本文關鍵詞： 真菌毒素 蛋白質微陣列 四環(huán)素 酶聯(lián)免疫吸附法　出處：《南昌大學》2008年碩士論文　論文類型：學位論文

【摘要】： 1真菌毒素(Mycotoxin)是真菌產生的次級代謝產物,通常存在于霉變的谷物中,一旦通過食物鏈進入人體后,將可能造成致畸、致突變和致癌等嚴重危害,因此在全球食品安全問題中備受關注。目前,真菌毒素的檢測一般采用高效液相色譜法(HPLC)或酶聯(lián)免疫吸附法(ELISA),前者檢測靈敏度高,但樣品前處理煩瑣、操作復雜、時間長,所需設備較昂貴;后者操作簡便、快速,但每次只能完成一種真菌毒素的檢測。因此,具有高通量優(yōu)點的蛋白質微陣列技術將是真菌毒素多殘留快速檢測的發(fā)展趨勢。本研究在前期已制備出玉米赤霉烯酮(Zearelenone,ZEN)、黃曲霉毒素B_1(Aflatoxin B_1,AFB_1)和脫氧雪腐鐮刀菌烯醇(Deoxynivalenol,DON)三種真菌毒素人工抗原及其相應單克隆抗體的基礎上,采用間接競爭的檢測原理,以熒光作為檢測信號,對ZEN、AFB_1和DON蛋白質微陣列檢測方法進行了初步研究。結果如下: (1)ZEN、AFB_1和DON蛋白質微陣列單指標檢測的研究 采用正交實驗分別確定了ZEN、AFB_1和DON人工抗原的最佳點陣濃度為40μg/mL、40μg/mL和20μg/mL,對人工抗原固定條件、封閉方式和二抗?jié)舛冗M行了單因素優(yōu)化,確定了4℃過夜固定、37℃20min封閉和2μg/mL羊抗鼠IgG-CY3二抗為三個單因素實驗的最佳條件,進而以ZEN、AFB_1和DON標品分別進行間接競爭抑制實驗,繪制了ZEN、AFB_1和DON蛋白質微陣列單指標檢測的標準曲線,靈敏度(IC_(50))分別為2.1ng/mL、0.29ng/mL和86.8ng/mL,線性檢測范圍分別為0.5-10ng/mL、0.125-1.0ng/mL和50-400ng/mL。 (2)同時檢測ZEN、AFB_1和DON蛋白質微陣列的初步研究 在研究結果(1)的基礎上,進行了同時檢測ZEN、AFB_1和DON蛋白質微陣列的初步研究,確定了羊抗鼠IgG-CY3二抗的最佳濃度為4μg/mL,以ZEN、AFB_1和DON標準品同時進行間接競爭抑制實驗,繪制了同時檢測ZEN、AFB_1和DON蛋白質微陣列的標準曲線,靈敏度(IC_(50))分別為1.3ng/mL、0.154ng/mL和70.4ng/mL,線性檢測范圍分別為0.75-6ng/mL、0.125-0.6ng/mL和25-200ng/mL,均滿足我國規(guī)定的最高允許限量的檢測要求。 2四環(huán)素(Tetracycline,TC)是由鏈霉菌產生的一種堿性廣譜抗生素,因價格低廉,抗菌效果較好,在畜禽類養(yǎng)殖業(yè)中廣泛使用,不可避免將造成動物體內四環(huán)素的殘留,即使殘留量很小,但長期食用,對人體健康仍具有很大的潛在危害。因此,對四環(huán)素殘留進行及時檢測是預防和控制其危害的有效手段。本研究進行了抗四環(huán)素多克隆抗體的制備研究,旨在建立四環(huán)素的免疫學檢測方法。結果如下: 采用甲醛一步連接法(Mannich反應)分別制備了四環(huán)素偶聯(lián)陽離子化牛血清白蛋白(TC-cBSA)人工抗原和四環(huán)素偶聯(lián)卵清白蛋白(TC-OVA)人工抗原。經紫外圖譜鑒定后,以TC-cBSA為免疫原對三只BALB/C雌性小鼠進行背部皮內免疫,經三次加強免疫后,以TC-OVA為檢測抗原采用間接ELISA檢測抗血清效價,并采用間接競爭ELISA檢測抗血清特異性及交叉反應。競爭抑制曲線表明三只小鼠均產生了抗四環(huán)素的特異性抗體,其中2號小鼠抗血清效價最高,為1:32000,半量抑制濃度(IC_(50))為173.78ng/mL,該抗血清僅與土霉素存在44.7%的交叉反應率,與鏈霉素、氯霉素和阿莫西林的交叉反應率均小于＜0.01%。
[Abstract]:1 (Mycotoxin) of mycotoxins are secondary metabolites produced by fungi, mildew is usually found in cereals, once enter the body through the food chain, may cause teratogenic, mutagenic and carcinogenic and other serious harm, therefore in the global food safety concern. At present, the detection of mycotoxin by HPLC method (HPLC) and enzyme-linked immunosorbent assay (ELISA), the former high detection sensitivity, but the sample pretreatment is cumbersome, complex operation, long time, the equipment required is expensive; the latter is simple and fast, but only can detect a mycotoxin. Therefore, protein microarray technology has the advantages of high throughput will be the development trend of mycotoxins multi residue detection. This study has been prepared in the early stage of zearalenone (Zearelenone, ZEN), aflatoxin B_1 (Aflatoxin B_1 AFB_1) and deoxynivalenol enol Alcohol (Deoxynivalenol, DON) based on three kinds of mycotoxins in artificial antigen and its corresponding monoclonal antibody, detection principle by indirect competition, as with the fluorescence detection signal of ZEN, AFB_1 and DON protein microarray detection method were studied. The results are as follows:
(1) ZEN, the detection of AFB_1 and DON protein micro array index list
By the orthogonal experiment were determined by ZEN, the optimal concentration of AFB_1 and dot matrix DON artificial antigen was 40 g/mL, 40 g/mL and 20 g/mL, the fixed condition of artificial antigen, closed and two anti concentration by single factor optimization, to determine the optimum conditions of 4 DEG C for fixed, closed at 37 for 20min and 2 g/ mL Goat anti mouse IgG-CY3 two anti three single factor experiments, and then to ZEN, AFB_1 and DON standard respectively by indirect competitive inhibition experiment, draw the standard curve of ZEN, detection of AFB_1 and DON protein micro array and single index, sensitivity (IC_ (50)) were 2.1ng/ mL, 0.29ng/mL and 86.8ng/mL the linear range of detection, respectively 0.5-10ng/mL, 0.125-1.0ng/mL and 50-400ng/mL.
(2) preliminary study on simultaneous detection of ZEN, AFB_1 and DON protein microarrays
In the results of the study (1) on the basis of the simultaneous detection of ZEN, AFB_1 and DON of protein microarray, to determine the optimum concentration of Goat anti mouse IgG-CY3 two antibody was 4 g/mL, ZEN, AFB_1 and DON standard and indirect competitive inhibition experiment, draw the simultaneous detection of ZEN, standard curve AFB_1 and DON protein microarray, sensitivity (IC_ (50)) were 1.3ng/mL, 0.154ng/mL and 70.4ng/mL, the linear detection range were 0.75-6ng/mL, 0.125-0.6ng/mL and 25-200ng/mL, can meet the requirements of China's maximum allowable limit testing requirements.
2 tetracycline (Tetracycline, TC) is a kind of alkaline broad-spectrum antibiotics produced by Streptomyces, because of low price, good antibacterial effect, widely used in livestock and poultry breeding industry, will inevitably cause the animal residues of tetracycline residues, even very small, but eaten for a long time, still has great potential harm to human health. Therefore, the timely detection of tetracycline residues is the effective way to prevent and control the harm. The study of anti tetracycline polyclonal antibody preparation of immunological detection methods to establish tetracycline. The results are as follows:
The formaldehyde one-step ligation (Mannich reaction) were prepared by the coupling of tetracycline cationic bovine serum albumin (TC-cBSA) artificial antigen and tetracycline coupling ovalbumin (TC-OVA) artificial antigen. By UV spectrum identification, using TC-cBSA as immunogen for the back subcutaneous immunization of three BALB/C female mice after three times to strengthen the immunity, the detection of TC-OVA antigen by indirect ELISA antibody titers and the indirect competitive ELISA for detection of antisera and cross reaction. The competitive inhibition curve showed that all the three mice produced specific antibodies against tetracycline, 2 mice antiserum titer was 1:32000, the highest, half inhibitory concentration (IC_ (50) 173.78ng/mL), and the antiserum only has cross reaction rate of 44.7% oxytetracycline and streptomycin, chloramphenicol, amoxicillin and cross reaction rate were less than 0.01%.

【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R379

【引證文獻】

相關博士學位論文 前2條

1 王希春;農產品中兩種真菌毒素蛋白質芯片檢測技術的研究[D];南京農業(yè)大學;2010年

2 宋慧君;真菌毒素及潛藏性產毒真菌液相芯片多重測試方法的研究[D];沈陽農業(yè)大學;2012年

相關碩士學位論文 前2條

1 王丹;建立同時檢測三種真菌毒素膜免疫芯片方法的研究與抗慶大霉素多克隆抗體的制備[D];南昌大學;2010年

2 王瑩;同時檢測多種真菌毒素的生物芯片技術研究[D];華中農業(yè)大學;2012年

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本文編號：1455267