牛乳酪蛋白双联胶体金免疫层析试纸条的研制
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摘要
原料羊乳中掺入牛乳会对其制品的食品安全造成损害.本研究的目的是建立一种免疫层析试纸条的检测方法检测羊乳原料乳的掺假.以牛乳αs1-CN及牛乳β-LG为检测对象,制备两者的特异性抗体,建立双联胶体金免疫层析试纸条.该实验制备的多克隆抗体α-CN IgG和β-LG IgG蛋白质浓度分别为4.86和5.99 mg/mL,两种抗体的效价为1∶25 600和1∶204 800,制备的胶体金溶液最适pH值为8.0,最佳蛋白标记量为10μL,且β-LG IgG中α-CN IgG的最适掺入量为20%.试纸条以玻璃纤维素膜VL68作为金标垫、硝酸纤维素膜SarteriesCN 95作为NC膜,分别用6μL,1 mg/mL的α-CN IgG,6μL,1 mg/mL的β-LG IgG包被NC膜T线、用6μL,1mg/mL二抗包被C线.该试纸条可特异性的检测出α-CN和β-LG,无明显的交叉反应,且可检出的牛乳最低掺入量为5%.
Aduteration of raw bovine milk to goat milk causes damage to food safety of its products,especially to consumers with bovine milk allergy.The purpose of this study was to establish a method of immunochromatographic strips to detect adulteration of raw goat milk.Bovine milkαs1-CN(αs1-Casein)and milkβ-LG(β-Lactoglobulin)was taking as the test object to develop double the colloidal gold immunochromatographic strip.Their specific ployclonal antibodies was prepared.The protein concentrations of polyclonal antibodies ofα-CN IgG andβ-LG IgG were 4.86 mg/mL and 5.99 mg/mL and the titers of these two antibodies were 1∶25 600 and 1∶204 800 respectively.In the preparation experiments of colloidal gold,the optimal pH was 8.0,and the best protein labeling amount was 10μL,and the optimal incorporation ratio ofα-CN IgG inβ-LG IgG was 20%.Test strips were coated with glass fiber VL68 as gold standard and Sarteries CN 95 as nitrocellulose NC membranes.NC membrane was coated with 6μL,1.0 mg/mLα-CN IgG and 6μL,1.0 mg/mLβ-LG IgG on T lines,and use 6μL,1.0 mg/mL secondary antibody to coated C line as well.The test strip can detectα-CN andβ-LG with no obvious cross-reaction,and the lowest detectable content of milk is5%.
Aduteration of raw bovine milk to goat milk causes damage to food safety of its products,especially to consumers with bovine milk allergy.The purpose of this study was to establish a method of immunochromatographic strips to detect adulteration of raw goat milk.Bovine milkαs1-CN(αs1-Casein)and milkβ-LG(β-Lactoglobulin)was taking as the test object to develop double the colloidal gold immunochromatographic strip.Their specific ployclonal antibodies was prepared.The protein concentrations of polyclonal antibodies ofα-CN IgG andβ-LG IgG were 4.86 mg/mL and 5.99 mg/mL and the titers of these two antibodies were 1∶25 600 and 1∶204 800 respectively.In the preparation experiments of colloidal gold,the optimal pH was 8.0,and the best protein labeling amount was 10μL,and the optimal incorporation ratio ofα-CN IgG inβ-LG IgG was 20%.Test strips were coated with glass fiber VL68 as gold standard and Sarteries CN 95 as nitrocellulose NC membranes.NC membrane was coated with 6μL,1.0 mg/mLα-CN IgG and 6μL,1.0 mg/mLβ-LG IgG on T lines,and use 6μL,1.0 mg/mL secondary antibody to coated C line as well.The test strip can detectα-CN andβ-LG with no obvious cross-reaction,and the lowest detectable content of milk is5%.
引文
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[11]雷颖颖.乳制品中蛋白质RP-HPLC指纹图谱的建立及其在乳源分析和乳制品质量控制中的应用[D].杭州:浙江大学,2017.
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[13]袁耀武,李聪,明若阳,等.利用竞争酶联免疫吸附法测定牛血清白蛋白鉴别掺假牛乳[J].食品科技,2017,42(3):290-293.
[14]褚晓红.牛乳中α-酪蛋白三种ELISA检测方法的研究[D].长春:吉林大学,2014.
[15]葛武鹏,李元瑞,陈瑛,等.牛、羊乳及其制品的脂肪酸组成分析[J].西北农林科技大学学报,2008,36(7):173-178.
[16]陈筱婷.乳及乳制品真实属性多重实时荧光PCR检测方法研究[D].广州:华南理工大学,2016.
[17]李洁,童锐,李向伟.实时荧光定量PCR法鉴别生鲜羊乳中牛源性成分[J].中国乳品工业,2016:44(10):38-39,42.
[18]宋蓓,宋桂雪,宋薇.羊乳制品中牛乳源成分的鉴别检测技术[J].食品科学技术学报,2016,34(6):69-74.
[19]宋宏新,刘静,张歌,等.变性与非变性电泳对牛羊乳蛋白质差异比较研究[J].陕西科技大学学报(自然科学版),2013,31(6):109-113.
[20]Lin S,Sun J,Cao D,et al.Distinction of different heattreated bovine milks by native-PAGE fingerprinting of their whey proteins[J].Food Chemistry,2010,121(3):803-808.
[21]姜潮,韩剑众,范佳利,等.低场核磁共振结合主成分分析法快速检测掺假牛乳[J].农业工程学报,2010,26(9):340-344.
[22]常玉清,王姝,王福利,等.基于多PCA模型的过程监测方法[J].仪器仪表学报,2014,35(4):901-908.
[23]A Di Pinto,V Terio,P Marchetti,et al.DNA-based approach for species identification of goat-milk products[J].Food Chemistry,2017,229:93-97
[24]L Dvorak,J Mlcek,K Sustova.Comparison of FT-NIR spectroscopy and ELISA for detection of adulteration of goat cheeses with cow′s milk[J].Journal of Aoac International,2016,99(1):180-186.
[25]P Galan Malo,I Mendiara,P Razquin,et al.Validation of a rapid lateral flow method for the detection of cows′milk in water buffalo,sheep or goat milk[J].Food Additives&Contaminants.Part A,2018,35(4):599-604.
[2]毛露甜,向军俭,张在军.牛乳中主要过敏原组分的分离纯化及鉴定[J].食品科学,2007,28(5):305-308.
[3]符海英.大肠杆菌O157∶H7和乳源蛋白胶体金免疫层析试纸条的研制[D].西安:陕西科技大学,2012.
[4]Mu`noz Martín T,de la Hoz Caballer B,Mara`nón Lizana F,et al.Selective allergy to sheep′s and goat′s milk proteins[J].Allergologiaet Immunopathologia,2004,32(1):39-42.
[5]张歌.牛乳和羊乳蛋白质差异比较及检测方法的研究[D].西安:陕西科技大学,2013.
[6]Tomotake Hiroyuki,Okuyama Ryoko,Katagiri Mitsuaki,et al.Comparison between Holstein cow′s milk and Japanese-saanen goat′s milk in fatty acid composition,lipid digestibility and protein profile[J].Bioscience,Biotechnology,and Bio-chemistry,2006,70(11):2 771-2 774.
[7]王逸斌,朱晗,朱凌燕,等.全羊乳蛋白配方羊奶粉对婴儿生长发育的影响研究[J].中国食物与营养,2013,19(8):78-81.
[8]张晓旭,葛武鹏,李宝宝,等.牛羊乳混掺检测鉴别技术研究进展[J].食品安全质量检测学报,2015,6(9):3 594-3 601.
[9]薛建龙.牛羊乳中蛋白质和脂肪的差异比较研究[D].西安:陕西科技大学,2015.
[10]Chen R K,Chang L W,Chung Y Y,et al.Quantification of cow milk adulteration in goat milk using high-performance liquid chromatography with electrospray ionization mass spectrometry[J].Rapid Commun Mass Spectr,2004,18(10):1 167-1 171.
[11]雷颖颖.乳制品中蛋白质RP-HPLC指纹图谱的建立及其在乳源分析和乳制品质量控制中的应用[D].杭州:浙江大学,2017.
[12]程妮.牛羊乳蛋白质的双向电泳分析及免疫检测技术研究[D].西安:陕西科技大学,2017.
[13]袁耀武,李聪,明若阳,等.利用竞争酶联免疫吸附法测定牛血清白蛋白鉴别掺假牛乳[J].食品科技,2017,42(3):290-293.
[14]褚晓红.牛乳中α-酪蛋白三种ELISA检测方法的研究[D].长春:吉林大学,2014.
[15]葛武鹏,李元瑞,陈瑛,等.牛、羊乳及其制品的脂肪酸组成分析[J].西北农林科技大学学报,2008,36(7):173-178.
[16]陈筱婷.乳及乳制品真实属性多重实时荧光PCR检测方法研究[D].广州:华南理工大学,2016.
[17]李洁,童锐,李向伟.实时荧光定量PCR法鉴别生鲜羊乳中牛源性成分[J].中国乳品工业,2016:44(10):38-39,42.
[18]宋蓓,宋桂雪,宋薇.羊乳制品中牛乳源成分的鉴别检测技术[J].食品科学技术学报,2016,34(6):69-74.
[19]宋宏新,刘静,张歌,等.变性与非变性电泳对牛羊乳蛋白质差异比较研究[J].陕西科技大学学报(自然科学版),2013,31(6):109-113.
[20]Lin S,Sun J,Cao D,et al.Distinction of different heattreated bovine milks by native-PAGE fingerprinting of their whey proteins[J].Food Chemistry,2010,121(3):803-808.
[21]姜潮,韩剑众,范佳利,等.低场核磁共振结合主成分分析法快速检测掺假牛乳[J].农业工程学报,2010,26(9):340-344.
[22]常玉清,王姝,王福利,等.基于多PCA模型的过程监测方法[J].仪器仪表学报,2014,35(4):901-908.
[23]A Di Pinto,V Terio,P Marchetti,et al.DNA-based approach for species identification of goat-milk products[J].Food Chemistry,2017,229:93-97
[24]L Dvorak,J Mlcek,K Sustova.Comparison of FT-NIR spectroscopy and ELISA for detection of adulteration of goat cheeses with cow′s milk[J].Journal of Aoac International,2016,99(1):180-186.
[25]P Galan Malo,I Mendiara,P Razquin,et al.Validation of a rapid lateral flow method for the detection of cows′milk in water buffalo,sheep or goat milk[J].Food Additives&Contaminants.Part A,2018,35(4):599-604.