基于点印迹的水产品过敏原检验方法的研究

发布时间：2018-05-23 23:43

  本文选题：食物过敏原 + 检验　； 参考：《中国海洋大学》2012年硕士论文

【摘要】：食品过敏已引起了全球多个国际组织和国家的高度重视，，但食物过敏原的诊断仍然是个挑战。由于体内实验的安全性问题，体外实验检验食物过敏原正受到越来越多的关注。目前已有很多体外过敏原特异性IgE检测设备及试剂盒被广泛应用，以Immuno CAP为代表的检测仪器准确率高但设备费用昂贵，普及率低。此外，食物过敏原由于地域性差异其种类也有很大的区别，现有的检验试剂盒多为进口，过敏原组合不符合中国国情、结果准确性及重现性较差。本研究立足上述问题，旨在研究出一种结果准确、适用于我国过敏人群、操作简便快捷、成本较低的食物过敏原检验方法。 本论文以我国过敏人群主要食物过敏原鱼类为研究对象，通过过敏原鉴定、交叉反应研究及加工对过敏原免疫活性的影响的研究探讨用以检验的过敏原的选择条件；建立了基于点印迹检验方法的肉眼可视化食物过敏原检验方法并进行了性能测定，最后通过与其他检验方法的结果比对验证其准确性。主要内容如下： 1、对青岛地区常见的7个鱼类品种进行了过敏原鉴定，发现真鲷、阿拉斯加狭鳕、鲤鱼、鲑鱼、蓝点马鲛、大菱鲆及大黄花鱼等的过敏原蛋白分子量为35~42kD、48~51kD、28~30kD及17kD，其中主要过敏原为41kD蛋白，并非国际公认的小清蛋白；7种鱼过敏原蛋白之间存在着严重的交叉反应，故可选择其中一种作为鱼肉过敏原代表进行检验，检验结果基本可表示鱼类过敏原总体情况。 2、研究发现经过热加工后，大菱鲆鱼肉蛋白组分中分子量较大的蛋白有很大程度的降解，免疫活性基本丧失；热加工使鱼肉过敏原蛋白的免疫活性降低，蒸5min后免疫活性降低67.9%，蒸30min后减少了89.2%的免疫活性；而鱼肉中小清蛋白及其免疫活性并未随着加热发生明显改变，耐热性好。而加热后，鱼肉蛋白中产生了一条新的具有致敏性的蛋白，分子量~18kD。实验结果表明，热加工对的鱼肉过敏原的免疫活性有较大的改变，对生鲜鱼肉产生过敏反应不一定会对热加工后的鱼肉产生相同反应，反之亦然。这对过敏原的检验方法的研究具有一定的指导意义，即：过敏原的检验中有必要将生鲜食物与经过不同加工方式的食物过敏原进行细分，从而得到更加准确的检验结果，也可为过敏患者提供一定的食用指导。 3、采用硝酸纤维素膜作为水产品过敏原载体，建立了肉眼可视化点印迹检验水产品过敏原的方法，即：过敏原蛋白点样浓度为1mg/mL，人血清用50%封闭液稀释10倍，于37℃孵育1h，二抗采用1:1000倍稀释的HRP-羊抗人IgE，于37℃孵育45min，显色液采用沉淀型TMB显色液避光显色15min，且过敏反应程度与点灰度值正相关。检验方法性能测定结果表明，硝酸纤维素膜内变异系数为1.9%~6.7%，膜间变异系数在3.3%~8.9%之间，稳定性良好。 4、通过与皮肤点刺试验、免疫印迹实验结果的比对表明，三种方法结果无显著性差异。其中，肉眼可视化点印迹检验的结果与免疫印迹结果一致性为82%、与皮肤点刺试验结果一致性为62%。此外，肉眼可视化点印迹检验方法与一种商业化的食物过敏原检验试剂盒相比，检验结果与免疫印迹和皮肤点刺试验结果更加接近，减少了假阴性反应的发生。 本研究建立了肉眼可视化的水产品过敏原检验方法，检验方便快捷，整个检验过程仅为2.5h；成本低廉，不需要特殊仪器设备即可判读结果；可制成高通量检验芯片，准确性较好，具有良好的实际应用价值。另外，首次从加工食品的角度提出了过敏原“细筛”的概念，为食物过敏原检验的发展提出了新思路。
[Abstract]:Food allergy has caused many international organizations and countries to attach great importance to food allergy , but the diagnosis of food allergens is still a challenge . In addition , many in vitro allergen specific IgE detection devices and kits are widely used , and many in vitro allergen specific IgE detection devices and kits are widely used .

In this paper , the main food allergen fishes of the allergic population in China were studied , and the selection conditions of allergen were studied by means of allergen identification , cross reaction and the effects of processing on the activity of allergen .
The visual visual food allergen test method based on the dot blot testing method is established and the performance measurement is carried out , and the accuracy is finally verified through comparison with the result of other inspection methods .

1 . It was found that the molecular weight of allergen protein was 35 - 42 kD , 48 - 51 kD , 28 - 30 kD and 17 kD . The major allergen was 41 kD protein and not internationally recognized little albumin . The molecular weight of allergen was 35 - 42 kD , 48 - 51 kD , 28 - 30 kD and 17 kD .
There is a serious cross reaction between the seven fish allergen proteins , so one of them can be selected as the fish allergen representative to carry out the test , and the test result can basically indicate the overall situation of fish allergen .

2 . It was found that the protein of the fish meat protein of turbot had a great degree of degradation and the immune activity was lost .
The immune activity of the fish allergen protein decreased by 67.9 % after 5 min , and 89.2 % of the immune activity was decreased after 30 min steaming .
The results show that there is a great change in the immune activity of the fish meat allergen . The results show that it is necessary to subdivide the fresh food from the food allergen in different processing ways , thus obtaining more accurate results and providing certain edible guidance for the allergic patients .

3 . Using nitrocellulose membrane as allergen carrier of aquatic products , the method to test the allergen of aquatic products by visual visual dot blot was established , namely : the concentration of allergen protein was 1mg / mL , human serum was diluted 10 times with 50 % blocking solution , incubated for 1 hour at 37 鈩

本文编号：1926855