冷冻鱼糜中微生物谷氨酰胺转胺酶ELISA检测方法的建立及应用  

ELISA-based Method to Detect Microbial Transglutaminase in Frozen Surimi

作　　者：李轶[1] 范大明[1] 顾震南[1] 黄建联 连惠章 赵建新[1] 陈卫[1] 张灏[1] 

机构地区：[1]江南大学食品学院食品科学与技术国家重点实验室,江苏无锡214122 [2]福建安井食品股份有限公司,福建厦门361022 [3]无锡华顺民生食品有限公司,江苏无锡214218

出　　处：《现代食品科技》2014年第9期275-279,共5页Modern Food Science and Technology

基　　金：厦门市重大产业技术攻关项目(3502Z20121034);"十二五"农村领域国家科技计划课题项目(2012BAD28B05-04);江苏省产学研联合创新资金项目(BY2013015-01)

摘　　要：本文研究了冷冻鱼糜中微生物谷氨酰胺转胺酶(MTG)的双抗体夹心酶联免疫检测方法(sandwich-ELISA法),以抗MTG兔多抗为包被抗体,抗MTG鼠单抗为检测抗体,再结合HRP标记的抗IgG1二抗,构成sandwich-ELISA检测方法,对冷冻鱼糜中是否添加MTG进行定量检测,防止冷冻鱼糜原料掺假。实验结果表明,包被抗体的最佳工作浓度是2.00μg/mL,检测抗体的最佳工作浓度为0.10μg/mL,酶标二抗的最佳稀释倍数为1:5000。方法的检测限为20 ng/mL,该检测方法在0.6 mg/mL^10 mg/mL范围内OD450值与MTG浓度的log值呈线性关系。建立掺假冷冻鱼糜样品模拟体系,测定其中MTG的添加量:MTG的添加回收率为94%以上,测定过程中回收率结果的板内变异系数为1.12%~4.02%,板间变异系数为5.43%~6.87%。经试验证明该方法灵敏度高,样品前处理简便,适用于冷冻鱼糜中MTG的定量检测。In this study, a sandwich-type enzyme-linked immunosorbent assay （ELISA） was developed to detect microbial tmnsglutaminase （MTG） in frozen sudmi, using rabbit anti-MTG polyclonal antibody as the capture antibody and mouse anti-MTG monoclonal antibody as the detection antibody. Combined with anti-IgG1 secondary antibody horseradish peroxidase （HRP） conjugate, the sandwich ELISA was developed to quantitatively detect MTG in frozen surimi samples, thus providing a means to prevent adulteration of frozen surimi. The results showed that the optimum working concentration of the capture antibody was 2 μg/mL whereas that of the detection antibody was 0.1 μg/mL, and the optimum dilution of HRP-conjugated secondary antibody was 1：5,000. The visual detection limit of the assay was 20 ng/mL, and alinear relationship between OD450 values and the log values of MTG concentration was observed in the range of 0.6-10 mg/mL. A simulation model of adulteration in frozen surimi was established to determine the amount of added MTG The recovery rate of MTG was over 94%, whereas the intm- and inter-plate coefficients of variation during the measurement were 1.12%-4.02% and 5.43%~6.87%, respectively. These results proved that this method was sensitive, required simple sample pretreatment, and was suitable for the quantitative detection of MTG added to frozen surimi.

关 键 词：微生物 谷氨酰胺转胺酶 冷冻鱼糜 双抗体夹心酶联免疫检测法 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]