【摘要】：[目的] 探討用致敏肥大細(xì)胞的組胺釋放率鑒定與評(píng)價(jià)食品過敏原的方法;建立能同時(shí)檢測(cè)多種食品過敏原的dot-ELISA診斷技術(shù)。 [方法] 以蝦、花生和雞蛋清蛋白浸液作為致敏原,氫氧化鋁為佐劑免疫BALB/c小鼠,建立IgE高反應(yīng)性食物過敏動(dòng)物模型。間接ELISA測(cè)定血清中特異性IgE(slgE);分離小鼠腹腔肥大細(xì)胞,過敏原體外激發(fā),電鏡觀察過敏原刺激前、后肥大細(xì)胞形態(tài)學(xué)變化,熒光法檢測(cè)肥大細(xì)胞的組胺定向釋放率。Western blot分析過敏原浸液中主要的過敏性組分,并用陰離子交換層析將其分離純化。dot-ELISA檢測(cè)過敏原sIgE,并探索其最佳反應(yīng)條件。 [結(jié)果] 模型組小鼠血清sIgE和肥大細(xì)胞組胺釋放率均比對(duì)照組明顯升高,提示過敏模型復(fù)制成功。過敏原激發(fā)后第14d slgE效價(jià)最高,蝦、花生和雞蛋清蛋白組分別達(dá)到1/6400,1/6400和1/3200;雞蛋清蛋白組肥大細(xì)胞的組胺釋放率達(dá)60.75%,河蝦組腹腔或皮下免疫分別達(dá)到71.53%和88.48%。電鏡觀察顯示,脫顆粒肥大細(xì)胞胞漿出現(xiàn)很多空泡,致密性顆粒減少。Western blot鑒定顯示蛋清中主要過敏性組分為OVA,相對(duì)分子量40kD;蝦中主要過敏性組分為Pen m2,相對(duì)分子量36kD。初步建立了能同時(shí)檢測(cè)多種過敏原的dot-ELISA實(shí)驗(yàn)方案,dot-ELISA中各種過敏原最適包被量為2μL(10μg蛋白),待檢抗血清最佳稀釋度為1:80,通過觀察NC膜上的斑點(diǎn)即可確定待檢血清中的sIgE類型,且三種過敏原之間無交叉反應(yīng)性。不同時(shí)間同批試劑重復(fù)檢測(cè)結(jié)果完全一致,包被過敏原的NC膜4℃可穩(wěn)定保存3個(gè)月。 [結(jié)論] 通過建立小鼠過敏模型,檢測(cè)致敏肥大細(xì)胞的組胺釋放率可能成為食品過敏原鑒定與評(píng)價(jià)的新方法。dot-ELISA可同時(shí)檢測(cè)多種過敏原sIgE,該法簡(jiǎn)便、特異、穩(wěn)定、重復(fù)性好,以期用于臨床過敏癥的診斷。
[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，

本文編號(hào)：2212539