三种真菌毒素蛋白质微阵列检测方法的初步研究以及抗四环素多克隆抗体的制备

发布时间：2018-01-22 17:14

本文关键词： 真菌毒素蛋白质微阵列四环素酶联免疫吸附法　出处：《南昌大学》2008年硕士论文　论文类型：学位论文

【摘要】： 1真菌毒素(Mycotoxin)是真菌产生的次级代谢产物,通常存在于霉变的谷物中,一旦通过食物链进入人体后,将可能造成致畸、致突变和致癌等严重危害,因此在全球食品安全问题中备受关注。目前,真菌毒素的检测一般采用高效液相色谱法(HPLC)或酶联免疫吸附法(ELISA),前者检测灵敏度高,但样品前处理烦琐、操作复杂、时间长,所需设备较昂贵;后者操作简便、快速,但每次只能完成一种真菌毒素的检测。因此,具有高通量优点的蛋白质微阵列技术将是真菌毒素多残留快速检测的发展趋势。本研究在前期已制备出玉米赤霉烯酮(Zearelenone,ZEN)、黄曲霉毒素B_1(Aflatoxin B_1,AFB_1)和脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)三种真菌毒素人工抗原及其相应单克隆抗体的基础上,采用间接竞争的检测原理,以荧光作为检测信号,对ZEN、AFB_1和DON蛋白质微阵列检测方法进行了初步研究。结果如下: (1)ZEN、AFB_1和DON蛋白质微阵列单指标检测的研究采用正交实验分别确定了ZEN、AFB_1和DON人工抗原的最佳点阵浓度为40μg/mL、40μg/mL和20μg/mL,对人工抗原固定条件、封闭方式和二抗浓度进行了单因素优化,确定了4℃过夜固定、37℃20min封闭和2μg/mL羊抗鼠IgG-CY3二抗为三个单因素实验的最佳条件,进而以ZEN、AFB_1和DON标品分别进行间接竞争抑制实验,绘制了ZEN、AFB_1和DON蛋白质微阵列单指标检测的标准曲线,灵敏度(IC_(50))分别为2.1ng/mL、0.29ng/mL和86.8ng/mL,线性检测范围分别为0.5-10ng/mL、0.125-1.0ng/mL和50-400ng/mL。 (2)同时检测ZEN、AFB_1和DON蛋白质微阵列的初步研究在研究结果(1)的基础上,进行了同时检测ZEN、AFB_1和DON蛋白质微阵列的初步研究,确定了羊抗鼠IgG-CY3二抗的最佳浓度为4μg/mL,以ZEN、AFB_1和DON标准品同时进行间接竞争抑制实验,绘制了同时检测ZEN、AFB_1和DON蛋白质微阵列的标准曲线,灵敏度(IC_(50))分别为1.3ng/mL、0.154ng/mL和70.4ng/mL,线性检测范围分别为0.75-6ng/mL、0.125-0.6ng/mL和25-200ng/mL,均满足我国规定的最高允许限量的检测要求。 2四环素(Tetracycline,TC)是由链霉菌产生的一种碱性广谱抗生素,因价格低廉,抗菌效果较好,在畜禽类养殖业中广泛使用,不可避免将造成动物体内四环素的残留,即使残留量很小,但长期食用,对人体健康仍具有很大的潜在危害。因此,对四环素残留进行及时检测是预防和控制其危害的有效手段。本研究进行了抗四环素多克隆抗体的制备研究,旨在建立四环素的免疫学检测方法。结果如下: 采用甲醛一步连接法(Mannich反应)分别制备了四环素偶联阳离子化牛血清白蛋白(TC-cBSA)人工抗原和四环素偶联卵清白蛋白(TC-OVA)人工抗原。经紫外图谱鉴定后,以TC-cBSA为免疫原对三只BALB/C雌性小鼠进行背部皮内免疫,经三次加强免疫后,以TC-OVA为检测抗原采用间接ELISA检测抗血清效价,并采用间接竞争ELISA检测抗血清特异性及交叉反应。竞争抑制曲线表明三只小鼠均产生了抗四环素的特异性抗体,其中2号小鼠抗血清效价最高,为1:32000,半量抑制浓度(IC_(50))为173.78ng/mL,该抗血清仅与土霉素存在44.7%的交叉反应率,与链霉素、氯霉素和阿莫西林的交叉反应率均小于＜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%.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R379

【引证文献】