花生、河虾过敏原抗体的制备及压电免疫传感同步检测

发布时间：2018-04-09 15:58

本文选题：花生　切入点：河虾　出处：《江南大学》2009年硕士论文

【摘要】： 花生、河虾属于八大类容易引起过敏的食品,相对其它食品过敏而言,它们的过敏发生率较高、临床症状更为严重,在公共卫生和食品安全领域倍受人们的关注。而现有的过敏原检测方法操作繁琐,无法满足快速、简便、在线检测的要求,因此,研究和建立一种方便、安全和快速的检测方法具有重要意义。研究主要内容包括花生、河虾过敏原的分离纯化,兔抗花生、河虾多克隆抗体的制备,探索胶体金是否可以作为一种新型的免疫佐剂;运用自组装的压电免疫传感器测定花生抗体和河虾抗体的免疫亲和常数,并同时包被两种抗体,对过敏原分别进行筛检。花生、河虾蛋白浸液经硫酸铵分级沉淀,等电点沉淀,AKTA蛋白纯化系统得到纯化,收集后经电泳检验。采用柠檬酸三钠还原法制备得到16nm左右的胶体金,以新西兰大白兔为模式动物制备抗体,免疫过程设立胶体金为佐剂和弗氏完全(不完全)佐剂作对照,发现胶体金能够显著提高大白兔体内抗花生、河虾过敏蛋白的抗体水平,花生与胶体金组的平均效价比弗氏佐剂组提高了一倍,其IC_(50)为34.28 ng/mL;河虾与胶体金组的平均效价比弗氏佐剂组提高了25%,其IC_(50)为43.95 ng/mL。运用自组装的压电免疫传感器测定得到花生抗体的亲和常数Kα=2.86×10~7 L/mol,混合抗体对花生抗原的Kα=1.37×10~7 L/mol;河虾抗体的Kα=5.16×10~7 L/mol,混合抗体对河虾抗原的Kα=2.57×10~7 L/mol。自组装的压电传感器同时包被花生和河虾混合抗体,分别加入一系列浓度的花生过敏原及河虾过敏原进行检测,绘制得到各自的标准曲线和其检测灵敏度。同时进行加标回收实验,回收率较高,重现性较好,实现了花生、河虾过敏原的同步筛检。本研究所建立的自组装的压电传感器同时包被花生、河虾两种抗体,实现了同步对花生、河虾过敏原的检测,快速、方便,具有较好的实用价值。
[Abstract]:However, the existing allergen detection methods are cumbersome and can not meet the requirements of rapid, simple and on-line detection. Therefore, it is of great significance to study and establish a convenient, safe and rapid detection method.The main contents of this study include the isolation and purification of peanut and shrimp allergen, the preparation of rabbit polyclonal antibody against peanut and river shrimp, and the exploration of whether colloidal gold can be used as a new immune adjuvant.Self-assembled piezoelectric immunosensor was used to detect the immune affinity constants of peanut antibody and shrimp antibody.The protein extracts of peanut and shrimp were purified by ammonium sulfate precipitation and isoelectric point precipitation.16nm colloidal gold was prepared by tri-sodium citrate reduction method, antibody was prepared from New Zealand white rabbits, and colloidal gold was used as adjuvant and Freund's complete (incomplete) adjuvant was used as contrast in immune process.The self-assembled piezoelectric sensor was coated with mixed antibodies of peanut and prawn, and a series of peanut allergens and shrimp allergens were added to detect them, and their standard curves and sensitivity were drawn.The recovery rate was high and the reproducibility was good. The simultaneous screening of peanut and shrimp allergens was realized.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

【引证文献】