基于碳纳米管的脂质体电化学发光免疫传感器检测人免疫球蛋白G

将人免疫球蛋白G(hIgG)抗体固定在多壁碳纳米管修饰的玻碳电极表面,制备了一种电化学发光(ECL)免疫传感器.以hIgG抗体标记的联吡啶钌脂质体为标记物,采用三明治型检测方式,成功建立了hIgG的ECL免疫检测技术.电化学发光强度与hIgG的浓度在0.01～0.8 μg/L范围内呈良好的线性关系; 线性回归方程为y=618.7x(μg/L)-23.9(n=6,r=0.995); 检出限为0.004 μg/L.用于人血清中hIgG的检测,结果令人满意.     

癌胚抗原毛细管电泳-化学发光均相免疫分析

设计了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测均相免疫分析新方法。采用四苯硼酸钠增强luminol-H2O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物。测定癌胚抗原的线性范围2.0~80.0μg/L(R=0.9921),检出限为0.1μg/L(绝对检出限为0.75 fg)。     

基于电化学聚合固定抗体的电化学发光免疫分析法检测人免疫球蛋白

基于电化学聚合在金电极表面固定兔抗人免疫球蛋白G抗体与人免疫球蛋白G及标记有Ru(bpy)2+3 的羊抗人免疫球蛋白G抗体之间发生特异性免疫反应,形成三明治结构,成功建立了用于测定人血清中免疫球蛋白G的电化学发光(ECL)免疫技术.利用此方法测定人免疫球蛋白G含量,浓度在50 μg/L～2 mg/L范围内与电化学发光强度呈良好的线性关系,线性回归方程为y(a. u.)=48.41+0.09x(μg/L) (n=7);检出限为20 μg/L (3σ).测得正常人血清中免疫球蛋白G平均含量为11.2 g/L ,结果令人满意.     

对克伦特罗具有高特异性的酶联免疫吸附分析法研究

合成了克伦特罗(CL)半抗原2-(1-(4-氨基-3,5-二氯苯基)-2-(叔丁胺基)乙氧基)乙酸,采用活性酯法将半抗原与牛血清白蛋白偶联合成免疫原,经免疫、分离、纯化获得抗CL多克隆抗体,建立了对CL具高特异性的直接竞争酶联免疫吸附分析(dc-ELISA)法。本方法对CL检测的线性浓度范围为1.0×10!4～1.0 mg/L,定量检测下限为0.13μg/L。在尿样中添加CL标准至含量分别为10.0和1.0μg/L,dc-ELISA法检测的回收率分别为90.0%～115.9%和80.5%～112.4%;相对标准偏差(RSD)分别为7.1%和12.6%(n=10)。特异性实验结果表明:抗体与CL结构类似物沙丁胺醇(SAL)的交叉反应率<0.3%,因此本研究所制备的抗体可有效避免现有克伦特罗ELISA试剂盒、胶体金检测试纸假阳性率高的问题。     

直接竞争酶联免疫吸附法测定饲料中沙丁胺醇

应用本实验室制备的对沙丁胺醇(SAL)具有高亲和力的多克隆抗体和酶标半抗原,采用包被抗体-酶标半抗原直接竞争模式建立沙丁胺醇的酶联免疫吸附分析(ELISA)法。在优化实验条件下,ELISA法检测沙丁胺醇的线性浓度范围为0.1~1.0×10~3μg/L,沙丁胺醇对抗体-酶标半抗原结合反应的抑制中浓度(Ic50)为14μg/L,相对标准偏差(RSD)为8.6%(n=5),定量检测下限(Ic_(10))为0.29μg/L。在猪饲料中分别添加沙丁胺醇标样10、50、250μg/kg,ELISA法检测的回收率分别为85%~108%、81%~92%和81%~102%,RSD(n=5)分别为9.1%、5.6%和8.9%,对饲料中沙丁胺醇的最低定量检测浓度为4.23μg/kg。利用高效液相色谱-紫外检测(HPLC-UV)法同步测定饲料中添加250μg/kg沙丁胺醇的平均回收率为89%(n=3),相对标准偏差为3.9%。     

微悬臂梁适配子传感器检测维埃克斯、沙林及动力学分析

采用生物素-亲和素法在微悬臂梁传感芯片上固定维埃克斯、沙林适配子,建立了压阻式微悬臂梁适配子传感器检测维埃克斯、沙林及动力学分析方法。传感器对维埃克斯检测的线性范围为2~60μg/L,线性回归方程为ΔUe=0.886C-1.039(n=5,R=0.984,p<0.001),检出限为2μg/L(S/N≥3);对沙林检测的线性范围为10~60μg/L,线性回归方程为ΔUe=0.716C-2.304(n=5,R=0.996,p<0.001),检出限为10μg/L(S/N≥3)。传感器具有良好的选择性和抗干扰能力,对毒剂类似物O-丁基甲基膦酰氯基本无响应。在此基础上,根据受体-配体结合特性与压阻式微悬臂梁传感器输出电压变化之间的关系,建立了传感器检测维埃克斯、沙林的反应动力学模型,根据拟合方程求出的传感器对不同浓度维埃克斯、沙林反应达到平衡时的响应电压(ΔUe)、响应时间(t0)均与实测值非常接近。     

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。     

基于磁性纳米颗粒的日本血吸虫抗体荧光免疫分析

研究了一种基于磁性纳米颗粒的日本血吸虫荧光免疫分析方法。日本血吸虫抗原通过共价吸附到核壳结构的磁性颗粒表面,与待测抗体结合后,再与酶标二抗夹心反应,最后以3,3′,5,5′-四甲基联苯胺(TMB)为底物,通过测定酶催化下TMB氧化生成无荧光的二聚体化合物,使溶液荧光强度降低来间接测定日本血吸虫抗体的浓度。应用本方法测定了兔血清中日本血吸虫抗体,荧光强度(If)与抗体浓度(C)在5.0~100μg/L之间呈线性关系,线性回归方程为If=225.8-1.6C(r=0.9976),检出限达1.5μg/L。方法简单实用,具有良好的检测灵敏度和重现性。     

基于金磁微粒与酶标噬菌体抗体的磁分离免疫分析法

基于金磁微粒(Gold-magnetic particle)兼有纳米金颗粒与磁微粒特性的优势,以相思子毒素(Abrin)为目标物,将蛋白A(SPA)包被金磁微粒偶联多抗作为功能化捕获探针,酶标噬菌体抗体作为特异信号检测探针,建立了一种检测相思子毒素的磁分离免疫分析法。该方法的线性范围为0.008~250μg/L,相关系数(r)为0.991 0,检出限为0.008μg/L,定量下限为0.008μg/L。该方法将蛋白A-金磁微粒功能化探针与酶标噬菌体抗体探针的优势结合,提高了检测灵敏度、特异性和抗干扰能力,适用于各种环境样品中微量相思子毒素样品的分析。     

癌胚抗原毛细管电泳-化学发光均相免疫分析

建立了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测的均相免疫分析新方法.采用四苯硼钠增强luminol-H2 O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物.测定癌胚抗原的线性范围2.0～80.0 μg/L(R=0.9921),检出限为0.1 μg/L(绝对检出限为0.75 fg).     

呋喃唑酮代谢物单克隆抗体制备及酶联免疫吸附分析方法

本研究针对呋喃唑酮代谢物(AOZ),设计合成了系列半抗原,进一步通过偶联牛血清白蛋白(BSA)免疫Balb/c小鼠、细胞融合、筛选和亚克隆等过程成功获得了源于新颖半抗原H3的具有高亲和力(亲和力常数6.68× 1010L/mol)和高特异性(与其它功能类似物交叉反应小于0.1％)抗AOZ单克隆抗体.同时,基于设计合成的系列同/异源半抗原/包被抗原,考察了不同结构包被原对ELISA方法灵敏度的影响.另外,采用最佳的特征结构异源包被原H5 -OVA,建立了以对硝基苯甲醛(p-NP)为衍生剂的AOZ间接竞争ELISA(icELISA)和直接竞争ELISA(deELISA)检测方法.结果表明:icELISA模式的AOZ检测IC50为0.503 μg/L,定量检测线性范围(IC20～IG80)为0.06～14.0 μg/L,检出限(IC10)达0.017 μg/L; dcELISA模式的AOZ检测IC50为1.19 μg/L,定量检测线性范围为0.14～23.6 μg/L,检出限为0.056 μg/L.两种方法对AOZ的检测灵敏度和定量线性范围均达到相关检测限量要求,可满足不同需求的实际样品检测.     

基于纳米金固定半抗原的电化学免疫传感器灵敏检测克伦特罗

研制了一种基于纳米金固定半抗原的间接竞争电化学免疫传感器,可灵敏检测克伦特罗.在金电极表面组装1,6-己二硫醇单分子膜,通过Au-S共价作用连接纳米金颗粒,通过吸附作用固定克伦特罗牛血清白蛋白偶联物.样品中的待测组分与固定化的克伦特罗偶联物竞争结合单克隆抗体,碱性磷酸酯酶标记的二抗选择性地与电极表面捕获的一抗反应,进而催化底物1-萘酚磷酸酯水解生成1-萘酚,在电极表面氧化产生电信号.在优化的实验条件下,克伦特罗浓度在0.1～1000 μg/L范围内与电流强度线性相关,线性方程为I(A)-8.79× 10-7-2.66× 10-7logC (μg/L),相关系数0.9960,检出限达20 ng/L.同时测定了猪肉及猪肝样品中克伦特罗含量,相对标准偏差平均值为7.0％,加标回收率在89.1％～105.6％之间,与传统的间接竞争酶联免疫吸附法对照,结果无显著性差异.     

Production of a monoclonal antibody and development of enzyme-linked immunosorbent assay for alkyl ethoxylates

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed for the determination of alkyl ethoxylates (AEs) that are the most widely used nonionic surfactants in the world. Three types of hapten, hemi-succinated AEs (C12EO7suc, C16EO23suc, and C18EO10suc), were synthesized and conjugated to bovine serum albumin (BSA) for mouse immunization. The mice immunized with the C12EO7suc-BSA that showed the high immune responses were used for cell fusion. The obtained monoclonal antibody (TFG2-76) was specific to AEs, which had alkyl (C) and ethoxy (EO) chain lengths of C10-12 and EO5-15, respectively. Two types of solid support, namely, a polystyrene tube and a 96-well microplate, were used for antibody immobilization. The working ranges of the tube-type and plate-type ELISAs were 2-100 and 20-1000 μg/L with IC₅₀ values of 12 and 71 μg/L AE (C12EO7), respectively. Moreover, the lowest quantification limit of plate-type ELISA could be lowered to 5 μg/L by decreasing the coated antibody concentration. Cross-reactivities with non-AE surfactants were determined, and the assay proved highly selective for AEs. The application of plate-type ELISA to determine spiked AEs in distilled water, tap water and river water provided good recoveries without matrix effects.     

Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively.The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.     

Enzyme-linked immunosorbent assay for okadaic acid: investigation of analytical conditions and sample matrix on assay performance

Okadaic acid (OA) is a lipophilic marine biotoxin. In this study, OA was coupled with the carrier proteins keyhole limpet hemocyanin and bovine serum albumin as immunity and detection antigens by an active ester method. The polyclonal antibody against OA was prepared successfully, an indirect competitive ELISA (ciELISA) developed for the detection of OA in shellfish, and the reactive conditions of ciELISA optimized. The LOD (15% inhibition concentration) for the microwell plates was 1.28 +/- 0.38 ng/mL, corresponding to 12.8 +/- 3.8 ng/g. Two extraction methods were used to remove shellfish matrix interference with high recovery of spiked samples, and the methanol extraction of shellfish mussel was analyzed after dilution in phosphate-buffered saline. For validation of the optimized ciELISA, spiked and natural samples were analyzed by ciELISA, and HPLC with fluorescence detection. The correlation of linear regression equation was y = 1.0064x - 10.234, and the correlation coefficient was 0.9347. From the results of the comparative study, the established ciELISA assay using polyclonal antibody against OA could be used in preliminary screening of suspicious shellfish samples.     

Development of an indirect competitive ELISA for the determination of papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K_aff) of 7.3 × 10⁷ L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2 μg/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000 ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06 ng/mL, respectively.     

Synthesis and application of a molecularly imprinted polymer in the solid-phase extraction of ketoprofen from wastewater

Ketoprofen is a nonsteroidal anti-inflammatory drug widely consumed by humans as it possesses analgesic activities. A selective molecularly imprinted polymer (MIP) for ketoprofen was synthesized and applied as a solid-phase extraction sorbent. MIP was synthesized using 2-vinylpyridine, ethylene glycol dimethacrylate, 1,1′-azobis(cyclohexanecarbonitrile), toluene/acetonitrile (9:1, v/v), and ketoprofen as a functional monomer, cross-linker, initiator, porogenic mixture, and template, respectively. The polymerization was performed at 60 °C for 16 h, and thereafter the temperature was increased to 80 °C for 24 h to achieve a solid monolith polymer. Nonimprinted polymer was synthesized in a similar manner with the omission of ketoprofen. Characterization with thermogravimetric analysis and X-ray diffraction showed that the synthesized polymers were thermally stable and amorphous. Solid-phase extraction cartridges packed with MIP were used with high-performance liquid chromatography for quantitative analysis of ketoprofen in wastewater. The analytical method gave detection limits of 0.23, 0.17, and 0.09 μg/L in wastewater influent, effluent, and deionized water, respectively. The recovery for the wastewater influent and effluent spiked with 5 μg/L of ketoprofen was 68%, whereas 114% was obtained for deionized water. The concentrations of ketoprofen in the influent and effluent samples were in the ranges of 22.5–34.0 and 1.14–5.33 μg/L, respectively. Overall, the analytical method for the analysis of ketoprofen in wastewater was rapid, affordable, accurate, precise, sensitive, and selective.     

酸性红73多克隆抗体的制备及其应用

在酸性红73分子的羟基上引入一个带有羧基的“间隔臂”,采用N-羟基琥珀亚胺活性酯法将酸性红73分别与牛血清白蛋白(BSA)、卵清蛋白(OA)偶联,合成免疫原和包被原,经免疫新西兰白兔获得多克隆抗体,所得抗体最大效价可达2.56×105,建立了酸性红73的间接竞争ELISA检测方法.本方法的半数抑制浓度(IC50)为181.2 μg/L,检出限(LOD)为7.9 μg/L.交叉反应实验表明,除苏丹红3号(1.13％)外,抗AR73抗体与其它竞争物均无交叉反应.在虾仁中的空白添加回收率为63.5％～90.7％,RSD＜6.8％.说明本方法可用于虾仁中酸性红73的残留检测.     

细交链孢菌酮酸酶联免疫吸附分析方法研究

采用水合肼和乙醛酸依次对细交链孢菌酮酸(Tenuazonic acid,TeA)进行衍生化,设计合成了含有氮杂共轭双键偶联手臂,可增强免疫效果的半抗原TeAHGA.通过偶联载体蛋白BSA后的免疫原TeAHGABSA免疫新西兰大白兔,成功制备了特异性识别TeA水合肼衍生物TeAH的多克隆抗体;优化确立了ELISA最佳反应条件(TeAH-OVA为异源包被原、包被浓度0.156 μg/L、药物稀释及反应缓冲液为PBS、一抗反应时间40 min、二抗反应时间20 min),建立了TeA间接竞争ELISA(icELISA)检测方法,其抑制中浓度(IC50)为1.61 μg/L,检出限(LOD)为0.08 μg/L,定量线性检测范围为0.19～12.89 μg/L (IC20～IC80).番茄、面粉样品平均添加回收率分别为67.2％～89.8％和74.8％～93.7％.     

Determination of β-adrenergic agonists by hapten microarray

The use of highly active β-agonists as growth promoters is not appropriate because of the potential hazard for human and animal health. To investigate the residue level of these β-agonists, hapten microarrays were employed for clenbuterol (CLB), ractopamine (RAC) and salbutamol (SAL) residue analysis. CLB, RAC and SAL conjugates were immobilized on the slides, which were precoated by agarose film to construct hapten microarrays, and then the corresponding monoclonal antibodies of these β-agonists and the standards or samples were introduced for indirect competitive immunoassay. Finally, Cy3-labeled secondary antibody was employed to indicate the antigen-antibody complex. The fluorescence intensity of each spot was imaged and recorded, and the calibration curve of each analyte was obtained by plot fluorescence intensity against different standard concentrations. Compared to the ELISA, the hapten microarray method was more sensitive, which got the detection limits 0.09 μg/L for CLB, 0.50 μg/L for RAC, and 0.01 μg/L for SAL. What's more, with the recovery rate between 96.5% and 106.4%, and the coefficient of variation below 10%, the proposed hapten microarray method was shown to be both quantitative and reproducible.