多种食品过敏原体外快速检测技术的研究
发布时间：2018-08-30 08:31
【摘要】：[目的] 探讨用致敏肥大细胞的组胺释放率鉴定与评价食品过敏原的方法;建立能同时检测多种食品过敏原的dot-ELISA诊断技术。 [方法] 以虾、花生和鸡蛋清蛋白浸液作为致敏原,氢氧化铝为佐剂免疫BALB/c小鼠,建立IgE高反应性食物过敏动物模型。间接ELISA测定血清中特异性IgE(slgE);分离小鼠腹腔肥大细胞,过敏原体外激发,电镜观察过敏原刺激前、后肥大细胞形态学变化,荧光法检测肥大细胞的组胺定向释放率。Western blot分析过敏原浸液中主要的过敏性组分,并用阴离子交换层析将其分离纯化。dot-ELISA检测过敏原sIgE,并探索其最佳反应条件。 [结果] 模型组小鼠血清sIgE和肥大细胞组胺释放率均比对照组明显升高,提示过敏模型复制成功。过敏原激发后第14d slgE效价最高,虾、花生和鸡蛋清蛋白组分别达到1/6400,1/6400和1/3200;鸡蛋清蛋白组肥大细胞的组胺释放率达60.75%,河虾组腹腔或皮下免疫分别达到71.53%和88.48%。电镜观察显示,脱颗粒肥大细胞胞浆出现很多空泡,致密性颗粒减少。Western blot鉴定显示蛋清中主要过敏性组分为OVA,相对分子量40kD;虾中主要过敏性组分为Pen m2,相对分子量36kD。初步建立了能同时检测多种过敏原的dot-ELISA实验方案,dot-ELISA中各种过敏原最适包被量为2μL(10μg蛋白),待检抗血清最佳稀释度为1:80,通过观察NC膜上的斑点即可确定待检血清中的sIgE类型,且三种过敏原之间无交叉反应性。不同时间同批试剂重复检测结果完全一致,包被过敏原的NC膜4℃可稳定保存3个月。 [结论] 通过建立小鼠过敏模型,检测致敏肥大细胞的组胺释放率可能成为食品过敏原鉴定与评价的新方法。dot-ELISA可同时检测多种过敏原sIgE,该法简便、特异、稳定、重复性好,以期用于临床过敏症的诊断。
[Abstract]:[objective] to study the method of identification and evaluation of food allergen by histamine release rate of sensitized mast cells, and to establish a dot-ELISA diagnostic technique for simultaneous detection of various food allergens. [methods] BALB/c mice were immunized with shrimp, peanut and egg white protein extract as allergen and aluminum hydroxide as adjuvant to establish IgE hyperreactive food allergic animal model. Indirect ELISA was used to detect the specific IgE (slgE); isolated from mouse peritoneal mast cells. The allergen was stimulated in vitro. The morphologic changes of mast cells before and after allergen stimulation were observed by electron microscope. The histamine specific release rate of mast cells was determined by fluorescence assay. Western blot was used to analyze the main anaphylactic components in the anaphylactic extract. The anion exchange chromatography was used to isolate and purify the allergen sIgE, and to explore the optimum reaction conditions. [results] Serum sIgE and mast cell histamine release rate in the model group were significantly higher than those in the control group, indicating that the allergic model was successfully duplicated. The titer of slgE in shrimp, peanut and egg white protein group reached 1 / 6400 and 1 / 3200.The histamine release rate of mast cells in egg white protein group was 60.75 and that in prawn group was 71.53% and 88.48, respectively. Electron microscopic observation showed that there were many vacuoles in the cytoplasm of degranulated mast cells. Western blot analysis showed that the main anaphylactic components of egg white were 40 kD of OVA, and the main allergic components of shrimp were Pen m2 and 36 KD. A dot-ELISA experimental scheme for simultaneous detection of multiple allergens was established. The optimal envelope of each allergen in dot-ELISA was 2 渭 L (10 渭 g protein), and the optimal dilution of antiserum was 1: 80. The type of sIgE in the serum could be determined by observing the spots on the NC membrane. There was no cross-reactivity among the three allergens. The results of repeated detection of the same batch of reagents at different times were identical. The NC membrane coated with allergen could be stably preserved for 3 months at 4 鈩，
本文编号：2212539
[Abstract]:[objective] to study the method of identification and evaluation of food allergen by histamine release rate of sensitized mast cells, and to establish a dot-ELISA diagnostic technique for simultaneous detection of various food allergens. [methods] BALB/c mice were immunized with shrimp, peanut and egg white protein extract as allergen and aluminum hydroxide as adjuvant to establish IgE hyperreactive food allergic animal model. Indirect ELISA was used to detect the specific IgE (slgE); isolated from mouse peritoneal mast cells. The allergen was stimulated in vitro. The morphologic changes of mast cells before and after allergen stimulation were observed by electron microscope. The histamine specific release rate of mast cells was determined by fluorescence assay. Western blot was used to analyze the main anaphylactic components in the anaphylactic extract. The anion exchange chromatography was used to isolate and purify the allergen sIgE, and to explore the optimum reaction conditions. [results] Serum sIgE and mast cell histamine release rate in the model group were significantly higher than those in the control group, indicating that the allergic model was successfully duplicated. The titer of slgE in shrimp, peanut and egg white protein group reached 1 / 6400 and 1 / 3200.The histamine release rate of mast cells in egg white protein group was 60.75 and that in prawn group was 71.53% and 88.48, respectively. Electron microscopic observation showed that there were many vacuoles in the cytoplasm of degranulated mast cells. Western blot analysis showed that the main anaphylactic components of egg white were 40 kD of OVA, and the main allergic components of shrimp were Pen m2 and 36 KD. A dot-ELISA experimental scheme for simultaneous detection of multiple allergens was established. The optimal envelope of each allergen in dot-ELISA was 2 渭 L (10 渭 g protein), and the optimal dilution of antiserum was 1: 80. The type of sIgE in the serum could be determined by observing the spots on the NC membrane. There was no cross-reactivity among the three allergens. The results of repeated detection of the same batch of reagents at different times were identical. The NC membrane coated with allergen could be stably preserved for 3 months at 4 鈩，
本文编号：2212539
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2212539.html
最近更新
教材专著
