酶标SPA免疫酶法检测宫颈癌患者血清中单纯疱疹病毒抗体

免疫酶法用于检测病人体液中病毒特异性抗体与其它方法相比。是一种简便易行、敏感性、特异性较好的方法,应用日广。用它检测病毒IgG抗体,通常以酶标抗人IgG(简称酶标抗体)作为第二抗体。本文将辣根过氧化物酶标记葡萄球菌A蛋白(简称酶标SPA)用于免疫酶法中检测宫颈癌患者血清中单纯疱疹病毒     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

血栓调节蛋白化学发光免疫分析检测方法的建立*

目的:建立并评价基于板式化学发光免疫分析(CLIA)平台的血栓调节蛋白(TM)定量检测方法。方法:以链霉亲和素包被微孔板,加入待检血浆,偶联生物素和辣根过氧化物酶的配对抗体组成分析体系,采用双抗体夹心模式,建立TM抗原定量检测方法,并对其进行条件优化和性能评价。结果:生物素化抗体和酶标抗体的工作浓度分别为0.5 μg/mL和0.75 μg/mL,加样后的孵育时间选为15 min,最低检测限为0.2 TU/mL,该检测方法的检测范围为1~200 TU/mL,批间和批内精密度(CV)均小于8%,37 ℃ 10天稳定性良好,207份临床血浆测值与希森美康测值相关性较高(R²>0.96)。结论:建立了TM板式化学发光定量检测方法,且各项性能指标良好,可满足临床检测的需要。     

改良双抗体夹心酶联免疫法测定重组人活性蛋白C含量的研究

研究用ELISA法直接定量检测细胞培养液中的重组人活性蛋白C(rhAPC)。用改良双抗体夹心酶联免疫检测法进行定量分析。用棋盘滴定法对包被抗体、反应一抗和辣根过氧化物酶(HRP)标记二抗的适宜工作浓度进行了选择,并对实验条件进行优化,参考品标准曲线线性相关性较好。改良双抗体夹心酶联免疫检测法可用于直接定量分析细胞培养液中的rhAPC水平。     

酶标免疫分析法鉴定蛇毒

蛇伤的治疗与蛇伤种类的快速诊断密切相关。我们用亲和层析法纯化了三种蛇毒的抗体Fab,它们之间无免疫交叉反应。然后用高碘酸钠法将辣根过氧化物酶标记到抗体Fab上。我们建立的三夹心式酶标免疫测定蛇毒的方法,可在90分钟内检测到5毫微克以下的蛇毒,人体血清对该测定无干扰现象,临床检测效果良好。     

抗鹅免疫球蛋白轻链单克隆抗体的鉴定及其应用

免疫球蛋白是机体固有免疫系统的组成部分,是机体防御的第一道防线。本研究对抗鹅免疫球蛋白轻链单克隆抗体进行了特征分析并将其应用到不同免疫试验中用以检测鹅免疫球蛋白。用此单克隆抗体制备的免疫亲和层析柱用以分离血清中的鹅免疫球蛋白;偶联辣根过氧化物酶 (Horseradish peroxidase,HRP) 后的单克隆抗体用作第二抗体来检测鹅特异性抗体。此外,该单克隆抗体可以识别和定位外周血淋巴细胞中的SIg+淋巴细胞。研究表明,该单克隆抗体可在多种条件下检测或分离鹅免疫球蛋白并作为研究鹅体液免疫的有力工具。     

研究报告：金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果。ELISA的本质是抗原与相应抗体之间的特异性相互作用。然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果。本课题组先前应用构象工程方法开发了一种基于金纳米粒子的人工抗体(简称金抗体)。金抗体可以像天然抗体一样特异性地与抗原相互作用,并且具备远优于天然抗体的稳定性。出色的稳定性使金抗体可能成为天然抗体更好的替代物,用于ELISA中。方法经过必要的优化并与辣根过氧化物酶(HRP)耦联后,制得酶标金抗体10HRP-(Au-400P1),然后用酶标金抗体代替天然酶标抗体用于ELISA检测中。结果通过一系列的实验证明,抗溶菌酶金抗体可用于ELISA特异性检测1～16 mg/L范围内的鸡蛋清溶菌酶(HEWL)样品。结论金抗体可以替代天然抗体用于ELISA检测,并具有优于传统ELISA法的检测准确性和一致性。     

辣根过氧化物酶标记抗体在免疫定位上的应用 Ⅱ.肝细胞内乙型肝炎表面抗原的电子显微镜定位方法

本文介绍辣根过氧化物酶标记抗体对肝细胞内乙型肝炎表面抗原(HBsAg)的免疫电镜定位方法。电镜样品制备,可采用酶标免疫显色组织切片的再包埋、或直接利用超薄切片进行酶标免疫显色定位。对实验中应注意的一些问题进行了讨论。在电子显微镜下初步观察了HBsAg 阳性肝细胞中HBsAg 的分布和形态,并发现在光学显微镜下,辣根过氧化物酶标免疫定位HBsAg 完全为阴性部位的肝细胞中,有的细胞胞浆内或扩大的内质网池内,尚可见到少量散在的HBsAg 与酶标抗体的沉积物,提示电镜观察有更好的灵敏性。     

蛋白质谷氨酰胺酶成熟肽基因mpg原核表达及其多克隆抗体的制备

蛋白质谷氨酰胺酶(Protein-glutaminase,简称PG)是一种新型蛋白质脱酰胺酶,在蛋白质改性领域具有广阔的应用前景。但是,目前关于蛋白质谷氨酰胺酶的测定方法主要是通过苯酚-次氯酸盐反应测定铵离子含量指示蛋白质谷氨酰胺酶的酶活,该方法精确度和灵敏度有限。因此,利用原核表达系统表达解朊金黄杆菌(Chryseobacterium proteolyticum)分泌的蛋白质谷氨酰胺酶成熟肽基因mpg,纯化后制备其多克隆抗体用于检测解朊金黄杆菌分泌的蛋白质谷氨酰胺酶的含量。首先以解朊金黄杆菌基因组为模板扩增出成熟肽基因mpg,将其与pET32a载体连接构建重组质粒pET32a-mpg,转化至大肠杆菌BL21(DE3)。在25℃条件下,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导6 h,目的蛋白表达量最高、呈可溶性蛋白。通过Ni-NTA柱纯化的重组蛋白mPG-His_6,将其作为抗原,免疫新西兰白兔制备多克隆抗体。间接酶联免疫法检测其效价,所得抗体效价为1∶5 000。蛋白质免疫印迹实验结果说明,该多克隆抗体特异性良好。最后使用该多克隆抗体对解朊金黄杆菌的发酵上清液进行蛋白质免疫印迹反应,检测天然蛋白质谷氨酰胺酶,结果表明该抗体特异性良好,可用于检测解朊金黄杆菌分泌的蛋白质谷氨酰胺酶,为深入研究蛋白质谷氨酰胺酶的生物学功能、建立蛋白质谷氨酰胺酶高产菌株的高通量筛选方法奠定了基础。     

食源性致病菌检测免疫传感器研究进展

食源性致病菌是造成食品安全事件的主要原因之一,因此其检测方法已成为人们研究的热点.食源性致病菌的检测方法主要有病原体培养法、免疫学方法、核酸检测和生物传感器等.其中,免疫传感器基于抗原抗体特异性结合,整合光学、电化学等多学科交叉技术,具有特异性强、检测速度快等特点.本文对比食源性致病菌传统检测方法,综述了近年来免疫传感...     

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.     

A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen

A new simple immunoassay method for carcinoembryonic antigen (CEA) detection using a disposable immunosensor coupled with a flow injection system was developed. The immunosensor was prepared by coating CEA/colloid Au/chitosan membrane at a screen-printed carbon electrode (SPCE). Using a competitive immunoassay format, the immunosensor inserted in the flow system with an injection of sample and horseradish peroxidase (HRP)-labeled CEA antibody was used to trap the labeled antibody at room temperature for 35 min. The current response obtained from the labeled HRP to thionine-H(2)O(2) system decreased proportionally to the CEA concentration in the range of 0.50-25 ng/ml with a correlation coefficient of 0.9981 and a detection limit of 0.22 ng/ml (S/N=3). The immunoassay system could automatically control the incubation, washing and current measurement steps with good stability and acceptable accuracy. Thus, the proposed method proved its potential use in clinical immunoassay of CEA.     

Electrochemical immune-biosensor for immunoglobulin G based bioelectrocatalytic reaction on micro-comb electrodes

A new amplification strategy of electrochemical signaling from antigen-antibody interactions was proposed via back-filling immobilization of horseradish peroxidase (HRP), immunoglobulin G antibodies (anti-IgG) and gold nanoparticles onto a three-dimensional sol-gel (3DSG)-functionalized biorecognition interface. The 3DSG sol-gel network was employed not only as a building block for the surface modification but also as a matrix for ligand functionalization. The signal-amplification was based on the bioelectrocatalytic reaction of the back-filling immobilization of HRP to H(2)O(2). With the non-competitive format, the formation of the antigen-antibody complex by a simple one-step immunoreaction between the immobilized anti-IgG and IgG in sample solution inhibited partly the active center of HRP, and decreased the immobilized HRP towards H(2)O(2) reduction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to IgG in a dynamic range of 1.12-162 ng/mL with a detection limit of 0.56 ng/mL (at 3delta). Moreover, the precision, reproducibility and stability of the as-prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of biomarkers and its metastasis.     

A chemiluminescence flow immunosensor based on a porous monolithic metacrylate and polyethylene composite disc modified with protein G

A generic, fast, sensitive and new type of flow immunosensor has been developed. The basis is a monolithic porous poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) polymer disc modified with protein G, placed in a fountain type flow cell compartment, in close proximity to a photomultiplier tube (PMT). Analyte and HRP labelled analyte derivative (tracer) compete for anti-analyte antibody binding sites. The mixture is then injected into the flow immunosensor system where the formed analyte- and tracer-antibody complexes are trapped by the monolithic protein G disc. The amount of bound tracer, inversely related to the concentration of analyte in the sample, is determined in a second step by injection of luminol, p-iodophenol and H2O2, generating enhanced chemiluminescence (CL) with horseradish peroxidase (HRP). A third and final step is need for regeneration of the protein G disc so that a new analysis cycle can take place. The performance of the disc immunosensor system was compared with a one step continuous flow injection immunoassay (FIIA) system, using the same reagents and a protein G column, in terms of assay sensitivity and influence of matrix effects from various water samples (millipore-, tap- and surface water). The detection limit for the analyte atrazine in PBS and surface water (SW) was 0.208 +/- 0.004 microg l(-1) (PBS) and 0.59 +/- 0.120 microg l(-1) (SW) for the FIIA and 0.033 +/- 0.003 microg l(-1) (PBS) and 0.038+/-0.003 microg l(-1) (SW) for the disc immunosensor. Statistical comparison of the two systems shows that the disc immunosensor results were significantly less influenced by the sample matrix, which is explained by the fact that the sample in the FIIA arrives simultaneously with the matrix to the detector, whereas these are separated in time in the disc immunosensor system.     

A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chitosan-membrane-modified screen-printed carbon electrode

A screen-printed three-electrode system is fabricated to prepare a novel disposable screen-printed immunosensor for rapid determination of alpha-1-fetoprotein (AFP) in human serum. The immunosensor is prepared by entrapping horseradish peroxidase (HRP)-labeled AFP antibody in chitosan membrane to modify the screen-printed carbon electrode. The membrane is characterized with scanning electron microscope and electrochemical methods. After the immunosensor is incubated with AFP at 30 degrees C for 35 min, the access of the active center of HRP catalyzing the oxidation reaction of thionine by H(2)O(2) is partly inhibited. In presence of 1.2 mM thionine and 6 mM H(2)O(2), the electrocatalytic current decreases linearly in two concentration ranges of AFP from 0 to 20 and from 20 to 150 ng/mL with a detection limit of 0.74 ng/mL. The immunosensor shows an acceptable accuracy compared with those obtained from immunoradiometric assays. The interassay coefficients of variation are 6.6 and 4.2% at 10 and 100 ng/mL, respectively. The storage stability is acceptable in pH 7.0 phosphate buffer solution at 4 degrees C for more than 10 days. The proposed method can detect the AFP through one-step immunoassay and would be valuable for clinical immunoassay.     

Amperometric immunosensor for diagnosis of BLV infection

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody-antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N',N'-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.     

Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybrids platform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a model protein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels and secondary antibodies (Ab₂) linked to GNPs at high HRP/Ab₂ molar ratio. The approach provided a linear response range between 0.125 and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.     

A disposable two-throughput electrochemical immunosensor chip for simultaneous multianalyte determination of tumor markers

A disposable two-throughput immunosensor array was proposed for simultaneous electrochemical determination of tumor markers. The low-cost immunosensor array was fabricated simply using cellulose acetate membrane to co-immobilize thionine as a mediator and two kinds of antigens on two carbon electrodes of a screen-printed chip, respectively. With two simultaneous competitive immunoreactions the corresponding horseradish peroxidase (HRP) labeled antibodies were captured on the membranes, respectively, on which the immobilized thionine shuttled electrons between HRP and the electrodes for enzymatic reduction of H2O2 to produce detectable signals. The electrochemical and electronic cross-talks between the electrodes could be avoided, which was beneficial to the miniaturization of the array without considering the distance between immunosensors. Under optimal conditions the immunosensor array could be used for fast simultaneous electrochemical detection of CA 19-9 and CA 125 with the limits of detection of 0.2 and 0.4 U/ml, respectively. The serum samples from clinic were assayed with the proposed method and the results were in acceptable agreement with the reference values. The proposed method for preparation of immunosensor array could be conveniently used for fabrication of disposable electrochemical biochip with high throughput and possessed the potential of mass production and commercialization.     

GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein

A new flow-through electrochemical immunosensor was designed for sensitive detection of alpha-fetoprotein (AFP) in human serum by using nanogold-functionalized magnetic graphene nanosheets as immunosensing probes. Initially, amino functionalized magnetic beads were covalently immobilized on the surface of graphene oxide nanosheets (MGPs), then nanogold particles were adsorbed on the amino groups of the MGPs to construct GoldMag nanocomposites functionalized graphene nanosheets (GMGPs), and then horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP) were assembled onto the surface of nanogold particles (bio-GMGP). With the aid of an external magnet, the formed bio-GMGPs were attached onto the base electrode in the flow system. With a non-competitive immunoassay format, the injected sample containing AFP antigens was produced transparent immunoaffinity reaction with the immobilized HRP-anti-AFP on the bio-GMGPs. The formed immunocomplex inhibited partly the active center of HRP, and decreased the labeled HRP toward the reduction of H(2)O(2). The performance and factors influencing the performance of the immunosensor were investigated in detail. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.01-200 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) AFP (at 3s(B)). Intra- and inter-assay coefficients of variation (CV) were below 10%. In addition, the methodology was validated with real serum samples, receiving a good correlation with the results obtained from commercially available electrochemiluminescence automated analyzer.     

A disposable electrochemical immunosensor based on carbon screen-printed electrodes for the detection of prostate specific antigen

A novel screen-printed electrode (SPEs) on sheets of vegetable parchment was prepared. The obtained SPEs were stable, convenient, inexpensive and suitable for large-area screen-printing. With these SPEs, we explored the fabrication of a novel, disposable and highly sensitive electro-analytical immunosensor using graphene nanosheets (GS) and horseradish peroxidase (HRP)-labeled signal antibody functionalized with gold nanoparticles (HRP-Ab(2)/Au NPs). GS was used to increase the conductivity and stability of this immunosensor due to its fast electron transportation and good biocompatibility. Au NPs could not only provide a large surface area for the immobilization of HRP-Ab(2) but also enhance the electroreduction between HRP and H(2)O(2) to amplify the electrochemical signal on the sandwich immuno-complexes modified SPEs. The proposed SPEs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical methods involving cyclic voltammetry (CV), and electrochemical impedence method. Using prostate specific antigen (PSA) as a model analyte, this immunosensor showed a wide linear range over 6 orders of magnitude with the minimum value down to 2pgmL(-1). In addition, this immunosensor could avoid the need of deoxygenation for the electrochemical immunoassay. Thus, it provided a promising potential in clinical applications.