荧光法检测鱼糜制品中大豆蛋白的研究  

Detection of soybean protein in surimi products by fluorescently-labeled monoclonal antibody

作　　者：江韬玲[1] 沈建东[1] 蔡秋凤[1] 张凌晶[1] 林岚[1] 曹敏杰[1] 

机构地区：[1]集美大学生物工程学院,福建厦门361021

出　　处：《食品工业科技》2013年第14期54-57,62,共5页Science and Technology of Food Industry

基　　金：福建省重大科研专项(2010NZ0001-3;2011NZ0002-1);福建省自然科学基金(2011J01227);厦门市科技计划项目(3502Z20113028)

摘　　要：鱼糜制品中大豆蛋白的过量添加已成为水产食品生产的一个新问题,建立有效的检测方法尤为重要。本研究通过丙酮浸泡、酸处理、硫酸铵盐析和柱层析等步骤分离纯化大豆胰蛋白酶抑制剂(Soybean trypsin inhibitor,STI)并制备抗STI单克隆抗体(A11-6)。经过Protein G Sepharose柱层析分离得到高纯度的免疫球蛋白(IgG)。采用SPDP(N-琥珀酰亚氨基-3-2-吡啶)和DTT(二硫苏糖醇)组合处理,成功将R-藻红蛋白偶联到抗STI单克隆抗体。利用该荧光标记的抗体建立的免疫荧光法能够检测出鱼糜制品中的STI,检测限为1.56μg/mL,灵敏度略高于传统的化学发光法。该免疫荧光法具有操作时间短、灵敏度高和使用安全等优点。The usage of soybean proteins in surimi products is increasing and becomes a new problem for aquatic products processing. Thus,establishment of an effective detection method for soybean proteins is essential. In the present study,soybean trypsin inhibitor（STI） was purified from soybean meal by a series of pretreatments,including acetone,H2O4 treatments,ammonium sulfate fractionation and sequential column chromatographies. For monoclonal antibody（All-6） preparation, purified STI was used to immunize mouse and the serum was purified by a Protein G Sepharose affinity column. Heherobifunctional reagent N-succinimidyl- 3-2- pyridyldithiopropionate （SPDP） and dithiothreitol （DTT） were used successfully to crosslink anti -STI monoclonal antibody with R-phycoerythrin（R-PE）. Western blot using this fluorescently-labeled antibody was then established and applied to analyze the existence of STI in surimi products. The limit of detection （LOD） of this method was 1.56μg/mL,which was slightly higher than traditional chemiluminescence method. This detection method is convenient,sensitive and safe in usage.

关 键 词：R-藻红蛋白 荧光探针 单克隆抗体 鱼糜制品 大豆蛋白 

分 类 号：TS254.4[轻工技术与工程—水产品加工及贮藏工程]