基于光激化学发光均相免疫检测技术的黄曲霉毒素检测方法研究
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摘要
黄曲霉毒素是一种毒性极强的生物毒素,广泛存在于各种农产品与食品中,对公众健康危害极大。提出了一种基于光激化学发光均相免疫检测技术的黄曲毒素B1检测方法,利用生物素亲和及抗原与抗体免疫反应,形成受体微球—AFB1-BSA完全抗原—AFB1单抗体—供体微球的聚合体系,在激发光作用下产生化学发光反应,根据化学发光值可定量分析样品中黄曲霉毒素浓度。通过分析并优化反应条件,包括各种反应物浓度、反应体系体积、反应温度与时间等,可实现最低检测限0.048ng/mL、最低定量限0.22ng/mL、批内与批间变异系数均小于10%、植物油样品中AFB1的加标回收率在87～105%之间。方法灵敏度高、特异性强、免清洗、检测速度快,在真菌毒素现场快速检测领域具有广阔的应用前景。
Aflatoxin is a highly toxic biological toxin that is widely existed in agricultural products and food.It endangers public health.In the manuscript,a method based on luminescent oxygen channeling immunoassay technology is used for aflatoxin B1 detection.By using the affinity of biotin and the immune reaction between the antigen and the antibody,a polymer system of acceptor beads-AFB1-BSA complete antigen-AFB1 monoclonal antibody-donor beads is formed.Then,with the excitation of the laser,the chemiluminescence reaction produces excitation light.The aflatoxin concentrations could be calculated by the intensity of excitation light.The experimental conditions are analyzed and optimized,including the volume,the concentration of various reagents,incubation temperature and time.However,the method can achieve the lower detection limit of 0.048ng/mL,the lower limit of quantification of 0.22ng/mL,the intra-and inter-assay variation both below 10%,and the recovery rates 87～105% with oil samples.Taking the advantages of high sensitivity,strong specificity,and wash-free,this method could be a promising tool for rapid detection of toxins.
Aflatoxin is a highly toxic biological toxin that is widely existed in agricultural products and food.It endangers public health.In the manuscript,a method based on luminescent oxygen channeling immunoassay technology is used for aflatoxin B1 detection.By using the affinity of biotin and the immune reaction between the antigen and the antibody,a polymer system of acceptor beads-AFB1-BSA complete antigen-AFB1 monoclonal antibody-donor beads is formed.Then,with the excitation of the laser,the chemiluminescence reaction produces excitation light.The aflatoxin concentrations could be calculated by the intensity of excitation light.The experimental conditions are analyzed and optimized,including the volume,the concentration of various reagents,incubation temperature and time.However,the method can achieve the lower detection limit of 0.048ng/mL,the lower limit of quantification of 0.22ng/mL,the intra-and inter-assay variation both below 10%,and the recovery rates 87～105% with oil samples.Taking the advantages of high sensitivity,strong specificity,and wash-free,this method could be a promising tool for rapid detection of toxins.
引文
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[18]Wen C L,Chen K Y,Chen C T,et al.Development of an alphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker[J].Journal of Proteomics,2012,75(13):3963.
[19]Wu F,Wang L,Guo Q,et al.A homogeneous immunoassay method for detecting interferon-gamma in patients with latent tuberculosis infection[J].Journal of Microbiology&Biotechnology,2016,26(3):588.
[20]Zhang Y,Huang B,Zhang J,et al.Development of a homogeneous immunoassay based on the AlphaLISA method for the detection of chloramphenicol in milk,honey and eggs[J].Journal of the Science of Food&Agriculture,2012,92(9):1944—1947.
[21]Zhao F,Yue Z F,Lv J Z,et al.Quantitative determination of clenbuterol using the amplified luminescent proximity homogeneous assay[J].Journal of Food Safety&Quality,2014,5(2):497—502.
[22]Hui Z,Lin G,Liu T,et al.Rapid quantitation of human epididymis protein 4in human serum by amplified luminescent proximity homogeneous immunoassay(AlphaLISA)[J].Journal of Immunological Methods,2016,437(8):64—69.
[23]洪小柳,吕敬章,张恒,等.AlphaLISA技术在食品安全检测中的研究进展[J].食品安全质量检测学报,2014,5(1):198—202.Hong Xiaoliu,Lv Jingzhang,Zhang Heng,et al.Research progress of AlphaLISA technology in food safety detection[J].Journal of Food Safety&Quality,2014,5(1):198—202.
[24]Zhou B,Ke W,Jian J,et al.Quantitative determination of deoxynivalenol(DON)using the amplified luminescent proximity homogeneous assay(AlphaLISA)[J].Food Analytical Methods,2011,4(2):228—232.
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[29]PerkinElmer,Inc.Exclusive AlphaScreen and AlphaLISA assay technology[EB/OL].(2009-02)/[2012-12].http://www.perkinelmer.com.cn/labsolutions/resources/docs/BRO_AlphaTechnologiesBrochure2009.pdf.
[30]张存亮.肺炎嗜衣原体AlphaLISA检测方法研究[D].重庆:重庆理工大学,2014.Zhang Cunliang.Research of AlphaLISA method for chlamydia pneumoniae detection[D].Chongqing:Chongqing University of Technology,2014.
[31]董志宁.乙型肝炎病毒表面抗原光激化学发光免疫测定试剂及人类免疫缺陷病毒时间分辨荧光免疫检测试剂的研制[D].南方医科大学,2012.Dong Zhinoing.Development of diagnostic reagents for hepatitis b surface antigen using amplified luminescent proximity homogeneous assay method and for human immunodefiency virus using time-resolved immunoassay method[D].Southern Medical University,2012.
[32]张兆威,李培武,张奇,等.农产品中黄曲霉毒素的时间分辨荧光免疫层析快速检测技术研究[J].中国农业科学,2014,47(18):3668—3674.Zhang Zhaowei,Li Peiwu,Zhang Qi,et al.Study on time-resolved fluorescence immunochromatography for afaltoxin determination in agricultural product[J].Scientia Agricultura Sinica,2014,47(18):3668—3674.
[2]Mukherjee M,Bhatt P,M H K.Fluorescent competitive aptasensor for detection of aflatoxin B1[J].Journal of Molecular Recognition,2017,30(12):e2650.
[3]Matabaro E,Ishimwe N,Uwimbabazi E,et al.Current immunoassay methods for the rapid detection of aflatoxin in milk and dairy products[J].Comprehensive Reviews in Food Science&Food Safety,2017,16(2):808—820.
[4]Li X,Li P,Zhang Q,et al.Multi-component immunochromatographic assay for simultaneous detection of aflatoxin B1,ochratoxin A and zearalenone in agro-food[J].Biosensors&Bioelectronics,2013,49(22):426—432.
[5]中华人民共和国卫生部.GB2761—2017食品安全国家标准-食品中真菌毒素限量[S].北京:中国标准出版社,2017.The ministry of health of people's republic of china.GB2761—2017National standard for food safety-The limits of mycotoxins in food[S].Beijing:China Standard Press,2017.
[6]Stroka J,Van O R,Anklam E.Immunoaffinity column clean-up prior to thin-layer chromatography for the determination of aflatoxins in various food matrices[J].Journal of Chromatography A,2000,904(2):251—256.
[7]Mochamad L,Hermanto B.High-performance liquid chromatography ultraviolet-photodiode array detection method for aflatoxin B1in cattle feed supplements[J].Veterinary World,2017,10(8):932—938.
[8]ChavarrǎA G,Granados Chinchilla F,Alfaro-Cascante M,et al.Detection of aflatoxin M1in milk,cheese and sour cream samples from costa rica using enzyme-assisted extraction and HPLC[J].Food Additives&Contaminants Part B Surveillance,2015,8(2):128—135.
[9]Shadbad M R S,Ansarin M,Tahavori A,et al.Determination of aflatoxins in nuts of Tabriz confectionaries by ELISA and HPLC methods[J].Adv Pharm Bull,2012,2(1):123—126.
[10]Gazioˇglu I,Kolak U.Method validation for the quantitative analysis of aflatoxins(B1,B2,G1,and G2)and ochratoxin A in processed cereal-based foods by hplc with fluorescence detection[J].Journal of Aoac International,2015,98(4):939—945.
[11]黎睿,谢刚,王松雪.高效液相色谱法同时检测粮食中常见8种真菌毒素的含量[J].食品科学,2015,36(6):206—210.Li Rui,Xie Gang,Wang Songxue.Simultaneous analysis of 8mycotoxins in grains by high performance liquid chromatography[J].Food Science,2015,36(6):206—210.
[12]Gross M,Asam S,Rychlik M.Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food[J].Mycotoxin Research,2017,33(1):1—4.
[13]Wang Z,Yue T,Yuan Y,et al.Development of polyclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of alicyclobacillus,strains in apple juice[J].Journal of Food Science,2012,77(11):M643—M649.
[14]马惠蕊,王玉坤,刘淑艳,等.食源性真菌毒素检测技术研究进展[J].福建分析测试,2011,20(1):40—43.Ma Huirui,Wang Yukun,Liu Shuyan,et al.Progress of researches on mycotoxin detection technology[J].Fujian Analysis&Testing,2011,20(1):40—43.
[15]Zhao H,Zhao J,Hou J,et al.AlphaLISA detection of alphasynuclein in the cerebrospinal fluid and its potential application in Parkinson’s disease diagnosis[J].Protein&Cell,2017,8(9):696—700.
[16]Pulidoolmo H,Rodríguezsánchez E,Barderas M G,et al.Rapid,Automated,and specific immunoassay to directly measure matrix metalloproteinase-9-tissue inhibitor of metalloproteinase-1interactions in human plasma using AlphaLISA technology:A new alternative to classical ELISA[J].Frontiers in Immunology,2017,8(7):853.
[17]Wang H,Nefzi A,Fields G B,et al.AlphaLISA-based highthroughput screening assay to measure levels of soluble amyloid precursor proteinα[J].Analytical Biochemistry,2014,459(8):24—30.
[18]Wen C L,Chen K Y,Chen C T,et al.Development of an alphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker[J].Journal of Proteomics,2012,75(13):3963.
[19]Wu F,Wang L,Guo Q,et al.A homogeneous immunoassay method for detecting interferon-gamma in patients with latent tuberculosis infection[J].Journal of Microbiology&Biotechnology,2016,26(3):588.
[20]Zhang Y,Huang B,Zhang J,et al.Development of a homogeneous immunoassay based on the AlphaLISA method for the detection of chloramphenicol in milk,honey and eggs[J].Journal of the Science of Food&Agriculture,2012,92(9):1944—1947.
[21]Zhao F,Yue Z F,Lv J Z,et al.Quantitative determination of clenbuterol using the amplified luminescent proximity homogeneous assay[J].Journal of Food Safety&Quality,2014,5(2):497—502.
[22]Hui Z,Lin G,Liu T,et al.Rapid quantitation of human epididymis protein 4in human serum by amplified luminescent proximity homogeneous immunoassay(AlphaLISA)[J].Journal of Immunological Methods,2016,437(8):64—69.
[23]洪小柳,吕敬章,张恒,等.AlphaLISA技术在食品安全检测中的研究进展[J].食品安全质量检测学报,2014,5(1):198—202.Hong Xiaoliu,Lv Jingzhang,Zhang Heng,et al.Research progress of AlphaLISA technology in food safety detection[J].Journal of Food Safety&Quality,2014,5(1):198—202.
[24]Zhou B,Ke W,Jian J,et al.Quantitative determination of deoxynivalenol(DON)using the amplified luminescent proximity homogeneous assay(AlphaLISA)[J].Food Analytical Methods,2011,4(2):228—232.
[25]Gong Q,Xi C,Nie F,et al.Determination of zearalanol and its analog zearalanone in muscle tissues by amplified luminescent proximity homogeneous assay(AlphaLISA)[J].Food Analytical Methods,2017,10(2):1—8.
[26]Du G,Yang X,Hu M,et al.Designing a novel high-throughput AlphaLISA assay to quantify plasma NHERF1as a nonsmall cell lung cancer biomarker[J].Rsc Advances,2015,5(102):84164—84171.
[27]Simard J R,Plant M,Emkey R,et al.Development and implementation of a high-throughput AlphaLISA assay for identifying inhibitors of EZH2methyltransferase[J].Assay&Drug Development Technologies,2013,11(3):152—162.
[28]范少娟,陈达,李奇峰,等.光激化学发光均相免疫分析的应用动态[J].纳米技术与精密工程,2014,12(2):151—156.Fan Shaojuan,Chen Da,Li Qifeng,et al.Current application status of luminescent oxygen channeling immunoassay(LOCI)technology[J].Nanotechnology&Precision Engineering,2014,12(2):151—156.
[29]PerkinElmer,Inc.Exclusive AlphaScreen and AlphaLISA assay technology[EB/OL].(2009-02)/[2012-12].http://www.perkinelmer.com.cn/labsolutions/resources/docs/BRO_AlphaTechnologiesBrochure2009.pdf.
[30]张存亮.肺炎嗜衣原体AlphaLISA检测方法研究[D].重庆:重庆理工大学,2014.Zhang Cunliang.Research of AlphaLISA method for chlamydia pneumoniae detection[D].Chongqing:Chongqing University of Technology,2014.
[31]董志宁.乙型肝炎病毒表面抗原光激化学发光免疫测定试剂及人类免疫缺陷病毒时间分辨荧光免疫检测试剂的研制[D].南方医科大学,2012.Dong Zhinoing.Development of diagnostic reagents for hepatitis b surface antigen using amplified luminescent proximity homogeneous assay method and for human immunodefiency virus using time-resolved immunoassay method[D].Southern Medical University,2012.
[32]张兆威,李培武,张奇,等.农产品中黄曲霉毒素的时间分辨荧光免疫层析快速检测技术研究[J].中国农业科学,2014,47(18):3668—3674.Zhang Zhaowei,Li Peiwu,Zhang Qi,et al.Study on time-resolved fluorescence immunochromatography for afaltoxin determination in agricultural product[J].Scientia Agricultura Sinica,2014,47(18):3668—3674.
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