基于噬菌体展示纳米抗体的绿色免疫PCR检测脱氧雪腐镰刀菌烯醇
|
摘要
目的:在获得脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)抗独特型纳米抗体的前期基础上,将纳米抗体作为酶标抗原的替代物,应用于荧光定量免疫聚合酶链式反应(polymerase chain reaction,PCR)体系,实现DON的高灵敏、绿色免疫分析。方法:将特异性结合DON抗体的噬菌体展示纳米抗体(P-28)作为竞争抗原,以编码P-28纳米抗体的DNA为靶标,设计特异性PCR扩增引物,优化荧光定量免疫PCR退火温度、抗DON抗体浓度、P-28投入量等参数,建立基于间接竞争模式的荧光定量免疫PCR检测DON的方法。结果:本研究建立的荧光定量免疫PCR方法线性检测范围为0.1～1 000 ng/mL,IC50值为(3.96±2.21)ng/mL,最低检出限为0.048 ng/mL,并与其他真菌毒素无交叉反应。结论:该方法直接使用噬菌体展示纳米抗体作为竞争抗原的替代物,应用于免疫PCR体系,避免了使用传统的化学合成酶标抗原所带来的环境污染、操作毒性等缺陷,并具有良好的特异性及灵敏度。
Objective: In order to develop a highly sensitive and green immunoassay for deoxynivalenol(DON)anti-idiotypic nanobody, obtained in our previous study, was used as an alternative to enzyme-labeled antigen in a?uorescence real-time immuno-PCR system. Methods: The phage displayed nanobody(P-28), which speci?cally binds with anti-DON antibody, was used as a competitive antigen and the DNA encoding P-28 Nanobody was used as a target for primer design. Important experimental parameters including annealing temperature, anti-DON antibody concentration and P-28 amount were optimized. Finally, we proposed a ?uorescence real-time immuno-PCR method based on indirect competition for the detection of DON. Results: The linear range of the immuno-fluorescence PCR method was 0.1–1 000 ng/mL with an IC50 of(3.96 ± 2.21) ng/mL, and the limit of detection(LOD) was 0.048 ng/mL. This method showed no crossreaction with other mycotoxins. Conclusion: This method avoids the defects of environmental pollution and operational toxicity caused by using traditional synthetic enzyme-labelled antigen and has good speci?city and sensitivity.
Objective: In order to develop a highly sensitive and green immunoassay for deoxynivalenol(DON)anti-idiotypic nanobody, obtained in our previous study, was used as an alternative to enzyme-labeled antigen in a?uorescence real-time immuno-PCR system. Methods: The phage displayed nanobody(P-28), which speci?cally binds with anti-DON antibody, was used as a competitive antigen and the DNA encoding P-28 Nanobody was used as a target for primer design. Important experimental parameters including annealing temperature, anti-DON antibody concentration and P-28 amount were optimized. Finally, we proposed a ?uorescence real-time immuno-PCR method based on indirect competition for the detection of DON. Results: The linear range of the immuno-fluorescence PCR method was 0.1–1 000 ng/mL with an IC50 of(3.96 ± 2.21) ng/mL, and the limit of detection(LOD) was 0.048 ng/mL. This method showed no crossreaction with other mycotoxins. Conclusion: This method avoids the defects of environmental pollution and operational toxicity caused by using traditional synthetic enzyme-labelled antigen and has good speci?city and sensitivity.
引文
[1]MARAGOS C M,MCCORMICK S P.Monoclonal antibodies for the mycotoxins deoxynivalenol and 3-acetyl-deoxynivalenol[J].Food&Agricultural Immunology,2000,12(3):181-192.DOI:10.1080/09540100050140722.
[2]LANCOVA K,HAJSLOVA J,POUSTKA J,et al.Transfer of fusarium mycotoxins and‘masked’deoxynivalenol(deoxynivalenol-3-glucoside)from field barley through malt to beer[J].Food Additives&Contaminants:Part A,2008,25(6):732-744.
[3]LORI G A,SISTERNA M N,HAIDUKOWSKI M,et al.Fusarium graminearum and deoxynivalenol contamination in the durum wheat area of Argentina[J].Microbiological Research,2003,158(1):29-35.DOI:10.1078/0944-5013-00173.
[4]VEGAHERRERA M,MADARIAGA R,ARANDA M,et al.Confirmation of deoxynivalenol presence in chilean wheat by highperformance thin-layer chromatography-mass spectrometry[J].Journal of the Chilean Chemical Society,2017,62(2):3435-3437.DOI:10.4067/S0717-97072017000200003.
[5]KIM D H,HONG S Y,JEON M H,et al.Simultaneous determination of the levels of deoxynivalenol,3-acetyldeoxynivalenol,and nivalenol in grain and feed samples from South Korea using a high-performance liquid chromatography-photodiode array detector[J].Applied Biological Chemistry,2016,59(6):1-7.
[6]HUANG Z B,XU Y,LI L S,et al.Development of an immunochromatographic strip test for the rapid simultaneous detection of deoxynivalenol and zearalenone in wheat and maize[J].Food Control,2012,28(1):7-12.DOI:10.1016/j.foodcont.2012.04.035.
[7]FOLLONI S,BELLOCCHI G,KAGKLI D M,et al.Development of an ELISA reverse-based assay to assess the presence of mycotoxins in cereal flour[J].Food Analytical Methods,2011,4(2):221-227.DOI:10.1007/s12161-010-9150-8.
[8]SINHA R C,SAVARD M E,LAU R.Production of monoclonal antibodies for the specific detection of deoxynivalenol and15-acetyldeoxynivalenol by ELISA[J].Journal of Agricultural&Food Chemistry,1995,43(6):1740-1744.DOI:10.1021/jf00054a061.
[9]WANG S,DU X,HUANG Y,et al.Detection of deoxynivalenol based on a single-chain fragment variable of the antideoxynivalenol antibody[J].FEMS Microbiology Letters,2007,272(2):214-219.DOI:10.1111/j.1574-6968.2007.00765.x.
[10]SMITH G P.Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J].Science,1985,228:1315-1317.DOI:10.1126/science.4001944.
[11]XU Y,XIONG L,LI Y P,et al.Citrinin detection using phagedisplayed anti-idiotypic single-domain antibody for antigen mimicry[J].Food Chemistry,2015,177:97-101.DOI:10.1016/j.foodchem.2015.01.007.
[12]XU Y,XIONG L,LI Y P,et al.Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library[J].Analytical&Bioanalytical Chemistry,2015,407(18):5333-5341.DOI:10.1007/s00216-015-8693-3.
[13]WANG X X,HE Q H,XU Y,et al.Anti-idiotypic VHH phage displaymediated immuno-PCR for ultrasensitive determination of mycotoxin zearalenone in cereals[J].Talanta,2016,147:410-415.DOI:10.1016/j.talanta.2015.09.072.
[14]JI Y W,HE Q H,XU Y,et al.Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A[J].Analytical Methods,2016,8(43).DOI:10.1039/C6AY01264G.
[15]HE Z Y,HE Q H,XU Y,et al.Ochratoxin A mimotope from secondgeneration peptide library and its application in immunoassay[J].Analytical Chemistry,2013,85(21):10304-10311.DOI:10.1021/ac402127t.
[16]HE Q H,XU Y,HUANG Y H,et al.Phage-displayed peptides that mimic zearalenone and its application in immunoassay[J].Food Chemistry,2011,126(3):1312-1315.DOI:10.1016/j.foodchem.2010.11.085.
[17]SHU M,XU Y,WANG D,et al.Anti-idiotypic nanobody:a strategy for development of sensitive and green immunoassay for fumonisin B1[J].Talanta,2015,143:388-393.DOI:10.1016/j.talanta.2015.05.010.
[18]WANG Y R,LI P W,MAJKOVA Z,et al.Isolation of alpaca antiidiotypic heavy-chain single-domain antibody for the aflatoxin immunoassay[J].Analytical Chemistry,2013,85(17):8298-8303.DOI:10.1021/ac4015885.
[19]SANO T,SMITH C L,CANTOR C R.Immuno-PCR:very sensitive antigen detection by means of specific antibody-DNA conjugates[J].Science,1992,258:120-122.DOI:10.1126/science.1439758.
[20]KUCZIUS T,BECKER K,FISCHER A,et al.Simultaneous detection of three CNS indicator proteins in complex suspensions using a single immuno-PCR protocol[J].Analytical Biochemistry,2012,431(1):4-10.DOI:10.1016/j.ab.2012.08.029.
[21]ASSUMP敲O A L F V,DA SILVA S R.Immuno-PCR in cancer and non-cancer related diseases:a review[J].Veterinary Quarterly,2016,36(2):63-70.DOI:10.1080/01652176.2016.1164912.
[22]LUBELLI C,CHATGILIALOGLU A,BOLOGNESI A,et al.Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay[J].Analytical Biochemistry,2006,355(1):102-109.DOI:10.1016/j.ab.2006.05.003.
[23]LIU Y Y,JIANG D J,LU X,et al.Phage-mediated immuno-PCRfor ultrasensitive detection of Cry1Ac protein based on nanobody[J].Journal of Agricultural and Food Chemistry,2016,64(41):7882-7889.DOI:10.1021/acs.jafc.6b02978.
[24]LEI J W,LI P W,ZHANG Q,et al.Anti-idiotypic nanobody-phage based real-time immuno-PCR for detection of hepatocarcinogen aflatoxin in grains and feedstuffs[J].Analytical Chemistry,2014,86(21):10841-10846.DOI:10.1021/ac5029424.
[25]QIU Y L,HE Q H,XU Y,et al.Deoxynivalenol-mimic nanobody isolated from a nae phage display nanobody library and its application in immunoassay[J].Analytica Chimica Acta,2015,887:201-208.DOI:10.1016/j.aca.2015.06.033.
[26]DE B P,RAHAOUI H,LEER R J,et al.Real-time PCR detection of Campylobacter spp.:a comparison to classic culturing and enrichment[J].Food Microbiology,2015,51:96-100.DOI:10.1016/j.fm.2015.05.006.
[27]KIM H J,MCCOY M,GEE S J,et al.Noncompetitive phage antiimmunocomplex real-time polymerase chain reaction for sensitive detection of small molecules[J].Analytical Chemistry,2011,83(1):246.
[28]NAKANO S,MORIZANE Y,MAKISAKA N,et al.Development of a highly sensitive immuno-PCR assay for the measurement ofα-galactosidase A protein levels in serum and plasma[J].PLoS ONE,2013,8(11):e78588-e78588.DOI:10.1371/journal.pone.0078588.
[29]ZHANG H,XU Y,HUANG Q,et al.Natural phage nanoparticlemediated real-time immuno-PCR for ultrasensitive detection of protein marker[J].Chemical Communications,2013,49(36):3778-3780.DOI:10.1039/c3cc40688a.
[30]GUAN D,LI P,CUI Y,et al.A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk[J].Analytica Chimica Acta,2011,703(1):64-69.DOI:10.1016/j.aca.2011.07.011.
[2]LANCOVA K,HAJSLOVA J,POUSTKA J,et al.Transfer of fusarium mycotoxins and‘masked’deoxynivalenol(deoxynivalenol-3-glucoside)from field barley through malt to beer[J].Food Additives&Contaminants:Part A,2008,25(6):732-744.
[3]LORI G A,SISTERNA M N,HAIDUKOWSKI M,et al.Fusarium graminearum and deoxynivalenol contamination in the durum wheat area of Argentina[J].Microbiological Research,2003,158(1):29-35.DOI:10.1078/0944-5013-00173.
[4]VEGAHERRERA M,MADARIAGA R,ARANDA M,et al.Confirmation of deoxynivalenol presence in chilean wheat by highperformance thin-layer chromatography-mass spectrometry[J].Journal of the Chilean Chemical Society,2017,62(2):3435-3437.DOI:10.4067/S0717-97072017000200003.
[5]KIM D H,HONG S Y,JEON M H,et al.Simultaneous determination of the levels of deoxynivalenol,3-acetyldeoxynivalenol,and nivalenol in grain and feed samples from South Korea using a high-performance liquid chromatography-photodiode array detector[J].Applied Biological Chemistry,2016,59(6):1-7.
[6]HUANG Z B,XU Y,LI L S,et al.Development of an immunochromatographic strip test for the rapid simultaneous detection of deoxynivalenol and zearalenone in wheat and maize[J].Food Control,2012,28(1):7-12.DOI:10.1016/j.foodcont.2012.04.035.
[7]FOLLONI S,BELLOCCHI G,KAGKLI D M,et al.Development of an ELISA reverse-based assay to assess the presence of mycotoxins in cereal flour[J].Food Analytical Methods,2011,4(2):221-227.DOI:10.1007/s12161-010-9150-8.
[8]SINHA R C,SAVARD M E,LAU R.Production of monoclonal antibodies for the specific detection of deoxynivalenol and15-acetyldeoxynivalenol by ELISA[J].Journal of Agricultural&Food Chemistry,1995,43(6):1740-1744.DOI:10.1021/jf00054a061.
[9]WANG S,DU X,HUANG Y,et al.Detection of deoxynivalenol based on a single-chain fragment variable of the antideoxynivalenol antibody[J].FEMS Microbiology Letters,2007,272(2):214-219.DOI:10.1111/j.1574-6968.2007.00765.x.
[10]SMITH G P.Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J].Science,1985,228:1315-1317.DOI:10.1126/science.4001944.
[11]XU Y,XIONG L,LI Y P,et al.Citrinin detection using phagedisplayed anti-idiotypic single-domain antibody for antigen mimicry[J].Food Chemistry,2015,177:97-101.DOI:10.1016/j.foodchem.2015.01.007.
[12]XU Y,XIONG L,LI Y P,et al.Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library[J].Analytical&Bioanalytical Chemistry,2015,407(18):5333-5341.DOI:10.1007/s00216-015-8693-3.
[13]WANG X X,HE Q H,XU Y,et al.Anti-idiotypic VHH phage displaymediated immuno-PCR for ultrasensitive determination of mycotoxin zearalenone in cereals[J].Talanta,2016,147:410-415.DOI:10.1016/j.talanta.2015.09.072.
[14]JI Y W,HE Q H,XU Y,et al.Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A[J].Analytical Methods,2016,8(43).DOI:10.1039/C6AY01264G.
[15]HE Z Y,HE Q H,XU Y,et al.Ochratoxin A mimotope from secondgeneration peptide library and its application in immunoassay[J].Analytical Chemistry,2013,85(21):10304-10311.DOI:10.1021/ac402127t.
[16]HE Q H,XU Y,HUANG Y H,et al.Phage-displayed peptides that mimic zearalenone and its application in immunoassay[J].Food Chemistry,2011,126(3):1312-1315.DOI:10.1016/j.foodchem.2010.11.085.
[17]SHU M,XU Y,WANG D,et al.Anti-idiotypic nanobody:a strategy for development of sensitive and green immunoassay for fumonisin B1[J].Talanta,2015,143:388-393.DOI:10.1016/j.talanta.2015.05.010.
[18]WANG Y R,LI P W,MAJKOVA Z,et al.Isolation of alpaca antiidiotypic heavy-chain single-domain antibody for the aflatoxin immunoassay[J].Analytical Chemistry,2013,85(17):8298-8303.DOI:10.1021/ac4015885.
[19]SANO T,SMITH C L,CANTOR C R.Immuno-PCR:very sensitive antigen detection by means of specific antibody-DNA conjugates[J].Science,1992,258:120-122.DOI:10.1126/science.1439758.
[20]KUCZIUS T,BECKER K,FISCHER A,et al.Simultaneous detection of three CNS indicator proteins in complex suspensions using a single immuno-PCR protocol[J].Analytical Biochemistry,2012,431(1):4-10.DOI:10.1016/j.ab.2012.08.029.
[21]ASSUMP敲O A L F V,DA SILVA S R.Immuno-PCR in cancer and non-cancer related diseases:a review[J].Veterinary Quarterly,2016,36(2):63-70.DOI:10.1080/01652176.2016.1164912.
[22]LUBELLI C,CHATGILIALOGLU A,BOLOGNESI A,et al.Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay[J].Analytical Biochemistry,2006,355(1):102-109.DOI:10.1016/j.ab.2006.05.003.
[23]LIU Y Y,JIANG D J,LU X,et al.Phage-mediated immuno-PCRfor ultrasensitive detection of Cry1Ac protein based on nanobody[J].Journal of Agricultural and Food Chemistry,2016,64(41):7882-7889.DOI:10.1021/acs.jafc.6b02978.
[24]LEI J W,LI P W,ZHANG Q,et al.Anti-idiotypic nanobody-phage based real-time immuno-PCR for detection of hepatocarcinogen aflatoxin in grains and feedstuffs[J].Analytical Chemistry,2014,86(21):10841-10846.DOI:10.1021/ac5029424.
[25]QIU Y L,HE Q H,XU Y,et al.Deoxynivalenol-mimic nanobody isolated from a nae phage display nanobody library and its application in immunoassay[J].Analytica Chimica Acta,2015,887:201-208.DOI:10.1016/j.aca.2015.06.033.
[26]DE B P,RAHAOUI H,LEER R J,et al.Real-time PCR detection of Campylobacter spp.:a comparison to classic culturing and enrichment[J].Food Microbiology,2015,51:96-100.DOI:10.1016/j.fm.2015.05.006.
[27]KIM H J,MCCOY M,GEE S J,et al.Noncompetitive phage antiimmunocomplex real-time polymerase chain reaction for sensitive detection of small molecules[J].Analytical Chemistry,2011,83(1):246.
[28]NAKANO S,MORIZANE Y,MAKISAKA N,et al.Development of a highly sensitive immuno-PCR assay for the measurement ofα-galactosidase A protein levels in serum and plasma[J].PLoS ONE,2013,8(11):e78588-e78588.DOI:10.1371/journal.pone.0078588.
[29]ZHANG H,XU Y,HUANG Q,et al.Natural phage nanoparticlemediated real-time immuno-PCR for ultrasensitive detection of protein marker[J].Chemical Communications,2013,49(36):3778-3780.DOI:10.1039/c3cc40688a.
[30]GUAN D,LI P,CUI Y,et al.A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk[J].Analytica Chimica Acta,2011,703(1):64-69.DOI:10.1016/j.aca.2011.07.011.