神经元特异性烯醇酶和癌胚抗原胶体金免疫层析联合检测

目的：应用双抗夹心胶体金免疫层析方法,实现对神经元特异性烯醇化酶（NSE）和癌胚抗原（CEA）两种肺癌肿瘤标志物的快速联合检测。方法：采用柠檬酸三钠还原法制备20nm胶体金颗粒,并分别对鼠抗NSE、CEA单克隆抗体进行标记,分别与之相配对的另一种单克隆抗体被喷在硝酸纤维素膜（NC膜）上,制成免疫层析检测试条。溶液中的抗原NSE、CEA与金标记抗体结合后沿着硝酸纤维素膜移动,与膜上固定的抗体结合形成肉眼可见的红色线条。结果：该试纸条只与NSE、CEA有特异性反应,与CA125、CYFRA21-1、TPA等肺癌标志物无交叉反应。标准样品中两种抗原的检测灵敏度分别可达到5ng/mL和3ng/mL。结论：胶体金免疫层析技术检测NSE、CEA特异性强、灵敏度高、简便快速,不需特殊仪器设备,有广泛应用价值。     

甲胎蛋白和癌胚抗原的胶体金免疫层析同步检测

目的:探讨建立一种同时快速检测甲胎蛋白(AFP)和癌胚抗原(CEA)两种肿瘤标志物的方法.方法:用胶体金分别标记鼠抗AFP单克隆抗体和鼠抗CEA单克隆抗体,喷于胶金垫上.与之配对的抗体喷在硝酸纤维素膜(NC膜)上分别做检测线T2和T1,兔抗鼠二抗喷在NC膜上做质控线,利用免疫层析技术进行检测.结果:采用Frens方法制备的胶体金粒径在20nm,紫外可见吸收峰在521 nm处,金标抗体的最适pH在9.0,最适抗体量为200μl,胶体金标记2.4μg抗体.测试结果表明同时检测AFP和CEA的试纸条灵敏度分别为20 ng.mL-1和5 ng.mL-1,检测时间仅需10min,与PSA、CA125、CA15-3、Ferritin、Human Albumin等没有交叉反应,稳定性好.结论:应用胶体金免疫层析技术,实现了AFP和CEA的同步检测.     

甲胎蛋白胶体金检测试剂的的研制

为建立一种快速,简易胶体金免疫层析法（GICA）用于检测血清中的甲胎蛋白（AFP）,将AFP单克隆抗体分别标记胶体金并包被硝酸纤维素膜,制成免疫检测层析试剂,血清中的AFP与金标记抗体结合,沿着硝酸纤维素膜移动,与膜上的固相抗体结合可形成肉眼可见的紫红色线条,此检测试剂对本室自制AFP参比品进行了检测,灵敏度达20ng/ml;对甘肃省肿瘤医院的176份癌症患者血清进行了检测。结果与ELISA法相一致,本法检测AFP快速,简便,结果准确,具有推广价值。     

胶体金渗滤方法检测乙肝患者血清中乙肝核心抗体的研究

目的:建立胶体金免疫渗滤法检测乙肝患者血清中乙肝核心抗体(anti-HBc Ig M)抗体的方法,评价其灵敏度、特异性和重复性。方法:预先在硝酸纤维素膜上包被乙肝核心抗原HBc Ag,将血清或者血浆加在硝酸纤维素膜上,血清或者血浆中的anti-HBc Ig M与HBc Ag结合。洗涤去掉非特异结合,加入胶体金标记的抗人-Ig M,洗涤去掉没有结合的抗人Ig M抗体。硝酸纤维素膜上的抗原-Ig M-抗人Ig M形成的"三明治"复合物呈现红色圆点,证实实验有效。结果:102份乙肝患者血清,anti-HBc Ig M阳性标本99份。检出率97%。50份正常血清中,有一份检测结果弱阳性,特异性98%。结论:胶体金免疫渗滤法检测anti-HBc Ig M敏感性高、特异性强、重复性好,且方便快捷,不需要特殊的仪器设备,在anti-HBc Ig M快速检测上有广阔的应用前景。     

一种快速检测甲型流感病毒的方法

目的 开发一种快速、简便的基于胶体金免疫层析法（GICA）的试剂盒,以用于对甲型流感病毒的检测。方法以柠檬酸三钠还原法制备胶体金颗粒,标记抗甲型流感病毒内部抗原的单克隆抗体。硝酸纤维素膜上包被两种抗甲型流感病毒单克隆抗体的混合液,制成免疫层析试纸。待测样品中的甲型流感病毒首先与胶体金标记抗体结合,后移动至硝酸纤维素上与固定的单克隆抗体发生反应,形成肉眼可见的红色带。结果GICA试纸条与甲1型和甲3型流感病毒共16种毒株均能发生特异性反应,与乙型流感病毒、副流感病毒、腺病毒和呼吸道合胞病毒无交叉反应。用三种不同甲型流感病毒毒株的不同浓度标本与美国同类经过FDA批准的产品比较,灵敏度相同。结论GICA试纸条灵敏度能够达到临床使用的要求,并具有简便快速、无需特殊仪器设备等优点,对甲型流感的诊断和流行病学调查具有十分重要的应用价值。     

青霉素结合蛋白2a单克隆抗体的研制和初步应用

目的研制青霉素结合蛋白2a(PBP2a)单克隆抗体(Mc Ab),为建立耐甲氧西林金黄色葡萄球菌(MRSA)免疫层析检测方法提供检测用抗体。方法以基因工程抗原r PBP2a免疫BALb/c小鼠,通过常规小鼠B淋巴细胞杂交瘤技术制备单克隆抗体,采用免疫印迹技术(Western blotting)分析单克隆抗体特异性。选取敏感、特异的单克隆抗体进行金标记和硝酸纤维膜包被,建立胶体金免疫层析检测方法。结果共获得11株分泌抗r PBP2a的杂交瘤细胞,其中6株分泌的单克隆抗体能够与天然PBP2a呈阳性反应。用其中2株单克隆抗体建立的PBP2a胶体金免疫层析方法,可在5~20 min内完成检测。结论获得了特异性针对PBP2a蛋白的单克隆抗体,并初步建立了检测PBP2a蛋白的胶体金免疫层析检测方法,为临床快速、简便检测产生PBP2a的细菌提供了检测方法。     

胶体金免疫层析法检测猪链球菌2型的研究

目的:制备胶体金免疫层析试纸检测猪链球菌2型.方法:用柠檬酸盐还原法制备胶体金颗粒,标记猪链球菌2型多克隆抗体,通过免疫层析作用对猪链球菌2型进行检测,并对试纸条的敏感性、特异性、稳定性进行评价.结果:每毫升胶体金最佳抗体标记量为22μg/mL,最佳包被抗体浓度为2 mg/mL,最佳BSA封闭浓度为1.5%,建立的胶体金免疫层析试纸条栓出猪链球菌2型的下限为106 CFU/mL,从检测到结果判断时间为5～15 min,与其他常见致病菌及链球菌属中15个群无交叉反应.结论:获得了检测猪链球菌2型的胶体金免疫层析试纸,该法操作简便,灵敏度高,特异性强,可用于猪链球菌的快速初筛和检测.     

纳米磁性颗粒标记的免疫层析法定量检测人乙型肝炎表面抗原

目的:应用纳米磁性颗粒标记的免疫层析法,研制可应用于乙肝表面抗原(HBsAg)快速定量检测的层析试纸条。方法:用1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)/N-羟基琥珀酰亚胺(NHS)交联的方法标记纳米磁珠,喷膜仪喷点硝酸纤维膜;根据双抗体夹心法原理建立免疫层析试纸条,对HBsAg特异性抗体捕获的磁信号进行检测,并对磁信号检测结果进行统计学分析和评价。结果:建立了HBsAg纳米磁性免疫层析试纸条,最低限度检测为0.1 ng/mL的HBsAg抗原,检测灵敏度达到了同类产品ELISA分析法的标准,且检测时间控制在5 min内;经检测临床血清标本证实,该方法可根据磁信号定量检测乙肝患者血清中HBsAg的浓度。结论:HBsAg纳米磁性免疫层析方法具有简单快速、灵敏度高的特点,可应用于临床血清样本中HBsAg的检测;该方法为体内极微量抗原抗体的快速检测建立了新模式。     

基于上转磷光颗粒的AFP免疫层析定量检测

目的：建立了上转磷光免疫层析快速定量检测AFP的方法,用于原发性肝癌的诊断。方法：利用上转磷光标记的双抗夹心免疫层析技术检测血清中AFP的含量。将AFP单克隆抗体分别标记上转磷光颗粒并包被硝酸纤维膜,制成免疫层析检测试纸。评价该试纸的灵敏度、特异性和精密度。并通过临床50例血清样本,探讨上转磷光技术与化学发光检测方法的一致性。结果：该方法能在20min内完成,检测灵敏度达5ng/ml。浓度范围从5-1000ng/ml作标准曲线,呈现较好的线性。通过三组平行实验探讨其精密度,变异系数都在10%以内。应用于其它肿瘤标志物如CEA、CA199、AFU和NSE的检测,无交叉反应。与化学发光相比,决定系数R2=0.9692,一致性较好。结论：该方法简便、快速、廉价,可以实现定量,尤其适合基层门诊和体检现场使用。     

心肌肌钙蛋白I和糖原磷酸化酶同工酶BB金标记免疫层析联合检测法的建立

目的：建立人心肌肌钙蛋白I（cTnI）及糖原磷酸化酶同工酶BB（GPBB）的胶体金免疫层析联合检测法。方法：以纯化的人心肌cTnI和GPBB为免疫原免疫小鼠,制备抗cTnI和抗GPBB单克隆抗体,并用胶体金标记cTnI和GPBB抗体,采用免疫层析技术建立快速准确检测cTnI和GPBB的胶体金免疫层析法。结果：建立的检测方法灵敏度高,可检出血液样品中1ng／mL的cTnI和7ng／mL的GPBB;特异性强,与心肌肌钙蛋白T、心肌肌钙蛋白C、肌酸激酶同工酶均无交叉反应。结论：该方法特异性强,灵敏度高,快速、简便,弥补了传统心肌梗死诊断方法的不足,对急性心肌梗死的早期筛查有重要意义,具有较高的临床应用价值和广泛的应用前景。     

Asia1型口蹄疫病毒胶体金免疫层析检测方法的建立

为建立一种快速、简便、灵敏检测Asia1型口蹄疫病毒的胶体金免疫层析方法(GICA)。本研究采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗Asia1型口蹄疫病毒的单克隆抗体,将该标记物与羊抗豚鼠IgG分别包被在硝酸纤维素膜(Nitrocellulose membrane)上,作为检测带和质控带。经条件优化,组装成检测Asia1型口蹄疫的诊断试纸条。用该试纸条分别对A、O、C和Asia1型口蹄疫病毒抗原以及猪水泡病病毒抗原等87份样品进行了检测,发现该试纸条不与口蹄疫病毒A、O、C型以及猪水泡病病毒抗原发生反应,特异性良好。用该试纸条对口蹄疫细胞毒(TCID50为6.25)的10倍系列稀释液进行了检测,最低可以检测到大约10?4。该试纸条与其他传统诊断方法的符合率为98.8%。初步实验确定该试纸条在4oC下可保存3个月、37oC和室温下大概可保存1周左右。该试纸条是一种快速、灵敏、特异的FMD抗原检测方法,对现场检测具有一定实用价值。     

Rapid detection of glycyrrhizin by immunochromatographic assay

An immunochromatographic assay was developed for detecting glycyrrhizin (1). The qualitative assay is based on a competitive immunoassay using anti-1 monoclonal antibody (MAb) and a detector reagent that contains colloidal gold particles coated with anti-1 MAb. The immunochromatographic strip test, which has a detection limit of 250 ng/mL, is useful as a rapid screening method for detecting glycyrrhizin in plants, biological fluids and food samples.     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

Novel electrochemical catalysis as signal amplified strategy for label-free detection of neuron-specific enolase

A label-free electrochemical immunoassay for neuron-specific enolase (NSE), a kind of lung cancer marker, was developed in this work via novel electrochemical catalysis for signal amplification. The new amplified strategy was based on the electrochemical catalysis of nickel hexacyanoferrates nanoparticles (NiHCFNPs) in the presence of dopamine (DA). NiHCFNPs, which were assembled on the porous gold nanocrystals (AuNCs) modified glassy carbon electrode (GCE), could exhibit a distinct pair of redox peaks corresponding to anodic and cathodic reactions of hexacyanoferrate (II/III). Subsequently, gold nanoparticles functionalized graphene nanosheets (Au-Gra) were coated on the surface of NiHCFNPs/AuNCs film. Then an enhanced amount of neuron-specific enolase antibody (anti-NSE) could be loaded to obtain a sensitive immunosensor of anti-NSE/Au-Gra/NiHCFNPs/AuNCs/GCE due to the strong adsorption capacity and large specific surface area of Au-Gra. More importantly, the oxidation peak current can be enormously enhanced towards the electrocatalytic oxidation of DA based on NiHCFNPs, resulting in the further improvement of the immunosensor sensitivity. Under optimal conditions, the electrochemical immunosensor exhibited a linear range of 0.001-100 ng/mL with a detection limit of 0.3 pg/mL (S/N=3). Thus, the proposed immunosensor provides a rapid, simple, and sensitive immunoassay protocol for NSE detection, which may hold a promise for clinical diagnosis.     

A novel electrochemical immunoassay based on diazotization-coupled functionalized bioconjugates as trace labels for ultrasensitive detection of carcinoembryonic antigen

In this article, a novel sandwich-type electrochemical immunosensor based on the signal amplification strategy of diazotization-coupling concept for ultrasensitive detection of carcinoembryonic antigen (CEA) was reported. It operates through physisorption of monoclonal anti-CEA on 4-aminothiophenol (4Atp) functionalized gold electrode interface as the detection platform. Diazo-4Atp-coupled-thionine (Thi)-conjugated gold nanoparticles (GNPs) were prepared for immobilization of horseradish peroxidase (HRP) and secondary anti-CEA to form core-shell bioconjugates that were used as electrochemical signal amplification reagent. The sensitivity of the immunosensor was greatly amplified by a dual amplification: one is that a large number of thionine and HRP was introduced on the electrode surface through sandwich immunoreaction, the other is that HRP as enhancer could catalyze the oxidation reaction of thionine by H(2)O(2), which results in great enhancement of the reduction peak current. Thus, the bioconjugates-based assay provided an amplification approach for detecting CEA at trace levels and led to a detection limit as low as 0.7 pg/mL (at a three times signal-to-noise ratio) that is well-below the threshold value of 2.5 ng/mL for clinical diagnosis. The assay was evaluated for clinical serum samples with various CEA concentrations and received in excellent accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA).     

Immunochromatographic assay for the detection of pseudojujubogenin glycosides

INTRODUCTION: Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. OBJECTIVE: The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. METHODOLOGY: The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. RESULTS: The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. CONCLUSION: The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.     

金标银染免疫渗滤法检测土拉弗朗西斯菌

目的:建立金标银染免疫渗滤法检测土拉弗朗西斯菌(土拉菌)的方法,评价其灵敏度、特异性、重复性及其应用。方法:以小鼠抗土拉菌脂多糖单克隆抗体作为捕获抗体包被硝酸纤维素膜、兔抗土拉菌多克隆抗体作为检测抗体标记胶体金,通过金标银染技术放大检测信号,建立金标银染免疫渗滤法检测土拉弗朗西斯菌体系;评价该方法的灵敏度、特异性和重复性;以经荧光定量PCR定量的土拉弗朗西斯菌为检测对象,比较金标银染免疫渗滤法和免疫层析法。结果:金标银染免疫渗滤法检测土拉弗朗西斯菌的最小检出量为1.0×103 CFU/mL,灵敏度高于免疫层析法;检测大肠杆菌、炭疽芽孢杆菌、布鲁菌和鼠疫耶尔森菌的结果均为阴性;密封保存的检测卡80 d内重复性良好,100 d后反应强度略有降低。结论:金标银染免疫渗滤法检测土拉弗朗西斯菌敏感性高、特异性强、重复性好,且方便快捷,不需要仪器设备,可作为快速检测土拉弗朗西斯菌的首选方法。     

Development of an immunochromatographic strip test for the rapid detection of okadaic acid in shellfish sample

A rapid detection technology for okadaic acid (OA) in shellfish with one-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe was developed. OA is one of the diarrhetic shellfish toxins. Firstly, OA was conjugated to bovine serum albumin, and the conjugations as immunogen were injected into mice to raise the polyclonal antibody against OA. Hybridoma cells fused between spleen cells from immunized mouse and myeloma cells (Sp2/0) were prepared and injected into mice intraperitoneally at 1?×?10⁶?cells to produce monoclonal antibody in the ascitic fluid. With the monoclonal antibody against OA, the idc-ELISA assay was established to detect OA. The calibration curve for OA was linear over the concentration range of 0.31–50 ng mL^?1, and the detection limit for OA was 0.45 ng mL^?1. On that basis, paper test strips for detecting OA were prepared, and a fast detection method for okadaic acid using gold-labeled immunological assay was established. With the paper test strips, the detection limit was 6.25 ng mL^?1, and whole detection process for OA in shellfish samples needed only about 40 min.