氟喹诺酮类药物的筛选及达氟沙星间接竞争ELISA检测方法的建立
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摘要
氟喹诺酮类(Fluoroquinolones, FQNs)药物是一种人工合成的新型抗菌药物,随着广泛用于动物和人类的多种感染性疾病的治疗[1～2],其残留问题已引起了人们的高度重视。目前,检测FQNs药物残留常用的方法有HPLC[3～5]、ELISA等,其中ELISA法操作简便,灵敏度又高,适用于大量快速检出残留药物。本研究旨在建立这种间接竞争ELISA残留检测的方法。
本实验首先是把常用的恩诺沙星(ENRO)、诺氟沙星(NFLX)、达氟沙星(DFLX) 3种FQNs类药物,用N-羟基琥珀酰亚胺法与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联成各自的完全抗原,并且进行紫外扫描光谱等鉴定。通过免疫新西兰白兔制备出了各自的抗血清。然后采用间接竞争ELISA分别与其它几种常用的FQNs类药物(恩诺沙星、沙拉沙星、诺氟沙星、达氟沙星、环丙沙星、洛美沙星、氧氟沙星、培氟沙星)做竞争反应。通过计算交叉反应率,成功的筛选出了达氟沙星(DFLX)能同时与本身及恩诺沙星(ENRO)、沙拉沙星(SALX)、诺氟沙星(NFLX)、洛美沙星(LMLX)、氧氟沙星(OFLX)5种FQNs类药物同时发生竞争反应,且交叉反应率较高。
优化ELISA条件后,理想的包被抗原浓度为1.25μg/L,多抗(DFLX-PcAb)的工作浓度为1:10000,酶标二抗的工作浓度为1:4000。并且得到回归方程(y=0.7975-0.125x R2=0.989)、标准曲线和最适检测范围(0.1～10ng /L)。通过评价这种方法,可得批内和批间变异系数分别为3.81%和6.25%;准确度(标准回收率)为104.41%;灵敏度(最低检测限)为0.8ng/mL。以上各项指标均符合ci-ELISA检测的要求,从而建立了DFLX药物残留的间接竞争ELISA检测法。
Fluoroquinolones(FQNs) medicine is a kind of artificial synthetic neotype antibacterials,along with its general use in healing infectious diseases of mankind and animals, the residue problem has caused much attention of people. At present, commonly used detective methods are HPLC,ELISA, et al. Among the total, ELISA is easy to operate and has high sensitivity, refer to detect residual medicine generously and quickly. This research intend to establish a method of the indirect competive ELISA to detect residue.
This experiment firstly coupled ENRO,NFLX,DFLX, which are three commonly used FNQs medicine, with BSA and OVA by NHS, formed complete antigen differently, then identified the complete antigen by ultraviolet scan spectrographical identification.Immuned New Zealand albino rabbit to prepare respective antiserum. Then operated competive reaction with other commonly used medicine(Enrofloxacin,Sarafloxacin,Norfloxacin,Danofloxacin,Ciprofloxacin,Lomefloxacin,Ofloxacin,Peflacine)respectively through indirect competive ELISA. Calculated consensual reaction rate, and bolting DFLX, which could synchronize contest reaction with itself and other five kinds of FQNs medicine: SALX,ENRO,NFLX,LMLX,OFLX simultaneously,and the consensual reaction rate was high.
After optimized the condition of ELISA, the optimal concentration of the coating antigen were 1.25μg/L, the working concentration of DFLX-PcAb and sheep anti- rabbit IgG were 1:10000 and 1:4000 respectively, then got the regression equation (y=0.7975-0.125x R2=0.989) and the most suitable detect range(0.1～10 ng /L). Through evaluated this method, gained the coefficients of variation of intra-assay and inter-assay were 3.81% and 6.25%; degree of accuracy (standard recovery rate)was 104.41%; sensibility(lowest detectable limit)was 0.8ng/mL. All the index accorded with the demand of indirect competitive ELISA, so the indirect competitive ELISA was established to detect the residues of DFLX.
本实验首先是把常用的恩诺沙星(ENRO)、诺氟沙星(NFLX)、达氟沙星(DFLX) 3种FQNs类药物,用N-羟基琥珀酰亚胺法与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联成各自的完全抗原,并且进行紫外扫描光谱等鉴定。通过免疫新西兰白兔制备出了各自的抗血清。然后采用间接竞争ELISA分别与其它几种常用的FQNs类药物(恩诺沙星、沙拉沙星、诺氟沙星、达氟沙星、环丙沙星、洛美沙星、氧氟沙星、培氟沙星)做竞争反应。通过计算交叉反应率,成功的筛选出了达氟沙星(DFLX)能同时与本身及恩诺沙星(ENRO)、沙拉沙星(SALX)、诺氟沙星(NFLX)、洛美沙星(LMLX)、氧氟沙星(OFLX)5种FQNs类药物同时发生竞争反应,且交叉反应率较高。
优化ELISA条件后,理想的包被抗原浓度为1.25μg/L,多抗(DFLX-PcAb)的工作浓度为1:10000,酶标二抗的工作浓度为1:4000。并且得到回归方程(y=0.7975-0.125x R2=0.989)、标准曲线和最适检测范围(0.1～10ng /L)。通过评价这种方法,可得批内和批间变异系数分别为3.81%和6.25%;准确度(标准回收率)为104.41%;灵敏度(最低检测限)为0.8ng/mL。以上各项指标均符合ci-ELISA检测的要求,从而建立了DFLX药物残留的间接竞争ELISA检测法。
Fluoroquinolones(FQNs) medicine is a kind of artificial synthetic neotype antibacterials,along with its general use in healing infectious diseases of mankind and animals, the residue problem has caused much attention of people. At present, commonly used detective methods are HPLC,ELISA, et al. Among the total, ELISA is easy to operate and has high sensitivity, refer to detect residual medicine generously and quickly. This research intend to establish a method of the indirect competive ELISA to detect residue.
This experiment firstly coupled ENRO,NFLX,DFLX, which are three commonly used FNQs medicine, with BSA and OVA by NHS, formed complete antigen differently, then identified the complete antigen by ultraviolet scan spectrographical identification.Immuned New Zealand albino rabbit to prepare respective antiserum. Then operated competive reaction with other commonly used medicine(Enrofloxacin,Sarafloxacin,Norfloxacin,Danofloxacin,Ciprofloxacin,Lomefloxacin,Ofloxacin,Peflacine)respectively through indirect competive ELISA. Calculated consensual reaction rate, and bolting DFLX, which could synchronize contest reaction with itself and other five kinds of FQNs medicine: SALX,ENRO,NFLX,LMLX,OFLX simultaneously,and the consensual reaction rate was high.
After optimized the condition of ELISA, the optimal concentration of the coating antigen were 1.25μg/L, the working concentration of DFLX-PcAb and sheep anti- rabbit IgG were 1:10000 and 1:4000 respectively, then got the regression equation (y=0.7975-0.125x R2=0.989) and the most suitable detect range(0.1～10 ng /L). Through evaluated this method, gained the coefficients of variation of intra-assay and inter-assay were 3.81% and 6.25%; degree of accuracy (standard recovery rate)was 104.41%; sensibility(lowest detectable limit)was 0.8ng/mL. All the index accorded with the demand of indirect competitive ELISA, so the indirect competitive ELISA was established to detect the residues of DFLX.
引文
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[3] Samuelsen O B. High-performance liquid chromatographic determination of furazolidone in Atlantic salmon(Salmo salar)tissue[J].Jour nal of Chromatography,1990,528(2):495 500.
[4] Smallidge R L,Rowe N B. L,et al.High-performance liquid chromatographic determina- tion of furazolidone in feed and feed premixes[J].Journal of AOAC International,1981,64(5):1100 1104.
[5] 葛宝坤,王云凤,贺信,等.高效液相色谱法测定鸡肉、水产品中呋喃西林和呋喃唑酮残留量的研究[J].中国卫生检验杂志 2002,12(6):661 662.
[6] 邱银生,操继跃,王大菊,等.盐酸沙拉沙星在肉鸡组织中的残留[J].中国兽医学报,2001,21(5):515-518.
[7] 丁焕中,冯淇辉,陈杖榴,等.沙拉沙星在猪体内的药动学研究[J].畜牧兽医学报,2002,33(1):55-58.
[8] Rolinski Z,owalski C W,Laz P.Distribution and elimination of norflox from broiler chicken tissue and eggs[J].Jvet Pharmacol Therap,1997,20(Suppl1): 200-201.
[9] 张国文,占春瑞,等.HPLC 法测定鳗鱼中多种喹诺酮[J].食品科技学报.2004(11): 69-71.
[10] 刘媛,谢孟峡,丁岚等. 高效液相色谱同时测定鸡蛋中 4 种氟喹诺酮类药物的残留[J].分析化学研究简报,2004,32(3):352-355.
[11] 长尾稳[日].肉中喹诺酮药物残留的 HPLC 简易分析[J]. 食品卫生学杂,1998, 39(5): 329-332.
[12] Rovhal T, Walker CC, Pfenning AP, et al. Concurrent determi-nation of four fluoroquinolones in catfish, shrimp, and salmon by liquid chromatography with fluorescence detection[J]. JAO AC Int, 2002,85( 6):1293- 1301.
[13] Cinqnina AL, Roberti P, Giannetti L,et al. Determination of enroflosacin and its meLaboliLc ciproflosacin in goal milk by high performance liquid chromatography with diode array detection Optimization and validation[J]. J Chromatogr A, 2003 ,987(12):221-226.
[14] Hernandez VI, Borrull F, Calull M.Using nonaqncons capillary electrophoresis to analyze several qninolones in pig kilney samples [J]. Electrophoresis,2002,23 (3):506- 511.
[15] Barron D, Jimenez-Lozano E, Bailace S, et al. Determination of diflesacin and saraflesacin in chicken muscle using solid-phase ex-traction and capillary electrophoresis[J].J Chromatogr B Analyt TechnolBiomed Life Sei, 2002,767(2):313-319
[16] Hernandez M,Aguilar Rorrull F Determinalinn of ciproflosacin, enroflosacin and flumequine in pig plasma samples by capillary isotachophoresis-capillary zone electrophoresis[J]. J ChromatogrB Analyt Technol Biomed Life Sei,2002,772(1):163 -172。
[17] 朱斌,蒋受军,胡昌勤.高效毛细管电泳法测定加替沙星含量[J].药分析杂志, 2002,5:397-400.
[18] Jeffery R M,Verdel K D,Willam H,et al.Liquid chromatographic determination of sarfloxacin residues in channel catfish muscle[J].AOAC Int,1994,77(4):871-875.
[19] Roybal J E,Pfenning A P,Turnipseed S B,et al.Determination of fourfluoroquinolones in milk by liquid chromatography[J].AOACInt,1997,80(5):982-987.
[20] Lesley Johnston, Lindsey Mackay, Meg Croft.J. ChromatogrA,2002,(982):97-109.
[21] 施冰,张志刚,吴抒怀,等.LC/MS/MS 测定水产品中 7 种氟喹诺酮类抗菌素残留量的方法研究[J].检验检疫科学,2004,14(增刊):25-30
[22]Toussaint G,Bordin A,Janosi A R Rodriguez.Validation of a liquid chromatog raphy tandem mass spectrometry methrod for the the simultaneousquantification of 11(fluro) quinolone antibiotics in swine kidney[J].Journal of Chromatogra phy,2002,976:195-206.
[23] Ckerman L,Hoof J V,Debeuckelaere W.Evaluation of the European Four-plate Test as a tool for screening antibiotic residues in meat samples from retailoutlets[J].AOAC Int,1998, 81(1):51-56.
[24] 沈建忠,何继红 兽药残留分析技术研究进展[].中国禽业导刊,2004,21(15):15-16.
[25] Carol K. Holtzapple,Sandra A.Buckley,Larry H.Stanker.Determin Tion of fluoroquinolones in using an on-line clean-up column coPled to high-performance immunoa ffinity-reversed-phase liquid chroatography.Journal of Chromatography B,2001,754:1-9.
[26] Holtzapple CK, Buc:klev SA, Stanker LH.Determination of fluo- roquinolonas in serum using an on-line clean-up column coupled to higl performance immuno- affinitv- reverse-phase liquid camomato-graphy[J].J Chromatogr B Biomed SciAppl, 2001, 754(1):l-9
[27] P.Tijissen. Practice and Theary of Enzyme Immunoassays, lsevier, Amsterdam, 1985, 83~85.
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[29] Vallejo RP, Bogus ER, Mumma RO. Effects of hapten structur and bridging groups on antisera specificity in parathion immunoassay development. J Agic Food Chem, 1982,30(3): 572-580.
[30] Tijssen P.Practice and Theory of Enzyme Immunoassay[M].Amsterdam:Elsever,1985.
[31] 石德时,周斌,覃雅丽等. 氯霉素间接竞争 ELISA(ciELISA)检测方法的建立. 中国兽医学报,2002,22(1):77-78
[32] 李俊锁,钱传范. 兽药残留免疫分析及其进展. 中国兽医学报,1998,18(4):411-415
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[2] 邱银生.兽用喹诺酮类抗菌剂甲磺酸达诺沙星的研究概况[J].中国兽药杂志,1994,28(1):51-53.
[3] Samuelsen O B. High-performance liquid chromatographic determination of furazolidone in Atlantic salmon(Salmo salar)tissue[J].Jour nal of Chromatography,1990,528(2):495 500.
[4] Smallidge R L,Rowe N B. L,et al.High-performance liquid chromatographic determina- tion of furazolidone in feed and feed premixes[J].Journal of AOAC International,1981,64(5):1100 1104.
[5] 葛宝坤,王云凤,贺信,等.高效液相色谱法测定鸡肉、水产品中呋喃西林和呋喃唑酮残留量的研究[J].中国卫生检验杂志 2002,12(6):661 662.
[6] 邱银生,操继跃,王大菊,等.盐酸沙拉沙星在肉鸡组织中的残留[J].中国兽医学报,2001,21(5):515-518.
[7] 丁焕中,冯淇辉,陈杖榴,等.沙拉沙星在猪体内的药动学研究[J].畜牧兽医学报,2002,33(1):55-58.
[8] Rolinski Z,owalski C W,Laz P.Distribution and elimination of norflox from broiler chicken tissue and eggs[J].Jvet Pharmacol Therap,1997,20(Suppl1): 200-201.
[9] 张国文,占春瑞,等.HPLC 法测定鳗鱼中多种喹诺酮[J].食品科技学报.2004(11): 69-71.
[10] 刘媛,谢孟峡,丁岚等. 高效液相色谱同时测定鸡蛋中 4 种氟喹诺酮类药物的残留[J].分析化学研究简报,2004,32(3):352-355.
[11] 长尾稳[日].肉中喹诺酮药物残留的 HPLC 简易分析[J]. 食品卫生学杂,1998, 39(5): 329-332.
[12] Rovhal T, Walker CC, Pfenning AP, et al. Concurrent determi-nation of four fluoroquinolones in catfish, shrimp, and salmon by liquid chromatography with fluorescence detection[J]. JAO AC Int, 2002,85( 6):1293- 1301.
[13] Cinqnina AL, Roberti P, Giannetti L,et al. Determination of enroflosacin and its meLaboliLc ciproflosacin in goal milk by high performance liquid chromatography with diode array detection Optimization and validation[J]. J Chromatogr A, 2003 ,987(12):221-226.
[14] Hernandez VI, Borrull F, Calull M.Using nonaqncons capillary electrophoresis to analyze several qninolones in pig kilney samples [J]. Electrophoresis,2002,23 (3):506- 511.
[15] Barron D, Jimenez-Lozano E, Bailace S, et al. Determination of diflesacin and saraflesacin in chicken muscle using solid-phase ex-traction and capillary electrophoresis[J].J Chromatogr B Analyt TechnolBiomed Life Sei, 2002,767(2):313-319
[16] Hernandez M,Aguilar Rorrull F Determinalinn of ciproflosacin, enroflosacin and flumequine in pig plasma samples by capillary isotachophoresis-capillary zone electrophoresis[J]. J ChromatogrB Analyt Technol Biomed Life Sei,2002,772(1):163 -172。
[17] 朱斌,蒋受军,胡昌勤.高效毛细管电泳法测定加替沙星含量[J].药分析杂志, 2002,5:397-400.
[18] Jeffery R M,Verdel K D,Willam H,et al.Liquid chromatographic determination of sarfloxacin residues in channel catfish muscle[J].AOAC Int,1994,77(4):871-875.
[19] Roybal J E,Pfenning A P,Turnipseed S B,et al.Determination of fourfluoroquinolones in milk by liquid chromatography[J].AOACInt,1997,80(5):982-987.
[20] Lesley Johnston, Lindsey Mackay, Meg Croft.J. ChromatogrA,2002,(982):97-109.
[21] 施冰,张志刚,吴抒怀,等.LC/MS/MS 测定水产品中 7 种氟喹诺酮类抗菌素残留量的方法研究[J].检验检疫科学,2004,14(增刊):25-30
[22]Toussaint G,Bordin A,Janosi A R Rodriguez.Validation of a liquid chromatog raphy tandem mass spectrometry methrod for the the simultaneousquantification of 11(fluro) quinolone antibiotics in swine kidney[J].Journal of Chromatogra phy,2002,976:195-206.
[23] Ckerman L,Hoof J V,Debeuckelaere W.Evaluation of the European Four-plate Test as a tool for screening antibiotic residues in meat samples from retailoutlets[J].AOAC Int,1998, 81(1):51-56.
[24] 沈建忠,何继红 兽药残留分析技术研究进展[].中国禽业导刊,2004,21(15):15-16.
[25] Carol K. Holtzapple,Sandra A.Buckley,Larry H.Stanker.Determin Tion of fluoroquinolones in using an on-line clean-up column coPled to high-performance immunoa ffinity-reversed-phase liquid chroatography.Journal of Chromatography B,2001,754:1-9.
[26] Holtzapple CK, Buc:klev SA, Stanker LH.Determination of fluo- roquinolonas in serum using an on-line clean-up column coupled to higl performance immuno- affinitv- reverse-phase liquid camomato-graphy[J].J Chromatogr B Biomed SciAppl, 2001, 754(1):l-9
[27] P.Tijissen. Practice and Theary of Enzyme Immunoassays, lsevier, Amsterdam, 1985, 83~85.
[28] 陈新建,陈梅英.免疫学技术在植物学中的应用[M].北京:中国农业出版社,1998,54-58.
[29] Vallejo RP, Bogus ER, Mumma RO. Effects of hapten structur and bridging groups on antisera specificity in parathion immunoassay development. J Agic Food Chem, 1982,30(3): 572-580.
[30] Tijssen P.Practice and Theory of Enzyme Immunoassay[M].Amsterdam:Elsever,1985.
[31] 石德时,周斌,覃雅丽等. 氯霉素间接竞争 ELISA(ciELISA)检测方法的建立. 中国兽医学报,2002,22(1):77-78
[32] 李俊锁,钱传范. 兽药残留免疫分析及其进展. 中国兽医学报,1998,18(4):411-415
[33] 周培,陆贻通. 农药残留的酶联免疫检测技术研究进展. 环境污染与防治,2002,24(4):248-251
[34] 王军,朱鲁生,林爱军等. 农药残留速测技术研究进展. 环境污染治理技术与设备,2001,2(1):17-24
[35] 常文保,张柏林,慈云祥.药物免疫分析及其进展.分析化学,1994, 22(11): 1167-1175
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