本文選題：食物過敏原 + 檢驗　； 參考：《中國海洋大學(xué)》2012年碩士論文

【摘要】：食品過敏已引起了全球多個國際組織和國家的高度重視，，但食物過敏原的診斷仍然是個挑戰(zhàn)。由于體內(nèi)實驗的安全性問題，體外實驗檢驗食物過敏原正受到越來越多的關(guān)注。目前已有很多體外過敏原特異性IgE檢測設(shè)備及試劑盒被廣泛應(yīng)用，以Immuno CAP為代表的檢測儀器準(zhǔn)確率高但設(shè)備費用昂貴，普及率低。此外，食物過敏原由于地域性差異其種類也有很大的區(qū)別，現(xiàn)有的檢驗試劑盒多為進口，過敏原組合不符合中國國情、結(jié)果準(zhǔn)確性及重現(xiàn)性較差。本研究立足上述問題，旨在研究出一種結(jié)果準(zhǔn)確、適用于我國過敏人群、操作簡便快捷、成本較低的食物過敏原檢驗方法。 本論文以我國過敏人群主要食物過敏原魚類為研究對象，通過過敏原鑒定、交叉反應(yīng)研究及加工對過敏原免疫活性的影響的研究探討用以檢驗的過敏原的選擇條件；建立了基于點印跡檢驗方法的肉眼可視化食物過敏原檢驗方法并進行了性能測定，最后通過與其他檢驗方法的結(jié)果比對驗證其準(zhǔn)確性。主要內(nèi)容如下： 1、對青島地區(qū)常見的7個魚類品種進行了過敏原鑒定，發(fā)現(xiàn)真鯛、阿拉斯加狹鱈、鯉魚、鮭魚、藍(lán)點馬鮫、大菱鲆及大黃花魚等的過敏原蛋白分子量為35~42kD、48~51kD、28~30kD及17kD，其中主要過敏原為41kD蛋白，并非國際公認(rèn)的小清蛋白；7種魚過敏原蛋白之間存在著嚴(yán)重的交叉反應(yīng)，故可選擇其中一種作為魚肉過敏原代表進行檢驗，檢驗結(jié)果基本可表示魚類過敏原總體情況。 2、研究發(fā)現(xiàn)經(jīng)過熱加工后，大菱鲆魚肉蛋白組分中分子量較大的蛋白有很大程度的降解，免疫活性基本喪失；熱加工使魚肉過敏原蛋白的免疫活性降低，蒸5min后免疫活性降低67.9%，蒸30min后減少了89.2%的免疫活性；而魚肉中小清蛋白及其免疫活性并未隨著加熱發(fā)生明顯改變，耐熱性好。而加熱后，魚肉蛋白中產(chǎn)生了一條新的具有致敏性的蛋白，分子量~18kD。實驗結(jié)果表明，熱加工對的魚肉過敏原的免疫活性有較大的改變，對生鮮魚肉產(chǎn)生過敏反應(yīng)不一定會對熱加工后的魚肉產(chǎn)生相同反應(yīng)，反之亦然。這對過敏原的檢驗方法的研究具有一定的指導(dǎo)意義，即：過敏原的檢驗中有必要將生鮮食物與經(jīng)過不同加工方式的食物過敏原進行細(xì)分，從而得到更加準(zhǔn)確的檢驗結(jié)果，也可為過敏患者提供一定的食用指導(dǎo)。 3、采用硝酸纖維素膜作為水產(chǎn)品過敏原載體，建立了肉眼可視化點印跡檢驗水產(chǎn)品過敏原的方法，即：過敏原蛋白點樣濃度為1mg/mL，人血清用50%封閉液稀釋10倍，于37℃孵育1h，二抗采用1:1000倍稀釋的HRP-羊抗人IgE，于37℃孵育45min，顯色液采用沉淀型TMB顯色液避光顯色15min，且過敏反應(yīng)程度與點灰度值正相關(guān)。檢驗方法性能測定結(jié)果表明，硝酸纖維素膜內(nèi)變異系數(shù)為1.9%~6.7%，膜間變異系數(shù)在3.3%~8.9%之間，穩(wěn)定性良好。 4、通過與皮膚點刺試驗、免疫印跡實驗結(jié)果的比對表明，三種方法結(jié)果無顯著性差異。其中，肉眼可視化點印跡檢驗的結(jié)果與免疫印跡結(jié)果一致性為82%、與皮膚點刺試驗結(jié)果一致性為62%。此外，肉眼可視化點印跡檢驗方法與一種商業(yè)化的食物過敏原檢驗試劑盒相比，檢驗結(jié)果與免疫印跡和皮膚點刺試驗結(jié)果更加接近，減少了假陰性反應(yīng)的發(fā)生。 本研究建立了肉眼可視化的水產(chǎn)品過敏原檢驗方法，檢驗方便快捷，整個檢驗過程僅為2.5h；成本低廉，不需要特殊儀器設(shè)備即可判讀結(jié)果；可制成高通量檢驗芯片，準(zhǔn)確性較好，具有良好的實際應(yīng)用價值。另外，首次從加工食品的角度提出了過敏原“細(xì)篩”的概念，為食物過敏原檢驗的發(fā)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

本文編號：1926855