一种新型莱克多巴胺-蛋白质载体结合技术在侧向流试纸条检测动物饲料中莱克多巴胺含量中的应用（英文）
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摘要
目的:开发一种新型简单快速的克莱多巴胺-蛋白质载体结合方法,用于侧向流试纸条检测动物饲料。创新点:基于曼尼希反应的克莱多巴胺-牛血清蛋白(RAC–BSA)结合方法,具有一步性、简单、快速,且所需试剂少的优点。方法:通过曼尼希反应产生RAC–BSA结合物,制备竞争型侧向流试纸条。将RAC标记于猪饲料中,以体积比10:90的甲醇:电泳缓冲液作为提取缓冲液,在25min内完成样品制备。通过裸眼观察定性评估RAC–BSA结合物的免疫应答以及检测灵敏性,通过智能手机和电脑软件(ImageJ)进行半定量分析。结论:在最佳条件下,侧向流试纸条检测的检出限(LOD)在5 min裸眼评估下为1 ng/g;半定量分析中,其线性范围为0.075～0.750ng/g,计算的LOD为0.10 ng/g,定量限为0.33 ng/g,且相关性较好(R2=0.992)。回收率为96.4%～103.7%,同实验内及不同实验间的相对标准偏差为2.5%～3.6%。通过与酶联免疫吸附法对比,该侧向流试纸条检测结果具有较高的准确性和特异性。因此,通过曼尼希反应制备RAC–BSA结合物是一步的简单的结合法,可用于侧向流试纸条检测法。
In this work, a novel conjugate of ractopamine and bovine serum albumin(RAC–BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC–BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC–BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection(LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075–0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%–103.7% with a relative standard deviation of 2.5%–3.6% for intra-and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC–BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.
In this work, a novel conjugate of ractopamine and bovine serum albumin(RAC–BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC–BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC–BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection(LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075–0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%–103.7% with a relative standard deviation of 2.5%–3.6% for intra-and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC–BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.
引文
Berlina AN,Zherdev AV,Xu CL,et al.,2017.Development of lateral flow immunoassay for rapid control and quantification of the presence of the colorant Sudan I in spices and seafood.Food Control,73:247-253.https://doi.org/10.1016/j.foodcont.2016.08.011
Buakeaw A,Puthong S,Kongkavitoon P,et al.,2016.Production of monoclonal antibodies for ractopamine residue detection in pork.Maejo Int J Sci Technol,10(2):175-186.
Dai MY,Gong YF,Liu AM,et al.,2015.Development of a colloidal gold-based lateral-flow immunoassay for the rapid detection of phenylethanolamine A in swine urine.Anal Methods,7(10):4130-4137.https://doi.org/10.1039/C5AY00641D
Dong JX,Li ZF,Lei HT,et al.,2012.Development of a singlechain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.Anal Chim Acta,736:85-91.https://doi.org/10.1016/j.aca.2012.05.033
Du W,Zhao G,Fu Q,et al.,2014.Combined microextraction by packed sorbent and high-performance liquid chromatographyultraviolet detection for rapid analysis of ractopamine in porcine muscle and urine samples.Food Chem,145:789-795.https://doi.org/10.1016/j.foodchem.2013.08.094
Feng Y,Zhou YX,Zou Q,et al.,2009.Preparation and characterization of bisphenol A-cationized bovine serum albumin.J Immunol Methods,340(2):138-143.https://doi.org/10.1016/j.jim.2008.10.011
Gao HF,Han J,Yang SJ,et al.,2014.Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol.Anal Chim Acta,839:91-96.https://doi.org/10.1016/j.aca.2014.05.024
Gu HY,Liu LQ,Song SS,et al.,2016.Development of an immunochromatographic strip assay for ractopamine detection using an ultrasensitive monoclonal antibody.Food Agric Immunol,27(4):471-483.https://doi.org/10.1080/09540105.2015.1126808
Haasnoot W,Stouten P,Lommen A,et al.,1994.Determination of fenoterol and ractopamine in urine by enzyme immunoassay.Analyst,119(12):2675-2680.https://doi.org/10.1039/AN9941902675
Hage DS,Thomas DH,Beck MS,1993.Theory of a sequential addition competitive binding immunoassay based on highperformance immunoaffinity chromatography.Anal Chem,65(11):1622-1630.https://doi.org/10.1021/ac00059a023
Han J,Gao HF,Wang WW,et al.,2013.Time-resolved chemiluminescence strategy for multiplexed immunoassay of clenbuterol and ractopamine.Biosens Bioelectron,48:39-42.https://doi.org/10.1016/j.bios.2013.03.033
He LM,Su YJ,Zeng ZL,et al.,2007.Determination of ractopamine and clenbuterol in feeds by gas chromatographymass spectrometry.Anim Feed Sci Technol,132(3-4):316-323.https://doi.org/10.1016/j.anifeedsci.2006.03.013
Hermanson GT,2008.Preparation of Hapten-Carrier Immunogen Conjugates.In:Hermanson GT(Ed.),Bioconjugate Techniques,2nd Ed.Academic Press,New York,p.743-782.https://doi.org/10.1016/B978-0-12-370501-3.00019-9
Holme DJ,Peck H,1998.Analytical Biochemistry,3rd Ed.Prentice Hall,Essex,Britain.
Hu LM,Luo K,Xia J,et al.,2017.Advantages of timeresolved fluorescent nanobeads compared with fluorescent submicrospheres,quantum dots,and colloidal gold as label in lateral flow assays for detection of ractopamine.Biosens Bioelectron,91:95-103.https://doi.org/10.1016/j.bios.2016.12.030
Lei YC,Tai YT,Hsieh KH,et al.,2013.A polyclonal antibodybased immunoassay for determination of growth stimulant ractopamine:comparative study with recent advances in immunoassay methods.Taiwan Vet J,39(4):212-224.
Li CL,Li JY,Jiang WX,et al.,2015.Development and application of a gel-based immunoassay for the rapid screening of salbutamol and ractopamine residues in pork.J Agric Food Chem,63(48):10556-10561.https://doi.org/10.1021/acs.jafc.5b04203
Li X,Zhang G,Deng R,et al.,2010.Development of rapid immunoassays for the detection of ractopamine in swine urine.Food Addit Contam Part A Chem Anal Control Expo Risk Assess,27(8):1096-1103.https://doi.org/10.1080/19440041003754985
Liang XW,Zhang K,Zhang JM,et al.,2016.Ractopamine residues in beef cattle hair during and after treatment.JAnal Toxicol,40(2):153-158.https://doi.org/10.1093/jat/bkv135
Lin XY,Ni YN,Li SZ,et al.,2012.A novel method for simultaneous analysis of threeβ2-agonists in foods with the use of a gold-nanoparticle modified glassy carbon electrode and chemometrics.Analyst,137(9):2086-2094.https://doi.org/10.1039/C2AN16062E
Liu LQ,Kuang H,Peng CF,et al.,2014.Fragment-based hapten design and screening of a highly sensitive and specific monoclonal antibody for ractopamine.Anal Methods,6(1):229-234.https://doi.org/10.1039/C3AY41827H
Liu XY,He XW,Moore C,et al.,2009.Highly sensitive and specific liquid chromatography-tandem mass spectrometry method for testing ractopamine in cow and sheep urine.J Anal Toxicol,33(6):289-293.https://doi.org/10.1093/jat/33.6.289
Ni?o AMM,Granja RHMM,Wanschel ACBA,et al.,2017.The challenges of ractopamine use in meat production for export to European Union and Russia.Food Control,72:289-292.https://doi.org/10.1016/j.foodcont.2015.10.015
Patience JF,Shand P,Pietrasik Z,et al.,2009.The effect of ractopamine supplementation at 5 ppm of swine finishing diets on growth performance,carcass composition and ultimate pork quality.Can J Anim Sci,89(1):53-66.https://doi.org/10.4141/CJAS07152
Preechakasedkit P,Pinwattana K,Dungchai W,et al.,2012.Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum.Biosens Bioelectron,31(1):562-566.https://doi.org/10.1016/j.bios.2011.10.031
Ren ML,Chen XL,Li CH,et al.,2014.Lateral flow immunoassay for quantitative detection of ractopamine in swine urine.Biomed Environ Sci,27(2):134-137.https://doi.org/10.3967/bes2014.029
Shelver WL,Smith DJ,2000.Development of an immunoassay for theβ-adrenergic agonist ractopamine.J Immunoassay,21(1):1-23.https://doi.org/10.1080/01971520009349496
Shelver WL,Smith DJ,2002.Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals.J Agric Food Chem,50(10):2742-2747.https://doi.org/10.1021/jf011372+
Shelver WL,Smith DJ,2003.Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:comparative study with HPLC and ELISA.JAgric Food Chem,51(13):3715-3721.https://doi.org/10.1021/jf021175q
Shen L,He PL,2007.An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine.Electrochem Commun,9(4):657-662.https://doi.org/10.1016/j.elecom.2006.10.049
Shi CY,Deng N,Liang JJ,et al.,2015.A fluorescent polymer dots positive readout fluorescent quenching lateral flow sensor for ractopamine rapid detection.Anal Chim Acta,854:202-208.https://doi.org/10.1016/j.aca.2014.11.005
Shishani E,Chai SC,Jamokha S,et al.,2003.Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry.Anal Chim Acta,483(1-2):137-145.https://doi.org/10.1016/S0003-2670(03)00120-X
Smith DJ,Shelver WL,2002.Tissue residues of ractopamine and urinary excretion of ractopamine and metabolites in animals treated for 7 days with dietary ractopamine.JAnim Sci,80(5):1240-1249.https://doi.org/10.2527/2002.8051240x
Tang RH,Yang H,Choi JR,et al.,2016.Improved sensitivity of lateral flow assay using paper-based sample concentration technique.Talanta,152:269-276.https://doi.org/10.1016/j.talanta.2016.02.017
Teerinen T,Lappalainen T,Erho T,2014.A paper-based lateral flow assay for morphine.Anal Bioanal Chem,406(24):5955-5965.https://doi.org/10.1007/s00216-014-8001-7
Wang WY,Zhang YL,Wang JY,et al.,2010.Determination ofβ-agonists in pig feed,pig urine and pig liver using capillary electrophoresis with electrochemical detection.Meat Sci,85(2):302-305.https://doi.org/10.1016/j.meatsci.2010.01.018
Wang ZH,Liu MX,Shi WM,et al.,2015.New haptens and antibodies for ractopamine.Food Chem,183:111-114.https://doi.org/10.1016/j.foodchem.2015.03.043
Zhang MZ,Wang MZ,Chen ZL,et al.,2009a.Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine.Anal Bioanal Chem,395(8):2591-2599.https://doi.org/10.1007/s00216-009-3181-2
Zhang Y,Wang FX,Fang L,et al.,2009b.Rapid determination of ractopamine residues in edible animal products by enzyme-linked immunosorbent assay:development and investigation of matrix effects.J Biomed Biotechnol,2009:579175.https://doi.org/10.1155/2009/579175
Zhou YX,Wu JJ,Yu W,et al.,2007.Preparation for aflatoxin B1-cationized bovine serum albumin based on Mannichtype reaction.J Immunol Methods,328(1-2):79-88.https://doi.org/10.1016/j.jim.2007.08.009
Buakeaw A,Puthong S,Kongkavitoon P,et al.,2016.Production of monoclonal antibodies for ractopamine residue detection in pork.Maejo Int J Sci Technol,10(2):175-186.
Dai MY,Gong YF,Liu AM,et al.,2015.Development of a colloidal gold-based lateral-flow immunoassay for the rapid detection of phenylethanolamine A in swine urine.Anal Methods,7(10):4130-4137.https://doi.org/10.1039/C5AY00641D
Dong JX,Li ZF,Lei HT,et al.,2012.Development of a singlechain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.Anal Chim Acta,736:85-91.https://doi.org/10.1016/j.aca.2012.05.033
Du W,Zhao G,Fu Q,et al.,2014.Combined microextraction by packed sorbent and high-performance liquid chromatographyultraviolet detection for rapid analysis of ractopamine in porcine muscle and urine samples.Food Chem,145:789-795.https://doi.org/10.1016/j.foodchem.2013.08.094
Feng Y,Zhou YX,Zou Q,et al.,2009.Preparation and characterization of bisphenol A-cationized bovine serum albumin.J Immunol Methods,340(2):138-143.https://doi.org/10.1016/j.jim.2008.10.011
Gao HF,Han J,Yang SJ,et al.,2014.Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol.Anal Chim Acta,839:91-96.https://doi.org/10.1016/j.aca.2014.05.024
Gu HY,Liu LQ,Song SS,et al.,2016.Development of an immunochromatographic strip assay for ractopamine detection using an ultrasensitive monoclonal antibody.Food Agric Immunol,27(4):471-483.https://doi.org/10.1080/09540105.2015.1126808
Haasnoot W,Stouten P,Lommen A,et al.,1994.Determination of fenoterol and ractopamine in urine by enzyme immunoassay.Analyst,119(12):2675-2680.https://doi.org/10.1039/AN9941902675
Hage DS,Thomas DH,Beck MS,1993.Theory of a sequential addition competitive binding immunoassay based on highperformance immunoaffinity chromatography.Anal Chem,65(11):1622-1630.https://doi.org/10.1021/ac00059a023
Han J,Gao HF,Wang WW,et al.,2013.Time-resolved chemiluminescence strategy for multiplexed immunoassay of clenbuterol and ractopamine.Biosens Bioelectron,48:39-42.https://doi.org/10.1016/j.bios.2013.03.033
He LM,Su YJ,Zeng ZL,et al.,2007.Determination of ractopamine and clenbuterol in feeds by gas chromatographymass spectrometry.Anim Feed Sci Technol,132(3-4):316-323.https://doi.org/10.1016/j.anifeedsci.2006.03.013
Hermanson GT,2008.Preparation of Hapten-Carrier Immunogen Conjugates.In:Hermanson GT(Ed.),Bioconjugate Techniques,2nd Ed.Academic Press,New York,p.743-782.https://doi.org/10.1016/B978-0-12-370501-3.00019-9
Holme DJ,Peck H,1998.Analytical Biochemistry,3rd Ed.Prentice Hall,Essex,Britain.
Hu LM,Luo K,Xia J,et al.,2017.Advantages of timeresolved fluorescent nanobeads compared with fluorescent submicrospheres,quantum dots,and colloidal gold as label in lateral flow assays for detection of ractopamine.Biosens Bioelectron,91:95-103.https://doi.org/10.1016/j.bios.2016.12.030
Lei YC,Tai YT,Hsieh KH,et al.,2013.A polyclonal antibodybased immunoassay for determination of growth stimulant ractopamine:comparative study with recent advances in immunoassay methods.Taiwan Vet J,39(4):212-224.
Li CL,Li JY,Jiang WX,et al.,2015.Development and application of a gel-based immunoassay for the rapid screening of salbutamol and ractopamine residues in pork.J Agric Food Chem,63(48):10556-10561.https://doi.org/10.1021/acs.jafc.5b04203
Li X,Zhang G,Deng R,et al.,2010.Development of rapid immunoassays for the detection of ractopamine in swine urine.Food Addit Contam Part A Chem Anal Control Expo Risk Assess,27(8):1096-1103.https://doi.org/10.1080/19440041003754985
Liang XW,Zhang K,Zhang JM,et al.,2016.Ractopamine residues in beef cattle hair during and after treatment.JAnal Toxicol,40(2):153-158.https://doi.org/10.1093/jat/bkv135
Lin XY,Ni YN,Li SZ,et al.,2012.A novel method for simultaneous analysis of threeβ2-agonists in foods with the use of a gold-nanoparticle modified glassy carbon electrode and chemometrics.Analyst,137(9):2086-2094.https://doi.org/10.1039/C2AN16062E
Liu LQ,Kuang H,Peng CF,et al.,2014.Fragment-based hapten design and screening of a highly sensitive and specific monoclonal antibody for ractopamine.Anal Methods,6(1):229-234.https://doi.org/10.1039/C3AY41827H
Liu XY,He XW,Moore C,et al.,2009.Highly sensitive and specific liquid chromatography-tandem mass spectrometry method for testing ractopamine in cow and sheep urine.J Anal Toxicol,33(6):289-293.https://doi.org/10.1093/jat/33.6.289
Ni?o AMM,Granja RHMM,Wanschel ACBA,et al.,2017.The challenges of ractopamine use in meat production for export to European Union and Russia.Food Control,72:289-292.https://doi.org/10.1016/j.foodcont.2015.10.015
Patience JF,Shand P,Pietrasik Z,et al.,2009.The effect of ractopamine supplementation at 5 ppm of swine finishing diets on growth performance,carcass composition and ultimate pork quality.Can J Anim Sci,89(1):53-66.https://doi.org/10.4141/CJAS07152
Preechakasedkit P,Pinwattana K,Dungchai W,et al.,2012.Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum.Biosens Bioelectron,31(1):562-566.https://doi.org/10.1016/j.bios.2011.10.031
Ren ML,Chen XL,Li CH,et al.,2014.Lateral flow immunoassay for quantitative detection of ractopamine in swine urine.Biomed Environ Sci,27(2):134-137.https://doi.org/10.3967/bes2014.029
Shelver WL,Smith DJ,2000.Development of an immunoassay for theβ-adrenergic agonist ractopamine.J Immunoassay,21(1):1-23.https://doi.org/10.1080/01971520009349496
Shelver WL,Smith DJ,2002.Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals.J Agric Food Chem,50(10):2742-2747.https://doi.org/10.1021/jf011372+
Shelver WL,Smith DJ,2003.Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:comparative study with HPLC and ELISA.JAgric Food Chem,51(13):3715-3721.https://doi.org/10.1021/jf021175q
Shen L,He PL,2007.An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine.Electrochem Commun,9(4):657-662.https://doi.org/10.1016/j.elecom.2006.10.049
Shi CY,Deng N,Liang JJ,et al.,2015.A fluorescent polymer dots positive readout fluorescent quenching lateral flow sensor for ractopamine rapid detection.Anal Chim Acta,854:202-208.https://doi.org/10.1016/j.aca.2014.11.005
Shishani E,Chai SC,Jamokha S,et al.,2003.Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry.Anal Chim Acta,483(1-2):137-145.https://doi.org/10.1016/S0003-2670(03)00120-X
Smith DJ,Shelver WL,2002.Tissue residues of ractopamine and urinary excretion of ractopamine and metabolites in animals treated for 7 days with dietary ractopamine.JAnim Sci,80(5):1240-1249.https://doi.org/10.2527/2002.8051240x
Tang RH,Yang H,Choi JR,et al.,2016.Improved sensitivity of lateral flow assay using paper-based sample concentration technique.Talanta,152:269-276.https://doi.org/10.1016/j.talanta.2016.02.017
Teerinen T,Lappalainen T,Erho T,2014.A paper-based lateral flow assay for morphine.Anal Bioanal Chem,406(24):5955-5965.https://doi.org/10.1007/s00216-014-8001-7
Wang WY,Zhang YL,Wang JY,et al.,2010.Determination ofβ-agonists in pig feed,pig urine and pig liver using capillary electrophoresis with electrochemical detection.Meat Sci,85(2):302-305.https://doi.org/10.1016/j.meatsci.2010.01.018
Wang ZH,Liu MX,Shi WM,et al.,2015.New haptens and antibodies for ractopamine.Food Chem,183:111-114.https://doi.org/10.1016/j.foodchem.2015.03.043
Zhang MZ,Wang MZ,Chen ZL,et al.,2009a.Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine.Anal Bioanal Chem,395(8):2591-2599.https://doi.org/10.1007/s00216-009-3181-2
Zhang Y,Wang FX,Fang L,et al.,2009b.Rapid determination of ractopamine residues in edible animal products by enzyme-linked immunosorbent assay:development and investigation of matrix effects.J Biomed Biotechnol,2009:579175.https://doi.org/10.1155/2009/579175
Zhou YX,Wu JJ,Yu W,et al.,2007.Preparation for aflatoxin B1-cationized bovine serum albumin based on Mannichtype reaction.J Immunol Methods,328(1-2):79-88.https://doi.org/10.1016/j.jim.2007.08.009