發(fā)布時(shí)間：2018-05-23 23:43

  本文選題：食物過敏原 + 檢驗(yàn)��； 參考：《中國(guó)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(gè)國(guó)際組織和國(guó)家的高度重視，，但食物過敏原的診斷仍然是個(gè)挑戰(zhàn)。由于體內(nèi)實(shí)驗(yàn)的安全性問題，體外實(shí)驗(yàn)檢驗(yàn)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(cè)設(shè)備及試劑盒被廣泛應(yīng)用，以Immuno CAP為代表的檢測(cè)儀器準(zhǔn)確率高但設(shè)備費(fèi)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(yàn)試劑盒多為進(jìn)口，過敏原組合不符合中國(guó)國(guó)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(guó)過敏人群、操作簡(jiǎn)便快捷、成本較低的食物過敏原檢驗(yàn)方法。 本論文以我國(guó)過敏人群主要食物過敏原魚類為研究對(duì)象，通過過敏原鑒定、交叉反應(yīng)研究及加工對(duì)過敏原免疫活性的影響的研究探討用以檢驗(yàn)的過敏原的選擇條件；建立了基于點(diǎn)印跡檢驗(yàn)方法的肉眼可視化食物過敏原檢驗(yàn)方法并進(jìn)行了性能測(cè)定，最后通過與其他檢驗(yàn)方法的結(jié)果比對(duì)驗(yàn)證其準(zhǔn)確性。主要內(nèi)容如下： 1、對(duì)青島地區(qū)常見的7個(gè)魚類品種進(jìn)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(diǎn)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(guó)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(jìn)行檢驗(yàn)，檢驗(yàn)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(shí)驗(yàn)結(jié)果表明，熱加工對(duì)的魚肉過敏原的免疫活性有較大的改變，對(duì)生鮮魚肉產(chǎn)生過敏反應(yīng)不一定會(huì)對(duì)熱加工后的魚肉產(chǎn)生相同反應(yīng)，反之亦然。這對(duì)過敏原的檢驗(yàn)方法的研究具有一定的指導(dǎo)意義，即：過敏原的檢驗(yàn)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(jìn)行細(xì)分，從而得到更加準(zhǔn)確的檢驗(yàn)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(diǎn)印跡檢驗(yàn)水產(chǎn)品過敏原的方法，即：過敏原蛋白點(diǎn)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(diǎn)灰度值正相關(guān)。檢驗(yàn)方法性能測(cè)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(diǎn)刺試驗(yàn)、免疫印跡實(shí)驗(yàn)結(jié)果的比對(duì)表明，三種方法結(jié)果無(wú)顯著性差異。其中，肉眼可視化點(diǎn)印跡檢驗(yàn)的結(jié)果與免疫印跡結(jié)果一致性為82%、與皮膚點(diǎn)刺試驗(yàn)結(jié)果一致性為62%。此外，肉眼可視化點(diǎn)印跡檢驗(yàn)方法與一種商業(yè)化的食物過敏原檢驗(yàn)試劑盒相比，檢驗(yàn)結(jié)果與免疫印跡和皮膚點(diǎn)刺試驗(yàn)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(yàn)方法，檢驗(yàn)方便快捷，整個(gè)檢驗(yàn)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(yàn)芯片，準(zhǔn)確性較好，具有良好的實(shí)際應(yīng)用價(jià)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(yàn)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(hào)：1926855