HUANG B, XIAO H L, ZHU L G, et a1.Time-resolved fluoroimmunoassay ofα-fetoprotein[J]. Asian J Nucl Med, 2001,1(1):40～42
[52]朱立平,陈学清.免疫学常用实验方法[M].北京:北京人民军医出版社,2000
[2] Hormazabal V, Yndestad M.Rapid Assay for Monitoring Residues of Enrofloxacin in Milk and Meat Tissues by HPLC [J] .Journal of LiquidChromatography,1994,17 (17):3775～3782
[3]李俊锁,邱月明,王超.兽药残留分析[M].上海:上海科学技术出版社,2002,257~258
[4]蔡勤仁.恩诺沙星单克隆抗体的制备及初步鉴定[D].广州:华南农业大学,2001
[5] WATANABE H,SATAKE A,KIDO Y,et a1.Monoclonal-based enzyme-linked immunosorbent assay and immumochromatographic assay for enrofloxacin in biological matrices[J].Analyst,2002,127(1):98～103
[6] Bassmen JD, Gilmer PR Jr, Gardner FH.Improved classification of anemias by MCV and RDW[J].Am J Clin Pathol ,1983,80(3):322～326
[7]丛玉隆.MCV/ROD贫血分类法临床应用价值初步探讨[J].中华医学检验杂志.1990;11:27
[8] Patel AR. The use of the technical H in the diagnosis of hovediary sphorocytrsis[J]. Blood, 1989;11:27
[9]刘国卿.药理学[M].北京:中国医药科技出版社,2002,385
[10] Krause JR.Automoted differentials in the hematology laboratory[J]. Am J Clin Pathol 1990,93(4 Suppl 1):S11～S16
[11] BROWN SA.Fluoroquinolones in animal health[J].J Vet Pharmacol Ther,1996,19(1):11～14
[12] Smith CR. The adverse effects of fluoroquinolones. J Antimicrob Chemother, 1987,19(6):709～711
[13]邓万俊.喹诺酮类抗菌药物的常见不良反应及救治.临床医药.1994;14(1):37
[14] Ross DW, Watson JS, Davis PH, et al. Evaluation of the Coulter three-part differential screen. Am J Clin Pathol, 1985,84(4):481～484
[15]张婷.氟喹诺酮类抗菌素的光遗传毒性.癌变·畸变·突变,2003,15 ( 2):124～128
[16]邱银生.喹诺酮类药物在动物组织中的残留[J].中国兽药杂志,1996,30 (1):47～49
[17]庄无忌.各国食品和饲料农药兽药残留限量大全[M].北京:中国对外经济贸易出版社,1995:982～1014
[18]蔡勤仁.恩诺沙星单克隆抗体的制备及初步鉴定[D].广州:华南农业大学,2001
[19]农业部公告第235号.动物性食品中兽药最高残留限量[S].2002
[20] Hormazabal V, Yndestad M.Rapid Assay for Monitoring Residues of Enrofloxacin in Milk and Meat Tissues by HPLC [J].Journal of Liquid Chromatography,1994, 17 (17):3775～3782
[21] Andon A,Martinez-Larranage M R.Diaz M J. Pharmacokinetics and Residues of Enrofloxacin in Chickens[J].Journal of American Veterinary Research, 1995,56 (4):50～506
[22] Waggoner T B,Bowman M C. Spectrofluorometric determination of BAY Vp 2674 residues in poultry tissues [J]. J Assoc Off Anal Chem,1987,70 (5)813～818
[23] Okerman L, De Wasch K, Van Hoof J.Analyst,1998,123(11):2 361～2 365
[24]杜黎明,卫洪清,张俊燕.反相高效液相色谱法同时测定6种氟喹诺酮类药物[J].色谱,2003,21(5):503～506
[25]刘嫒,谢孟峡,丁岚.高效液相色谱同时测定鸡蛋中4种氟喹诺酮类药物残留[J].分析化学,2004,32(3):352～355
[26] TYCZKOWSKA K,HEDEEN KM,AUCOIN DP,et a1.High-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue[J]. J Chromatogr, 1989, 493(2):337～346
[27] Turnipseed SB, Walker CC, Roybal JE,et a1.Confirmation of fluoroquinolones in catfish muscle by electrospray liquid chromatography/mass spectrometry[J].J AOAC Int,1998,81(3)554～562
[28]邓国东,杨桂香,陈杖榴.氟喹诺酮类药物在食品动物中的残留检测研究进展[J].中国兽药杂志,2000,34(3):53～58
[29] Horstk?tter C, Jiménez-Lozano E, Barrón D,et a1.Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection[J].Electrophoresis,2002,23(17):3078～3083
[30] Hassapoglidou S, Diamandis E P, Sutherland D J A.Quantification of p53 protein in tumor cell lines, breast tissue extracts and serum with time-resolved immunofluorometry[J]. Oncogene,1993,8:1501～1509
[31] Yalow R S,BersonS A.Assay of Plasma Insulin in Human Subjects by Immunological Methods[J].Nature,1959,184:1648～1649
[32]蔡勤仁,曾振灵,杨桂香,等.恩诺沙星单克隆抗体的制备及鉴定[J].中国农业科学,2004,37(7): 1060～1064
[33]郑晶,黄晓蓉,李耀平,等.鳗鱼中恩诺沙星残留量的酶联免疫检测方法[J].食品科学,2004,25(10):247～250
[34] Hiroo W,Atsuko S,Yasumasa K, et.Monoclonal based enzyme- lined immunosorbent assay and immunoc hromatographic assay for enrofloxacin in biological matrices[J].AriaI,2002,12(1):98～103
[35] Ambrose WP,Goodwin PM,Jett JH,et al. Single molecule fluorescence spectroscopy at ambient temperature [J].Chem Rev,1999,99(10):2929～2956
[36] Ekins R.More sensitive immunoassays[J].Nature,l980,284 (5751):14～15
[37] Ito K,Oda M,Tsuji A,et a1. Simultaneous determination of alpha-fetoprotein, human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay[J].J Pharm Biomed Anal,1999,20(1-2):169～178
[38]田振,郭周义,贾雅丽.时间分辨荧光免疫分析及其在临床检测中的应用[J].激光生物学报,2002,11(4):290～293
[39]李振甲,陈泮藻,高平.时间分辨荧光分析技术与应用[M].北京:科学出版社,1996
[40] Alfthan H.Comparison of immunoradiometric and immunofluorometric assays for serum hCG[J].J Immunol Methods,1986,88(2):239～244.
[41] Mitrunen K,Pettersson K,Piironen T,et al.Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum[J].Clin Chem,1995,41(8 Pt 1):1115～1120
[42] Ito K,Oda M,Tsuji A,et a1. Simultaneous determination of alpha-fetoprotein, human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay[J].J Pharm Biomed Anal,1999,20:169～175
[43] Szekeres P, Leong K, Day T, Kingston A, and Karran E Development of homogeneous 384-well high-throughput screening assays for Ab 1-40 and Ab 1-42 using AlphaScreen technology[J].J Biomol Screen, 2008, 13(2):101～111
[44] Beasley JR, Swanson R, Yang J, and Dunn D. Conversion of ELISA to no-wash technologies.Poster: SBS 2007 Conference, Montreal
[45] Sehr P, Pawlita M, and Lewis J. Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen[J].J Biomol Screen, 2007, 12(4):560～567
[46] Peppard J, Glickman F, He Y, Hu SI, Doughty J, and Goldberg R. Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen)[J].J Biomol Screen, 2003,8(2):149～156
[47] Poulsen F and Jensen KB A. luminescent oxygen channeling immunoassay for thedetermination of insulin in human plasma[J].J Biomol Screen, 2007,12(2):240～247
[48] HUANG B, XIAO H L, ZHANG J, et a1.Dual-label time-resolved fluoroimmunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A[J]. Arch Toxicol, 2009,83(6):619～624
[49]刘红,曾振灵.恩诺沙星ELIS快速检测方法的建立[J].中国兽药,2006,40(11):13～15
[50] Hemmila I,Holttinen S,Pettersson K,et al.Double-label time-resolved immunofluorometry of lutropin and follitropin in serum[J].Clin Chem,1987,33(12):2281- 2283
[51] HUANG B, XIAO H L, ZHU L G, et a1.Time-resolved fluoroimmunoassay ofα-fetoprotein[J]. Asian J Nucl Med, 2001,1(1):40～42
[52]朱立平,陈学清.免疫学常用实验方法[M].北京:北京人民军医出版社,2000