本文選題：酪蛋白 + α-乳白蛋白　； 參考：《上海師范大學》2016年碩士論文

【摘要】：牛乳制品含有豐富的營養(yǎng),同時乳及乳制品也是WHO認定的導致人類過敏的八大類食物之一。其中牛乳中的酪蛋白(casein,CN)及α-乳白蛋白(α-lactalbumin,α-LA)被視為主要的過敏原,因此開展針對食品中酪蛋白及α-乳白蛋白強特異性、高靈敏度的檢測具有重要的理論和現(xiàn)實意義。具體研究內(nèi)容如下:1、酪蛋白、α-乳白蛋白單克隆抗體的制備。以CN、α-LA各免疫4只BALB/c小鼠,用間接ELISA法測小鼠抗血清效價,獲得效價最高的小鼠的脾細胞與骨髓瘤細胞進行細胞融合,獲得雜交瘤細胞。間接ELISA法篩選陽性孔,有限稀釋法進行3次亞克隆得到分泌同質(zhì)的雜交瘤細胞株。最終篩選到1株分泌酪蛋白單抗、2株分泌α-乳白蛋白單抗的雜交瘤細胞株分別為3D3、11G10、11F8。采用小鼠體內(nèi)誘生腹水法大量制備單抗。2、酪蛋白、α-乳白蛋白單抗的鑒定采用辛酸-硫酸銨沉淀法純化單抗腹水,經(jīng)SDS-PAGE電泳表征單抗純度,采用間接競爭ELISA、Western-blotting、非競爭性ELISA法分別檢測單抗效價、特異性及親和力,結(jié)果顯示CN、α-LA單抗?jié)舛确謩e為8.425 mg/m L和8.591mg/m L�？笴N 3D3單抗,抗α-LA 11G10、11F8單抗效價分別為1:256000、1:512000、1:128000。并檢測兩類單抗亞類均為Ig G1型。酪蛋白、α-乳白蛋白單抗親和常數(shù)分別為0.32×108 L/mol和0.37×108 L/mol。3、金磁(Fe3O4/Au)復合微粒與酶標抗原的制備。采用一鍋法制備氨基化的磁性微粒Fe3O4-PEI,之后在其表面包覆兩層Au顆粒,制備金磁(Fe3O4/Au)復合微粒,金磁復合微粒形貌呈圓形,粒徑均一,粒徑約170-185 nm。采用過碘酸鈉法制備辣根過氧化物酶(HRP)標記CN/α-LA,計算CN-HRP和α-LA-HRP蛋白濃度分別為1.991 mg/m L,2.816 mg/m L;CN-HRP與α-LA-HRP的酶活損失率分別為8.7%、7.63%。4、金磁酶聯(lián)免疫法檢測牛乳過敏原CN/α-LA體系的建立。按照最佳條件制備得到金磁免疫探針和CN/α-LA酶標抗原后,將它們與CN/α-LA標準溶液競爭結(jié)合建立金磁酶聯(lián)免疫檢測體系,初步探究了該體系在牛乳中CN/α-LA檢測的可行性。研究結(jié)果顯示CN濃度在4 ng/m L~256 ng/m L范圍內(nèi),CN濃度對數(shù)與抑制率有較好的線性關系,標準曲線為y=32.654x-5.17143,R2=0.993;IC50為50ng/m L,LOD為2.9 ng/m L。α-乳白蛋白濃度在2 ng/m L~256ng/m L檢測范圍內(nèi)有較好的線性關系,標準曲線為y=35.718x-2.8429,R2=0.99545;IC50為30.2 ng/m L,LOD為2.3 ng/m L。此外,對該檢測體系進行方法學評價,結(jié)果顯示該方法精密度、特異性、穩(wěn)定性均較好。CN、α-LA回收率分別在82%~105%和87.1%~111%范圍內(nèi),滿足對CN/α-LA檢測的要求。在食品安全檢測中有重要的實際應用價值。
[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 偽 -lactalbumin (偽 -LAA) in milk are regarded as the main allergens. Therefore, the detection of casein and 偽 -lactalbumin in food has important theoretical and practical significance. The specific research contents are as follows: 1, casein, 偽-lactoalbumin monoclonal antibody preparation. Four BALB/c mice were immunized with CN-LA and 偽 -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 偽 -lactoalbumin monoclonal antibody hybridoma cell lines were screened. The monoclonal antibody. 2, casein and 偽 -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-偽 -LA McAbs was 8.425 mg/m L and 8.591mg/m L, respectively. The titers of anti-CN 3D3 McAb and 偽 -LA11G10 / 11F8 McAbs were 1: 256000 and 1: 512000.The titers of 偽 -LA11G10 / 11F8 McAbs were 1: 128000. The two monoclonal subclasses were detected to be Ig G 1 type. The affinity constants of casein and 偽 -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/ 偽 -LA-HRP was prepared by sodium periodate method. The protein concentrations of CN-HRP and 偽 -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 偽 -HRP and 偽 -LA-HRP were 8.7 ~ 7.63 路4 respectively. The establishment of CN/ 偽 -LA system for detection of bovine milk allergen by gold-magnetic enzyme-linked immunosorbent assay. The gold magnetic immunosorbent probe and CN/ 偽 -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/ 偽 -LA standard solution. The feasibility of this system in the detection of CN/ 偽 -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 路偽 -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 偽 -LA were in the range of 82105% and 87.1%, respectively, which met the requirements for the detection of CN/ 偽 -LA. It has important practical application value in food safety detection.
【學位授予單位】：上海師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R155.5

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本文編號：1970967