金磁酶联免疫法检测牛乳过敏原酪蛋白、α-乳白蛋白的研究

发布时间：2018-06-03 01:38

本文选题：酪蛋白 + α-乳白蛋白　；参考：《上海师范大学》2016年硕士论文

【摘要】：牛乳制品含有丰富的营养,同时乳及乳制品也是WHO认定的导致人类过敏的八大类食物之一。其中牛乳中的酪蛋白(casein,CN)及α-乳白蛋白(α-lactalbumin,α-LA)被视为主要的过敏原,因此开展针对食品中酪蛋白及α-乳白蛋白强特异性、高灵敏度的检测具有重要的理论和现实意义。具体研究内容如下:1、酪蛋白、α-乳白蛋白单克隆抗体的制备。以CN、α-LA各免疫4只BALB/c小鼠,用间接ELISA法测小鼠抗血清效价,获得效价最高的小鼠的脾细胞与骨髓瘤细胞进行细胞融合,获得杂交瘤细胞。间接ELISA法筛选阳性孔,有限稀释法进行3次亚克隆得到分泌同质的杂交瘤细胞株。最终筛选到1株分泌酪蛋白单抗、2株分泌α-乳白蛋白单抗的杂交瘤细胞株分别为3D3、11G10、11F8。采用小鼠体内诱生腹水法大量制备单抗。2、酪蛋白、α-乳白蛋白单抗的鉴定采用辛酸-硫酸铵沉淀法纯化单抗腹水,经SDS-PAGE电泳表征单抗纯度,采用间接竞争ELISA、Western-blotting、非竞争性ELISA法分别检测单抗效价、特异性及亲和力,结果显示CN、α-LA单抗浓度分别为8.425 mg/m L和8.591mg/m L。抗CN 3D3单抗,抗α-LA 11G10、11F8单抗效价分别为1:256000、1:512000、1:128000。并检测两类单抗亚类均为Ig G1型。酪蛋白、α-乳白蛋白单抗亲和常数分别为0.32×108 L/mol和0.37×108 L/mol。3、金磁(Fe3O4/Au)复合微粒与酶标抗原的制备。采用一锅法制备氨基化的磁性微粒Fe3O4-PEI,之后在其表面包覆两层Au颗粒,制备金磁(Fe3O4/Au)复合微粒,金磁复合微粒形貌呈圆形,粒径均一,粒径约170-185 nm。采用过碘酸钠法制备辣根过氧化物酶(HRP)标记CN/α-LA,计算CN-HRP和α-LA-HRP蛋白浓度分别为1.991 mg/m L,2.816 mg/m L;CN-HRP与α-LA-HRP的酶活损失率分别为8.7%、7.63%。4、金磁酶联免疫法检测牛乳过敏原CN/α-LA体系的建立。按照最佳条件制备得到金磁免疫探针和CN/α-LA酶标抗原后,将它们与CN/α-LA标准溶液竞争结合建立金磁酶联免疫检测体系,初步探究了该体系在牛乳中CN/α-LA检测的可行性。研究结果显示CN浓度在4 ng/m L~256 ng/m L范围内,CN浓度对数与抑制率有较好的线性关系,标准曲线为y=32.654x-5.17143,R2=0.993;IC50为50ng/m L,LOD为2.9 ng/m L。α-乳白蛋白浓度在2 ng/m L~256ng/m L检测范围内有较好的线性关系,标准曲线为y=35.718x-2.8429,R2=0.99545;IC50为30.2 ng/m L,LOD为2.3 ng/m L。此外,对该检测体系进行方法学评价,结果显示该方法精密度、特异性、稳定性均较好。CN、α-LA回收率分别在82%~105%和87.1%~111%范围内,满足对CN/α-LA检测的要求。在食品安全检测中有重要的实际应用价值。
[Abstract]:Cow dairy products are rich in nutrition, milk and dairy products are also identified by WHO as one of the eight categories of food that cause human allergies. Among them, casein (CNN) and 伪 -lactalbumin (伪 -LAA) in milk are regarded as the main allergens. Therefore, the detection of casein and 伪 -lactalbumin in food has important theoretical and practical significance. The specific research contents are as follows: 1, casein, 伪-lactoalbumin monoclonal antibody preparation. Four BALB/c mice were immunized with CN-LA and 伪 -LA respectively. The antiserum titer of mice was measured by indirect ELISA method. The spleen cells of mice with the highest titer were fused with myeloma cells and hybridoma cells were obtained. Indirect ELISA method was used to screen positive holes, and 3 times subclone with limited dilution method was used to obtain homogenous hybridoma cell lines. Finally, one casein monoclonal antibody and two 伪 -lactoalbumin monoclonal antibody hybridoma cell lines were screened. The monoclonal antibody. 2, casein and 伪 -lactoalbumin monoclonal antibody were prepared in mice by induced ascites in vivo. The monoclonal antibody was purified by octanoic acid-ammonium sulfate precipitation method. The purity of the monoclonal antibody was characterized by SDS-PAGE electrophoresis. The titer, specificity and affinity of McAbs were detected by indirect competitive ELISAL Western-blotting. The results showed that the concentration of CN-伪 -LA McAbs was 8.425 mg/m L and 8.591mg/m L, respectively. The titers of anti-CN 3D3 McAb and 伪 -LA11G10 / 11F8 McAbs were 1: 256000 and 1: 512000.The titers of 伪 -LA11G10 / 11F8 McAbs were 1: 128000. The two monoclonal subclasses were detected to be Ig G 1 type. The affinity constants of casein and 伪 -lactoalbumin monoclonal antibody were 0.32 脳 10 ~ 8 L/mol and 0.37 脳 10 ~ 8 L / mol 路3, respectively. The magnetic particles Fe _ 3O _ 4-PEI were prepared by one-pot method, and then coated with two layers of au particles on the surface of the particles. The au _ 3O _ (4 / Au) composite particles were prepared. The morphology of the composite particles was circular, the particle size was uniform, and the particle size was about 170-185 nm. Horseradish peroxidase (horseradish peroxidase) labeled CN/ 伪 -LA-HRP was prepared by sodium periodate method. The protein concentrations of CN-HRP and 伪 -LA-HRP were calculated to be 1.991 mg/m / L ~ (2.816) mg/m / L ~ (-1) CN-HRP, respectively, and the loss rates of enzyme activity of 伪 -HRP and 伪 -LA-HRP were 8.7 ~ 7.63 路4 respectively. The establishment of CN/ 伪 -LA system for detection of bovine milk allergen by gold-magnetic enzyme-linked immunosorbent assay. The gold magnetic immunosorbent probe and CN/ 伪 -LA enzyme labeled antigen were prepared according to the optimum conditions. The gold magnetic enzyme linked immunosorbent assay system was established by combining them with the CN/ 伪 -LA standard solution. The feasibility of this system in the detection of CN/ 伪 -LA in milk was preliminarily explored. The results showed that the logarithm of CN concentration in the range of 4 ng/m L ~ (256 ng/m / L) had a good linear relationship with the inhibition rate, and the standard curve was: yz32.654x-5.17143N / 0.993C _ (50), 50ng/m _ L _ L _ (LD) = 2.9 ng/m L 路伪 -lactoalbumin in the range of 2 ng/m L~256ng/m / L. The standard curve was: yak 35.718x-2.8429 R2X 0.99545L IC50 was 30.2 ng/m / L and LOD was 2.3 ng/m / L. In addition, the method was evaluated. The results showed that the precision, specificity and stability of the method were good. The recoveries of 伪 -LA were in the range of 82105% and 87.1%, respectively, which met the requirements for the detection of CN/ 伪 -LA. It has important practical application value in food safety detection.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R155.5

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