三种试剂盒检测盐酸克伦特罗残留的比较研究
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摘要
盐酸克伦特罗(clenbuterol hydrochloride,CL)是一种β_2-肾上腺素受体激动剂,20世纪70年代起,被作为“营养重分配剂”及“促生长剂”用于饲料生产中,借以提高动物瘦肉率和减少脂肪率。但由于盐酸克伦特罗具有吸收快、分布广、脂溶性高以及残留性积累和半衰期长的特性,易在动物体内积蓄残留,造成人食用后引起中毒,我国和欧州等国家都明令禁止在畜牧业生产中使用此药。
目前,检测盐酸克伦特罗的方法很多,但应用最广的是酶联免疫吸附测定法(ELISA)。但盐酸克伦特罗试剂盒对温度和反应时间的要求很严格,本文就是在这种背景下,研究不同的ELISA试剂盒(德国拜发试剂盒、比利时BIX-O试剂盒和北京望尔试剂盒),在不同的温度和反应时间下的差别。
试验结果表明:温度对ELISA试剂盒的检测结果影响显著,但在一定温度范围内(20℃-37℃),对检测结果的判断没有影响;反应时间对ELISA试剂盒的影响不显著(反应时间的变化范围为30±5min);3种试剂盒之间差异不显著,这说明不同竞争类型的试剂盒对检测结果没有影响。
Clenbuterol hydrochloride(CL) is a kind ofβ_2-adrenoceptor agonists,which was regarded as "nutritional repartition agent" or "growth promoter" applied in feed production in order to make an increase on animal mean factor and to reduce fat ratio. However,CL was banned in the production of animal husbandry in view of causing food poisoning after being taken by human,for the reason that it is prone to be remained accumulatively in animals concerning some of its characteristics such as rapid absorption, wide distribution,high liposolubility,accumulated residue,and prolonged half life.
At present,ELISA is very broadly used among many of methods in detecting CL. Nonetheless,CL kit is strictly demanded for temperature and reaction time.Under such circumstances,our work is to study the differences among differed ELISA kits(German R-Biopharm ELISA Kit,Belgian BIX-O ELISA kit and Beijing Wanger ELISA Kit) in different reaction time and temperature.
Research results indicate that the temperature will have a remarkable influence on the results whenever it goes out of a certain temperature range(20℃-37℃);reaction time makes no significant difference to ELISA kit(reaction time range:30±5min);Since there are no obvious difference among these three kits,kits of different competitive types make no difference to detected results.
目前,检测盐酸克伦特罗的方法很多,但应用最广的是酶联免疫吸附测定法(ELISA)。但盐酸克伦特罗试剂盒对温度和反应时间的要求很严格,本文就是在这种背景下,研究不同的ELISA试剂盒(德国拜发试剂盒、比利时BIX-O试剂盒和北京望尔试剂盒),在不同的温度和反应时间下的差别。
试验结果表明:温度对ELISA试剂盒的检测结果影响显著,但在一定温度范围内(20℃-37℃),对检测结果的判断没有影响;反应时间对ELISA试剂盒的影响不显著(反应时间的变化范围为30±5min);3种试剂盒之间差异不显著,这说明不同竞争类型的试剂盒对检测结果没有影响。
Clenbuterol hydrochloride(CL) is a kind ofβ_2-adrenoceptor agonists,which was regarded as "nutritional repartition agent" or "growth promoter" applied in feed production in order to make an increase on animal mean factor and to reduce fat ratio. However,CL was banned in the production of animal husbandry in view of causing food poisoning after being taken by human,for the reason that it is prone to be remained accumulatively in animals concerning some of its characteristics such as rapid absorption, wide distribution,high liposolubility,accumulated residue,and prolonged half life.
At present,ELISA is very broadly used among many of methods in detecting CL. Nonetheless,CL kit is strictly demanded for temperature and reaction time.Under such circumstances,our work is to study the differences among differed ELISA kits(German R-Biopharm ELISA Kit,Belgian BIX-O ELISA kit and Beijing Wanger ELISA Kit) in different reaction time and temperature.
Research results indicate that the temperature will have a remarkable influence on the results whenever it goes out of a certain temperature range(20℃-37℃);reaction time makes no significant difference to ELISA kit(reaction time range:30±5min);Since there are no obvious difference among these three kits,kits of different competitive types make no difference to detected results.
引文
[1]李俊杰.浅谈饲有瘦肉精猪肉对人体危害及其检疫.福建畜牧兽医.2001.23(5):53
[2]Ricks C.A.,Baker P.K.,Dalrymple R.H.,Doscher M.E.,D.L.Ingle and J.Pankavish.1984.Use of clenbuterol to alter muscle and fat accretion in swine.Fed.Proc.43:851-859
[3]Baker P.K,Dalrymple R.H.,Ingle D.L.,and Ricks C.A.1984.use of a B-adrenergic agonist to alter muscle and fat deposition in lambs.J.Anim.Sci.59:1356-1364
[4]Williams P.E.V.The use of β-agonists as a mean of altering body composition in livestock species.Nutr.Abstr.Rev.,Sci.1987.57:453-464
[5]Salas OA,Romo FP,Gonzalez EA,Sanchez W,Coello CL,Camberos LO.1993.Effect of a beta-adrenergic agonist in the feeding of broiler chickens.Veterinaria Mexico.24(1):37-41
[6]Pulce CD,Lamaison D,Keck Getai.Collective human food poisonings by Clenbutenol residues in Vealliver[J].VetHum Toxicol,1995,27:1118-1121.
[7]陈忠熙.一起克伦特罗食物中毒的调查.中国卫生监督杂志.2001.8(5):205-506
[8]王选平.盐酸克伦特罗食物中毒[J].预防医学文献信息,2004,10(1):73-75.
[9]王选年,张改平.盐酸克伦特罗在我国的非法使用及安全评价[J].中国畜牧杂志,2004,40(3):52-55.
[10]张琪玉,李海云,等.盐酸克伦特罗中毒40例分析[J].中国医师杂志,2002,4(9):1035-1036.
[11]李慧,苏晓鸥.盐酸克伦特罗在动物组织中的残留及检测[J].检测分析,2004,15:21-24.
[12]凌明亮,黄仁术.瘦肉精的危害及检测方法[J].安徽农学通报,2003,9(4):87-88.
[13]Yamamoto L,Iwata K.1982.Enzyme immunoassay for clenbuterol,an beta-2-adrenergic stimlant.J.Immnoassay.3(2):155-171
[14]Fujino A.,Shibata K.,Ohyama T.,Thuj itaand N.,Eikawa N.1984.Metabolic fate of clenbuterol(NAB365)(Ⅱ),absorption and excretion in dogs.NRI Life Science,4-7-1,Kajiwara,Kamakurashi,Kangawa,247 Japan,Iyakuhin Kenky 15(3):254-268
[15]Zimmer A.,1976.Pharmakokinetik and Metaboliten-muster von Clenbuterol beim Kaninchen and beim Hund.Arzneim-Forsch.261-442
[16]Merwe PJ van der,Toerien S Burger WP,van der Merwe PJ,Peteghem C van.1998.Pharmacokinetics of clenbuterol in the ostrich.Third International Symposium on Hormone and Veterinary Drug Residue Analysis,Bruges,Belgium,June 2-5,Analyst.123(12):2715-2717
[17]Meyer H.H.D.,and L.M.Rinke.1991.The pharmacokinetics and residues of Clenbuterol in veal calves.J.Anim.Sci.69:4538-4546
[18]Malucelli,A.,F.Ellendorff and H.H.D.Meyer.1994.Tissue Distribution and Residues of Clenbuterol,Salbutamol,and Terbutaline in Tissues of Treated Broiler Chickens.J.Anim.Sci.72:1555-1560
[19]Zalko D.,Bories G.,Tulliez J.1998.Metabolic fate ofclenbuterol in calves.J agric food chem.Washington,D.C.American Chemical Society.May v.46(5):1935-1943.Hund.Arzneim-Forsch.26:1446-1453
[20]Smith D.J,Paulson G D.1997.Distribution,elimination,and residues of[(14)~C]clenbuterol HCL in HolstEin calves.J Anim Sci.75:454-461
[21]王选年,康晓迪,潘耀谦,等.盐酸克伦特罗的残留及检测[J].动物医学进展,2002,23(3):41-46.
[22]Smith D.J.1998.Total Radioctive Residues and Clenbuterol Residues in Edible Tissues and the Stererochemical Composition of Clenbterol in Lives of Dietary After Exposure to Three Levels of Ditary[(14)~C]Clenbuterol HCL and Three Preslaughter Withdrawal Periods.J.Anim.Sci.76:3043-3053
[23]古艳丽,曲志娜,郑增忍,等.畜产品中盐酸克伦特罗残留的风险评估[B].畜牧与兽医,2006,38(2):35-38
[24]中华人民共和国农业部.动物性食品中兽药最高残留限量[Z].1997,农牧发7号.
[25]Smith.D.J.2000.Total radioactive residues and clenbuterol residues in swine after dietary administration of[(14)~C]Clenbuterol for seven days and prestaughter wtthdrawal periods of zero,three,or seven days.J.Anim.Sci.78:2903-2912
[26]朱淑琴,张庆生,肖论征,等.盐酸克伦特罗在猪尿液中残留时间初探[A].江西畜牧兽医杂志,2004,6:8
[27]Gaillard Y,Balland A.,Doucet R,Pepin.1997.Detection of illegal clenbuterol use in Calves using hair analysis.Application in meat quality control.Chromatogr B Biomed Sci Appl.1997.703(1-2):85-95.
[28]Fente CA.,1999.Determination ofclenbuterol residues in bovine hair by using diphasic,dialysis and gas chromatography-mass spectrometry.J Chromatogr B Biomed Sci Appl.,726(1-2):133-9
[29]O'Keeffe MJ,O'Keeffe M,Glennon JD,Lightfield AR,Maxwell RJ.1998.Supercritical fluid extraction of clenbuferol from bovine liver tissue.Analyst.123 (12):2711-2714
[30]Hooijerink H,Schilt R,Haasnoot W,Courtheijn.1991.Determination of clenbuterol in urine of calves by high-performance liquid chromatography with in series ultraviolet and electrochemical detection.Journal of Pharmaceutical band Biomedical Analysis.9(6):485-492;18
[31]Tsai CE,1994.Kondo FLiquid chromatographic determination of salbutamol and clenbuterol residues in swine serum and muscle.Microbios.80:325,251-258
[32]Hauck HE,Mack M.,Reuke S.,Herbert H.1989.Hydrophilic surface modified HPTLC precoated layers in pesticide analysis,Journal of Planar Chromatography.1989,2(4):268-275;25
[33]Elbert A.Hogendoom,Piet van Zoonen.1998.The potential of restricted access medis columns as applied in coupled in coupled-column LC/LC-TSP/MS/MS for the highspeed determination of target ompounds in serum.Application to the direct trace analysis of salbutamol and clenbuterol.Analchem.70:1362-1368
[34]GiauIt,J and Fourtillan,J.B.1990.Determination of clenbuterol in booing plasma and tissue by gas chromatography-negative chemical ionization mass spectrometry.Journal of chromatography.518:41-52
[35]Gausephol C,Blaschke G.Stereoselective determination of clenbuterol residues in human urine by capillary electropho-resis[J].Journal of chromatography,1998,713:1175-1176.
[36]Pou K.,Ong H.,Adam A.1994.Combined immunoextraction approach coupled to a chemiluminescenec enzyme immunoassay for the determination of trace levels of salbtmol and clenbuterolin tessue sample.Analyst.119(12):2659-2662
[37]Sawaya W N,Lone K,Husain A,et al.Screening for β-agonists in sheep urine and eyes by anenzyme-linked immunosorbent assay in the state of Kuwait[J].Food control,2000,11:1-5.
[38]柳其芳.饲料与尿液中盐酸克伦特罗测定方法的研究[R].职业与健康,2005,21(12):1951-1952.
[39]芮冬妹.滴金免疫法快速检测活动性肺结核IgG抗体[J].现代中西医结合杂志,2001,10(12):1175-1176.
[40]农牧发[2001]38号文.猪尿中盐酸克伦特罗的检测--酶联免疫吸附测定法.
[2]Ricks C.A.,Baker P.K.,Dalrymple R.H.,Doscher M.E.,D.L.Ingle and J.Pankavish.1984.Use of clenbuterol to alter muscle and fat accretion in swine.Fed.Proc.43:851-859
[3]Baker P.K,Dalrymple R.H.,Ingle D.L.,and Ricks C.A.1984.use of a B-adrenergic agonist to alter muscle and fat deposition in lambs.J.Anim.Sci.59:1356-1364
[4]Williams P.E.V.The use of β-agonists as a mean of altering body composition in livestock species.Nutr.Abstr.Rev.,Sci.1987.57:453-464
[5]Salas OA,Romo FP,Gonzalez EA,Sanchez W,Coello CL,Camberos LO.1993.Effect of a beta-adrenergic agonist in the feeding of broiler chickens.Veterinaria Mexico.24(1):37-41
[6]Pulce CD,Lamaison D,Keck Getai.Collective human food poisonings by Clenbutenol residues in Vealliver[J].VetHum Toxicol,1995,27:1118-1121.
[7]陈忠熙.一起克伦特罗食物中毒的调查.中国卫生监督杂志.2001.8(5):205-506
[8]王选平.盐酸克伦特罗食物中毒[J].预防医学文献信息,2004,10(1):73-75.
[9]王选年,张改平.盐酸克伦特罗在我国的非法使用及安全评价[J].中国畜牧杂志,2004,40(3):52-55.
[10]张琪玉,李海云,等.盐酸克伦特罗中毒40例分析[J].中国医师杂志,2002,4(9):1035-1036.
[11]李慧,苏晓鸥.盐酸克伦特罗在动物组织中的残留及检测[J].检测分析,2004,15:21-24.
[12]凌明亮,黄仁术.瘦肉精的危害及检测方法[J].安徽农学通报,2003,9(4):87-88.
[13]Yamamoto L,Iwata K.1982.Enzyme immunoassay for clenbuterol,an beta-2-adrenergic stimlant.J.Immnoassay.3(2):155-171
[14]Fujino A.,Shibata K.,Ohyama T.,Thuj itaand N.,Eikawa N.1984.Metabolic fate of clenbuterol(NAB365)(Ⅱ),absorption and excretion in dogs.NRI Life Science,4-7-1,Kajiwara,Kamakurashi,Kangawa,247 Japan,Iyakuhin Kenky 15(3):254-268
[15]Zimmer A.,1976.Pharmakokinetik and Metaboliten-muster von Clenbuterol beim Kaninchen and beim Hund.Arzneim-Forsch.261-442
[16]Merwe PJ van der,Toerien S Burger WP,van der Merwe PJ,Peteghem C van.1998.Pharmacokinetics of clenbuterol in the ostrich.Third International Symposium on Hormone and Veterinary Drug Residue Analysis,Bruges,Belgium,June 2-5,Analyst.123(12):2715-2717
[17]Meyer H.H.D.,and L.M.Rinke.1991.The pharmacokinetics and residues of Clenbuterol in veal calves.J.Anim.Sci.69:4538-4546
[18]Malucelli,A.,F.Ellendorff and H.H.D.Meyer.1994.Tissue Distribution and Residues of Clenbuterol,Salbutamol,and Terbutaline in Tissues of Treated Broiler Chickens.J.Anim.Sci.72:1555-1560
[19]Zalko D.,Bories G.,Tulliez J.1998.Metabolic fate ofclenbuterol in calves.J agric food chem.Washington,D.C.American Chemical Society.May v.46(5):1935-1943.Hund.Arzneim-Forsch.26:1446-1453
[20]Smith D.J,Paulson G D.1997.Distribution,elimination,and residues of[(14)~C]clenbuterol HCL in HolstEin calves.J Anim Sci.75:454-461
[21]王选年,康晓迪,潘耀谦,等.盐酸克伦特罗的残留及检测[J].动物医学进展,2002,23(3):41-46.
[22]Smith D.J.1998.Total Radioctive Residues and Clenbuterol Residues in Edible Tissues and the Stererochemical Composition of Clenbterol in Lives of Dietary After Exposure to Three Levels of Ditary[(14)~C]Clenbuterol HCL and Three Preslaughter Withdrawal Periods.J.Anim.Sci.76:3043-3053
[23]古艳丽,曲志娜,郑增忍,等.畜产品中盐酸克伦特罗残留的风险评估[B].畜牧与兽医,2006,38(2):35-38
[24]中华人民共和国农业部.动物性食品中兽药最高残留限量[Z].1997,农牧发7号.
[25]Smith.D.J.2000.Total radioactive residues and clenbuterol residues in swine after dietary administration of[(14)~C]Clenbuterol for seven days and prestaughter wtthdrawal periods of zero,three,or seven days.J.Anim.Sci.78:2903-2912
[26]朱淑琴,张庆生,肖论征,等.盐酸克伦特罗在猪尿液中残留时间初探[A].江西畜牧兽医杂志,2004,6:8
[27]Gaillard Y,Balland A.,Doucet R,Pepin.1997.Detection of illegal clenbuterol use in Calves using hair analysis.Application in meat quality control.Chromatogr B Biomed Sci Appl.1997.703(1-2):85-95.
[28]Fente CA.,1999.Determination ofclenbuterol residues in bovine hair by using diphasic,dialysis and gas chromatography-mass spectrometry.J Chromatogr B Biomed Sci Appl.,726(1-2):133-9
[29]O'Keeffe MJ,O'Keeffe M,Glennon JD,Lightfield AR,Maxwell RJ.1998.Supercritical fluid extraction of clenbuferol from bovine liver tissue.Analyst.123 (12):2711-2714
[30]Hooijerink H,Schilt R,Haasnoot W,Courtheijn.1991.Determination of clenbuterol in urine of calves by high-performance liquid chromatography with in series ultraviolet and electrochemical detection.Journal of Pharmaceutical band Biomedical Analysis.9(6):485-492;18
[31]Tsai CE,1994.Kondo FLiquid chromatographic determination of salbutamol and clenbuterol residues in swine serum and muscle.Microbios.80:325,251-258
[32]Hauck HE,Mack M.,Reuke S.,Herbert H.1989.Hydrophilic surface modified HPTLC precoated layers in pesticide analysis,Journal of Planar Chromatography.1989,2(4):268-275;25
[33]Elbert A.Hogendoom,Piet van Zoonen.1998.The potential of restricted access medis columns as applied in coupled in coupled-column LC/LC-TSP/MS/MS for the highspeed determination of target ompounds in serum.Application to the direct trace analysis of salbutamol and clenbuterol.Analchem.70:1362-1368
[34]GiauIt,J and Fourtillan,J.B.1990.Determination of clenbuterol in booing plasma and tissue by gas chromatography-negative chemical ionization mass spectrometry.Journal of chromatography.518:41-52
[35]Gausephol C,Blaschke G.Stereoselective determination of clenbuterol residues in human urine by capillary electropho-resis[J].Journal of chromatography,1998,713:1175-1176.
[36]Pou K.,Ong H.,Adam A.1994.Combined immunoextraction approach coupled to a chemiluminescenec enzyme immunoassay for the determination of trace levels of salbtmol and clenbuterolin tessue sample.Analyst.119(12):2659-2662
[37]Sawaya W N,Lone K,Husain A,et al.Screening for β-agonists in sheep urine and eyes by anenzyme-linked immunosorbent assay in the state of Kuwait[J].Food control,2000,11:1-5.
[38]柳其芳.饲料与尿液中盐酸克伦特罗测定方法的研究[R].职业与健康,2005,21(12):1951-1952.
[39]芮冬妹.滴金免疫法快速检测活动性肺结核IgG抗体[J].现代中西医结合杂志,2001,10(12):1175-1176.
[40]农牧发[2001]38号文.猪尿中盐酸克伦特罗的检测--酶联免疫吸附测定法.