【作者】 孙颖；

【导师】 李会强；

【作者基本信息】 天津医科大学 ， 医学检验诊断学， 2012， 硕士

【摘要】 目的:TPO抗体是甲状腺自身免疫性疾病的重要诊断指标,本研究首先建立天然TPO抗原和TPO抗体的制备工艺,并以此为原料,分别建立新型酶联免疫试剂盒和均相化学发光免疫试剂盒,用于定量检测TPO抗体,并对其临床应用性能进行考察,为自身免疫性甲状腺疾病的诊断提供检测试剂。方法:1.TPO抗原的制备:收集外科手术切除的Grave’s病患者的甲状腺组织,经过组织匀浆,超声粉碎,反复离心,胰蛋白酶的水解,透析等方法制备TPO抗原,粗提物通过亲和层析柱获得TPO纯品,经SDS-PAGE及免疫印迹进行蛋白纯度和免疫活性鉴定。2.TPO抗体的制备:收集TPO抗体>1000IU/mL临床标本,混合,经50%饱和硫酸铵溶液沉淀获得丙种球蛋白,再通过Sephadex 4B-Potein A亲和层析获得IgG,经Abbott公司AXSYM微粒子酶免疫法鉴定TPO抗体的活性,定量作为制备标准品储存液。3.新型酶联免疫试剂盒:将生物素化的牛血清白蛋白包被在微孔板上,再加入链霉亲合素,获得BBA酶标反应板;将生物素标记TPO抗原,制备出生物素化的抗原;经棋盘滴定的方式确定生物素化TPO抗原与酶标抗人-IgG抗体工作浓度;并对反应条件进行优化,同时测定主要方法学参数。4.TPO抗体均相发光免疫测定(竞争法)试剂盒:将TPO抗体包被于受体微球上;制备生物素化TPO抗原;对反应条件进行优化,对试剂盒进行方法学评价。结果:1.TPO抗原的制备:从甲状腺组织中获得了 TPO抗原纯品,经SDS-PAGE鉴定为一条分子量约为55 KD的单一条带,免疫印迹显示能与TPO抗体发生反应。2.TPO抗体的制备:从阳性的血清标本中获得了 TPO抗体的IgG成分,测定蛋白总量512mg,TPO抗体活性为4408 IU/mL。3.新型酶联免疫试剂盒:棋盘滴定结果显示,生物素化TPO抗原的最佳稀释比例为1:6000,IgG-HRP的最佳稀释比例为1:8000。本方法的最适反应温度为37℃,最佳反应时间为30min。本方法的检测灵敏度为0.165IU/mL,批间变异9.0%～9.2%、批内变异6.6%～9.1%,回收率为96%～104%,试剂盒37。℃放置5d保持稳定。4.TPO抗体均相发光免疫测定(竞争法)试剂盒:本实验采用平衡法,包被TPO抗体的受体微球的最佳稀释比例为1:100,包被亲合素的供体微球采用150 ul的加样量。待测抗体与TPO抗体-受体微球的体积比为2:1,最佳反应时间为30min。本方法的检测灵敏度为0.135IU/mL,批间变异6.9%～8.0%、批内变异3.4%～6.2%,回收率为95.6%-103.3%,包被板37℃放置5d保持稳定。结论:1.本实验将TPO抗原成功地从人的甲状腺组织提取纯化出来,建立了一整套较为完善的提取纯化工艺,获得了较高纯度的TPO抗原,通过SDS-PAGE、免疫印迹技术及Abbott公司AXSYM微粒子酶免疫法对TPO抗原的纯度、特异性及免疫活性进行了鉴定,为研制新型的TPO抗体定量测定酶联免疫试剂盒和TPO抗体均相发光免疫测定(竞争法)试剂盒奠定了基础。2.本实验将人血清中的TPO抗体进行了蛋白纯化,提出了 IgG组分,排除了其他组分对本实验的干扰,通过Abbott公司AXSYM微粒子酶免疫法进行了免疫活性鉴定并定量,一方面制备出了 TPO抗体的标准品,另一方面在TPO抗体均相发光免疫测定(竞争法)试剂盒中,我们用从天然的人血清提纯出的TPO抗体包被受体微球。3.新型的TPO抗体定量测定酶联免疫试剂盒适合TPO抗体的测定,不仅节省抗原,降低成本,而且特异性强,操作简单,适合基层医院开展。4.TPO抗体均相发光免疫测定(竞争法)试剂盒无需分离与洗涤步骤,精密度较高,又节省了检测时间,采用了化学发光法,具有很强抗干扰能力,较低背景信号,自动化程度高,适合大型医院开展。更多还原

【Abstract】 Objective:TPO antibody is an important diagnostic indexe of thyroid autoimmune diseases.This study to establish the natural TPO antigen and TPO antibody preparation process,and use them as raw materials to establish a new enzyme-linked immunosorbent assay(ELISA)kit and homogeneous chemiluminescent immunoassay(LICA)kits for the quantitative detection of TPO antibodies.We investigate the kits clinical application performance which provide detection reagents for the diagnosis of antoimmune thyroid disease.Methods:1.TPO antigen preparation:Collect thyroid tissue of Graves’ patients which were surgically removed.After tissue homogenates,sonication,and repeated centrifuge,trypsin hydrolysis,dialysis and other methods of preparation of the TPO antigen.The crude extracts go through affinity chromatographycolum and then get the purified TPO antigen.The protein purity and immunological activity were identified by SDS-PAGE and Western Blot.2.TPO antibody preparation:Collect clinical Specimens:TPO antibody>1000 IU/mL,mix them.Get gamma globulin by 50%ammonium sulfate saturation solution precipitation.Get IgG through the Sephadex 4B-Potein A affinity chromatography.The activity of TPO antibody was identified by Abbott company AXSYM microparticle enzyme immunoassay.Determine the concentration and prepare the standare stock solution.3.New enzyme-linked immunosorbent assay(ELISA)kit:The microtiter plate was coated with biotinylated bovine serum albumin,then add streptavidin,get the BBA microtiter plate;Get the biotinylated TPO antigen by labeled TPO antigen with biotin;Determined the working concentration of biotinylated TPO antigen and enzyme labeled anti-human-IgG antibody by checkerboard titration Optimize the reaction conditions and identify the main methodological parameters.4.TPO antibody homogeneous luminescence immunoassay(competition method)Kit:.The TPO antibody was coated on receptor particles;Prepare the biotin TPO antigen;Optimize the reaction conditions such as reaction order,the dilution of the receptor particles labeled with TPO antibody,the addition of donor particles,the volume ratio of the measured antibody and the receptor particles labeled TPO antibody,as well as the reaction time of the first step and finally did method evaluation.Results:1.TPO antigen preparation:We obtained the purified TPO antigen from thyroid tissue.A simple protein band whose molecular mass is about 55KD is visible on SDS-PAGE,Western Blot shows that the protein band can react with TPO antigen.2.TPO antibody preparation:The purified IgG composition of TPO antibody was extracted from the positive clinical specimens.The total protein is 512 mg;The activity of protein is 4408 IU/ml.3.New enzyme-linked immunosorbent assay(ELISA)kit:According to the results of chessboard titration,the optimal dilution of biotinylated TPO antigen is 1:6000,the optimal dilution of IgG-HRP is 1:8000.The optimal temperature of the method is 37℃;the optimal reaction time is 30 min.The result of method evaluation shows that the detection sensitivity of the assay was 0.165 IU/ml,the coefficient of variations(CV)of inter-assay between high and low concentration of quality control serum were 9.2%～9.0%,CV of intra-assay were 4.6%～5.6%,the recovery rate was between 96%～104%,the kit remained stable for 5 days in 37℃.4.TPO antibody homogeneous luminescence immunoassay(competition method)Kit:This study uses the balance method.The optimize dilution ration of the microspheres coated with TPO antibody is 1:100;The sample amount of donor microspheres coated with avidin is 150 ul;The volume ratio of test antibody and receptor microspheres coated with TPO antibody is 2:1;The optimal reaction time is 30min.The sensitivity of this method is 1.35IU/ml,intra-assay CV is 6.9%～8.0%,inter-assay CV is 3.4%-6.2%,the recovery rate is 5.6%～103.3%,the kit remained stable for 5 days in 37℃.Conclusion:1.In this study,We extracted TPO antigen from the thyroid tissue successfully and established a set of relatively perfect extraction and purification process,;obtained higher purity TPO antigen.Purity,specificity and immunological activity of TPO antigen were identified by SDS-PAGE,Western blot and Abbott microparticle enzyme immunoassay.which lay the foundation for the development of new enzyme-linked immunosorbent assay(ELISA)kit kits for quantitative detection TPO antibody and light induced chemiluminescent immunoassay(LICA)kits for quantitative detection(competition method)of TPO antibody.2.In this study,We purified TPO antibody from the patients’ serum and extracted the IgG component,exclude the interference from other components to this experiment.The immunological activity identification and quantitative of TPO antibodies were done by the Abbott ASCYM microparticle.On the one hand,we prepared the TPO antibody standard preparation,on the other hand,we used purified TPO antibody from natural human serum to coat the receptor particles in TPO antibody homogeneous luminescence immunoassay kit(competition method).3.The new enzyme-linked immunosorbent assay(ELISA)kit is suit to detect TPO antibodies quantitatively,which can reduce dosage of TPO antigen,and cut down the cost.The new ELISA kit is sensitive and fit for basic-level hospital.4.The light induced chemiluminescent immunoassay(LICA)kits for quantitative detection(competition method)of TPO antibody don’t need separation and washing steps,high precision,and save testing time.Because of the method of chemical luminescence,it has a very strong ability of anti-interference,lower background signal,and a high degree of automation,and fit to large hospital.更多还原