基于酶标噬菌体抗体的磁分离免疫分析方法

以磁微粒偶联多抗为磁性捕获探针,酶标噬菌体抗体为特异信号检测探针,采用"磁性捕获探针-待测物-酶标噬菌体抗体探针"的检测模式,成功建立了一种基于酶标噬菌体抗体的磁分离免疫分析方法。本方法检测β-银环蛇毒素线性范围为0.016~62.5μg/L,回归方程为Y=0.641X+1.355(R=0.9925,n=13,p<0.0001),检出限为0.016μg/L。本方法比传统ELISA法检测灵敏度提高了10倍,与采用酶标单抗复合物探针的双抗体夹心磁分离免疫分析法相比,检测灵敏度提高4倍。本方法灵敏度高,具有较好重现性与特异性,在毒素的痕量检测方面具有广阔的应用前景。     

一种基于金磁微粒的电化学发光免疫检测方法

基于金磁微粒(GoldMag particles)的磁性分离富集性能与良好的生物相容性,直接在金磁微粒表面吸附固定相思子毒素多抗制备捕获探针,以三联吡啶钌标记相思子毒素单抗作为电化学发光探针,两者与相思子毒素发生特异性免疫反应形成夹心复合物,成功建立了一种简单、快速、灵敏的相思子毒素电化学发光免疫检测方法。利用此方法检测相思子毒素,其浓度在0.2～1 500μg/L范围内与电化学发光强度成良好的对数线性关系,检出限为0.2μg/L。与生物素-亲和素固定法相比,该法的检出限相当,其线性范围更宽、操作更简化,具有通用性,可以此为基础发展生物毒素及其它蛋白的检测方法,用于临床诊断、环境监测及生物防护等领域。     

基于噬菌体展示抗体的电化学发光免疫传感器检测相思子毒素研究

以多克隆抗体包被磁性微球制备捕获探针,以表面负载大量三联吡啶钌标记物的噬菌体展示抗体为发光探针有效放大信号,成功建立了相思子毒素电化学发光免疫传感检测方法。利用本方法检测相思子毒素,浓度在0.005～100μg/L范围内与电化学发光强度呈良好的对数线性关系,拟合方程为lgY=0.701lgX+1.767(R=0.9964,N=7,p<0.0001),检测限达0.005μg/L(S/N=3)。与采用单克隆抗体制备标记探针相比,检测灵敏度提高20倍,可用于相思子毒素的痕量检测。     

组装型金磁微粒的制备及其在免疫学检测中的应用

以化学共沉淀和柠檬酸还原法分别合成纳米级Fe₃O₄和Au粒子, 将经3-巯丙基三乙氧基硅烷修饰形成的Fe3O4聚集体与纳米金粒子相互作用, 制备得到组装型Fe₃O₄/Au磁性复合微粒(简称金磁微粒), 并对其形成过程、形貌特征、磁学性质等进行表征. 此外, 对金磁微粒作为新型免疫学检测载体的特性包括抗体固定化、质量控制及其在免疫学检测中的应用开展研究. 结果表明: 组装型金磁微粒形状不规则、表面粗糙, 平均粒径约为2~3 μm; 具有超顺磁性, 比饱和磁化强度达41 A·m²/kg; 1 mg金磁微粒最多可固定人IgG的量为330 μg, 以含有500 ng人IgG的金磁微粒为一个检测单位, 与辣根过氧化物酶标记羊抗人IgG的每批5个重复特异性反应测定结果的相对标准偏差均小于6%, 5批特异性反应测定结果的批间相对标准偏差小于7%(以每批5次测定结果的平均值计算), 符合免疫学检测载体的质量要求; 将抗乙肝表面抗原单克隆抗体和抗白介素-8单克隆抗体分别固定于金磁微粒表面, 采用双抗体夹心法对乙肝表面抗原和白介素-8分别进行定性和定量检测, 结果表明金磁微粒是一种较好的免疫学检测载体.     

基于金纳米微粒的化学发光金属免疫分析

建立了基于金纳米微粒溶解的化学发光反应体系,并探讨了金纳米微粒溶解及化学发光测定的最佳条件。首次将金纳米微粒引入生物素和IgG的化学发光金属免疫分析,比较了不同粒径金纳米微粒、不同检测系统对IgG测定的影响。在(一抗-IgG-二抗修饰金纳米微粒)检测系统中,基于10 nm和30 nm金纳米微粒测定IgG的线性范围分别为1~75 ng和0.5~25 ng,检出限分别为0.5 ng和0.1 ng。在(一抗-IgG-生物素化抗体-链霉亲和素修饰金纳米微粒)检测系统中,5 nm和10 nm金纳米微粒测定IgG的线性范围分别为10~250 ng和1~250 ng;检出限分别为5 ng和1 ng。     

基于酶联金纳米复合探针的磁分离免疫传感器检测莱克多巴胺

构建了一种基于新型酶联金纳米复合探针(E-GNPs)的磁分离竞争免疫传感器,用于检测莱克多巴胺(Ractopamine, RAC)。通过链霉亲和素与生物素的特异性相互作用,在抗体上标记辣根过氧化物酶(Horseradish peroxidase, HRP),采用静电组装法将抗体修饰于金纳米颗粒表面得到E-GNPs,引入卵清蛋白(Ovalbumin, OVA)-RAC半抗原(Hapten)复合物包被的磁珠进行竞争反应,通过显色反应实现目标物的定量分析。当目标分子RAC浓度变化时,酶催化底物显色随之改变,并且RAC的浓度与显色信号强度呈线性关系。紫外-可见吸收光谱表征结果表明,一个E-GNPs可携带11个HRP分子,使得该免疫传感器具有较高的灵敏度,检出限低至1.75 pg/mL,较传统酶联免疫方法灵敏度提高了10倍。交叉反应实验结果表明,此传感器对RAC的选择性良好,可应用于猪肉、牛肉和羊肉等实际样品中RAC的检测,加标回收率为88.3%～103.4%。本方法为动物源食品中RAC的快速筛查提供了一种新思路。     

基于包被包膜糖蛋白抗体纳米金磁性微粒的无试剂安培免疫传感器

在玻碳电极(GCE)表面固定对H2O2有催化还原活性的富马酸二甲酯联吡啶铜(GCE|CuL);再在GCE|CuL表面修饰一层金磁微粒-壳聚糖复合膜(nano Au/Fe3O4/Chit), 进而固定艾滋病毒(HIV)诊断标志物--包膜糖蛋白(gp160)抗体(anti gp160), 由此构建了一类快速检测 gp160的无试剂安培免疫传感器.当该传感器在含gp160溶液中37 ℃下温育30 min后, 传感器表面生成的免疫复合物随gp160浓度的增大而增加, 导致CuL 对H2O2 电催化还原效果降低, 催化电流呈现下降趋势.在PBS溶液(pH 7.0)和-300 mV下, 催化电流的降低值ΔIo与gp160浓度在1～400 μg/L 呈线性关系; 检出限为0.5 μg/L(3σ).研制的免疫传感器检测gp160时, 一步免疫反应即可得结果, 较相同条件下包被gp160抗体的纳米金单分子层修饰电极灵敏度更高, 检测范围更宽, 有望用于艾滋病人血清标志物gp160快速筛测.     

基于铜增强纳米金标记物结合磁分离的溶出伏安免疫分析法用于蛋白质的高灵敏测定

应用铜原位化学放大纳米金颗粒的信号增强特性, 并结合磁分离技术, 提出了一种高灵敏的溶出伏安免疫分析方法. 实验中以人IgG为模式蛋白质, 将抗体修饰的SiO₂@Fe₃O₄核壳型磁性纳米颗粒和纳米金标抗体悬浊液混合, 用以均相免疫识别人IgG, 借助外加磁场分离纯化, 在免疫复合物悬浊液中加入铜增强试剂进行沉积放大反应, 再将铜用稀硝酸溶解并进行溶出伏安分析检测. 结果表明, 与基于固相反应的金属免疫分析法相比, 所提出的基于均相反应和磁分离原理的方法具有操作简单、分析时间短等优点. 该方法显示出明显增强的人IgG检测性能, 其线性检测范围为01~1000 ng/mL, 检出限为73 pg/mL. 此外, 将其用于实际样品的回收率测定, 结果令人满意.     

氧氟沙星噬菌体库的构建、筛选及抗体结构模拟

应用噬菌体展示和重组抗体技术制备抗氧氟沙星单链抗体(scFv)库,筛选获得氧氟沙星特异性噬菌体scFv以及同源模拟其三维结构。将从氧氟沙星杂交瘤细胞提取的总RNA,用RT-PCR反转录合成cDNA,以针对鼠源重链可变区(VH)及轻链可变区(VL)基因的兼并引物,扩增获得VH和VL可变区基因,通过SOE-PCR法将VH基因和VL基因通过柔性多肽Linker(Gly4Ser)3拼接成全长scFv基因片段,将双酶切后的scFv基因片段插入T7噬菌体,经体外包装后转化宿主菌BLT5403,成功构建库容量为3×105pfu/mL的抗体库,经4轮吸附-洗脱-扩增的富集,采用直接竞争ELISA筛选到4个特异性噬菌体scFv,运用Expasy软件模拟特异性scFv的三维结构。为进一步大量表达氧氟沙星单链抗体奠定了基础。     

基于胱胺/壳聚糖/酶标纳米金自组装的的酪氨酶传感器的研制

通过自组装技术构制了一种简单有效的酪氨酸酶传感器。该法先通过戊二醛交联将壳聚糖固定在胱胺修饰的金丝电极上,进而通过氨基与纳米金的强力相互作用将酶标纳米金固定在壳聚糖层上。结果表明,酪氨酸酶能很好地保持其生物活性,所构制的传感器达到95%稳定状态电极的时间在15s以内。酚类化合物是通过酶催化产生的醌在-100mV(相对饱和甘汞电极)直接还原而测定的,传感器对邻苯二酚、苯酚、对甲基苯酚测定的灵敏度依次为2.216,4.828,4.885μA·μmol-1L.cm-2,检测限依次为0.32,0.60,0.18μmolL-1。二周后活性仍保持原有活性的75%。     

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT

A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.     

红细胞标记抗体的化学发光免疫分析

用红细胞代替辣根过氧化物酶作为双抗体夹心免疫分析中第二抗体的标记物, 建立了一种红细胞标记抗体的免疫化学发光测定乙型肝炎病毒表面抗原的新方法. 在免疫反应完成后, 结合了抗原-抗体免疫复合物的致敏红细胞在低渗溶液中溶血, 释放出血红蛋白. 基于血红蛋白对鲁米诺-H2O2体系化学发光具有催化作用的原理, 采用化学发光法测定血红蛋白含量. 测得的血红蛋白发光强度与待测抗原浓度呈线性关系. 采用这种方法可检测出0.5 ng/mL的乙型肝炎病毒表面抗原. 将该方法与酶联免疫吸附分析(ELISA)结合起来对乙型肝炎患者血清乙肝病毒表面抗原(HBsAg)进行检测, 两者符合率均为97%, 表明本法具有良好的灵敏度和特异性, 可用于临床标本测试.     

磁性珠状纤维素亲和吸附剂的制备与应用

采用反相悬浮包埋技术制备了粒径小于300um、粒径分布窄和湿态孔度高(85%～90%)的高顺磁性珠状纤维素,经高碘酸钠活化后,与具有生物活性的绒毛膜促性腺激素偶联,得磁性亲和吸附剂(每克磁性珠状纤维素上固载300～400IU绒毛膜促性腺激素).     

Kinetic Analysis of Prostate-Specific Antigen Interaction with Monoclonal Antibodies for Development of a Magnetic Immunoassay Based on Nontransparent Fiber Structures

Prostate cancer is the second most common cancer diagnosed in men worldwide. Measuring the prostate-specific antigen (PSA) is regarded as essential during prostate cancer screening. Early diagnosis of this disease relapse after radical prostatectomy requires extremely sensitive methods. This research presents an approach to development of an ultrasensitive magnetic sandwich immunoassay, which demonstrates the limit of PSA detection in human serum of 19 pg/mL at a dynamic range exceeding 3.5 orders of concentration. Such attractive performance stems, inter alia, from the kinetic analysis of monoclonal antibodies (mAbs) against free PSA to select the mAbs exhibiting best kinetic characteristics and specificity. The analysis is carried out with a label-free multiplex spectral-correlation interferometry compatible with inexpensive single-use glass sensor chips. The high sensitivity of developed PSA immunoassay is due to electronic quantification of magnetic nanolabels functionalized by the selected mAbs and three-dimension porous filters used as an extended solid phase. The assay is promising for PSA monitoring after radical prostatectomy. The proposed versatile approach can be applied for the rational design of highly sensitive tests for detection of other analytes in many fields, including in vitro diagnostics, veterinary, food safety, etc.     

Immobilization of Antibodies on Magnetic Carbonaceous Microspheres for Selective Enrichment of Lysine‐acetylated Proteins and Peptides

Lysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttranslational modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be detected before enrichment. In this study, for the first time, antibody‐immobilized magnetic carbonaceous microspheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, α‐casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody‐immobilized magnetic particles were successfully applied to analyze mouse mitochondrial proteins. After database search, 29 acetylated sites in 26 proteins were identi?ed.     

Biosensors Based on Ion-Sensitive Field-Effect Transistors for HLA and MICA Antibody Detection in Kidney Transplantation

This work demonstrates the ability of the Ion-Sensitive Field-Effect Transistor (ISFET)-based immunosensor to detect antibodies against the human leukocyte antigen (HLA) and the major histocompatibility complex class-I-related chain A (MICA). The sensing membrane of the ISFET devices was modified and functionalized using an APTES-GA strategy. Surface properties, including wettability, surface thickness, and surface topology, were assessed in each module of the modification process. The optimal concentrations of HLA and MICA proteins for the immobilization were 10 and 50 μg/mL. The dose-response curve showed a detection range of 1.98–40 µg/mL for anti-HLA and 5.17–40 µg/mL for anti-MICA. The analytical precision (%CV) was found to be 10.69% and 8.92% for anti-HLA and -MICA, respectively. Moreover, the electrical signal obtained from the irrelevant antibody was considerably different from that of the specific antibodies, indicating the specific binding of the relevant antibodies without noise interference. The sensitivity and specificity in the experimental setting were established for both antibodies (anti-HLA: sensitivity = 80.00%, specificity = 86.36%; anti-MICA: sensitivity = 86.67%, specificity = 88.89%). Our data reveal the potential of applying the ISFET-based immunosensor to the detection of relevant anti-HLA and -MICA antibodies, especially in the field of kidney transplantation.     

Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.     

A Novel Fluorescence Immunoassay Based on Two-time Amplified Fluorescence Signal by Hemin and Magnetic Nanoparticles for the Detection of Antigen

Abstract

A novel, selective, and sensitive magnetic-mimetic enzyme fluorescence immunoassay method for antigen detection has been developed by taking advantage of a magnetic separation process and the amplification feature of the hemin label. This method is based on a twice amplified fluorescence signal. The signal is first amplified due to the ultrasmall size and the high surface-to-volume ratio of the silica-coated magnetite nanoparticles, which enable the nanoparticles to carry much more antibodies. Second, the mimetic enzyme (hemin) as a labeling reagent catalyzes the reaction of p-hydroxyphenyl acetic acid and H₂O₂ can further amplify the fluorescence signal. This protocol was also evaluated for a sandwich-type immunoassay of human IgG, and the calibration graph for human IgG was linear over the range of 0–100 ng mL^?1 with a detection limit of 9.8 ng mL^?1. This method can easily separate magnetic nanoparticles from the solution, which simplified the process and played a promising role for various applications in immunoassay.