基于荧光体系的酶联免疫吸附分析法的研究进展

酶联免疫吸附分析（Enzyme linked immunosorbent assay, ELISA）作为一种重要的免疫分析方法,具有高通量、信号读取方便、操作简单和成本低廉等优点,广泛应用于环境监测、食品安全检测以及医疗诊断等领域。然而,由于受比色显色原理的限制,传统ELISA的检测灵敏度难以提高。为了解决此问题,研究者开发了多种类型的新型检测体系。其中,荧光方法由于具有灵敏度高、操作简单以及响应快速等优势,引起了广泛关注。目前,多种新型荧光材料已被广泛用于构建ELISA,促进了ELISA在分析化学和生物医疗检测中的应用。本文详细介绍了有机小分子、硅/碳纳米粒子、金属纳米簇和量子点等荧光材料构建的ELISA,根据使用标记酶的不同,介绍了以碱性磷酸酶、辣根过氧化物酶等酶作为标记酶的ELISA,评述了近五年来荧光材料在构建ELISA方面的研究进展,并对其应用前景与发展趋势进行了展望。     

新版《高分子物理》述评

分析了何曼君教授等主编的第三版高分子物理与修订版比较在讲授内容上的增加和删减,某些概念上的变化,章节编排上的构成和变化,就教学和试用情况进行了分析,感觉其很好地跟踪了高分子物理的时代发展步伐,而且结构合理,是一部很好的教材。并就这本教材中分子量及其测定方法、高分子物理中近期的理论研究进展、聚合物的弹性效应、聚合物仪器分析和研究方法以及聚合物的凝聚态结构等有关内容和章节结构提出了自己的一些意见和建议。     

稻瘟灵单克隆抗体的制备及评价

该文制备了农药稻瘟灵的单克隆抗体,并建立了稻瘟灵的酶联免疫吸附（ELISA）检测方法。在完全保留稻瘟灵结构的基础上从二硫杂环戊烷结构中衍生不同长度的活性手臂制备了2个半抗原,并分别与载体蛋白偶联合成免疫原与包被原。通过小鼠免疫、细胞融合、淘筛、腹水制备等步骤获得特异性识别稻瘟灵的单克隆抗体mAb－DWL。结果显示,基于mAb－DWL构建的间接竞争ELISA法的半抑制浓度（IC50）为55.2 ng/mL,线性范围为4.6 ~ 530.2 ng/mL,其与结构类似物的交叉反应可忽略不计。所建立的ELISA方法对蔬菜及粮食等样品的加标回收率为77.2% ~ 116%,可用于实际样品的快速检测。     

酶联免疫吸附法和液相色谱-质谱联用法分析海洋生物中记忆缺失性贝毒

采用酶联免疫吸附法(ELISA)和液相色谱-质谱联用法(LC-MS)分析检测我国大连周边海域生物样品中的记忆缺失性贝毒(ASP)。实验结果表明,4个浮游生物样品和2个贝类样品中检出ASP,两种方法检测结果基本一致,相关系数为0.996。ELISA方法适合初步筛选阳性样品;LC-MS方法适合深入研究样品中的ASP结构信息。使用商业ELISA试剂盒以及LC-MS两种方法,证明在某些海域的浮游生物样品和贝类样品中含有ASP,报道了在浮游生物中存在记忆缺失性贝毒主要成分软骨藻酸。     

电化学石英晶体微天平的应用

电化学石英晶体微天平（EQCM）即石英晶体微天平（QCM）与电化学检测相结合的测试技术。电化学石英晶体微天平以其简单、快速,可以在纳克级水平上对活性物质在石英晶振片上发生的沉积、吸附或溶解等过程进行动态检测等优势而成为表界面反应研究的有效手段之一。由于EQCM测试技术为原位测试方法,可以实现在线实时监测,利用其高精度和高灵敏度可以进一步对表界面上发生反应的过程及深层次的机理进行分析。本文就EQCM在电化学、生物医学及油田化学等领域以及研究机理及动力学等方面的应用进行了总结阐述,提出了EQCM的研究新方向以及发展中面临的问题。     

水溶性硼酸亲和富集材料的制备及基于酶联免疫吸附法检测人肝微粒体糖蛋白的应用

生物样品中的糖蛋白丰度低,且在检测中易受到其它非糖蛋白的抑制和干扰,需在分析检测前对糖蛋白进行富集,但常规的基于固相材料的糖蛋白富集方法不易与生物技术中最经典的酶联免疫吸附法(ELISA)兼容.本研究以树枝状聚合物PAMAM 4.0为载体, 结合硼酸亲和技术,制备了新型水溶性硼酸亲和富集材料(DBC),并将其应用于基于ELISA的人肝微粒体中糖蛋白的检测.采用标准糖蛋白对DBC富集条件进行优化,然后考察其灵敏度和抗干扰能力,将优化后的方法应用于复杂样品人肝微粒体糖蛋白富集.结果表明,DBC对糖蛋白的富集选择性可高达100000倍,可将糖蛋白的富集信号提高100倍.以DBC为富集材料,与ELISA分析技术相结合,只需一步简单的孵育,即可实现生物样品中糖蛋白的高灵敏度、高选择性检测,为疾病相关的糖蛋白组学研究提供了一种有效的检测手段.     

稀土掺杂氟化物纳米材料的上转换发光特征及其生物应用

稀土掺杂氟化物纳米材料由于具有低的声子能,可以获得较高的上转换发光效率,使其在太阳能电池、生物医学、光电子学、信息等领域有着广泛而重要的应用前景。本文就当前稀土氟化物上转换纳米材料的改性、光性能研究,以及在生物检测,生物成像标记,免疫分析,疾病治疗方面的最新研究进展做一综述,并对上转换纳米颗粒在生物应用过程中存在的主要问题进行了讨论。     

POSS/聚氨酯复合材料结构与性能的研究进展

多面体低聚倍半硅氧烷(POSS)是新兴的有机/无机杂化材料,其特殊的笼状结构可以从纳米尺度上影响聚氨酯体系的结晶行为、微相分离程度、交联密度等结构特征。少量添加即可大幅提高聚氨酯的耐水性、热稳定性、力学性能,以及阻燃性能、黏结性能、韧性等。本文从POSS/聚氨酯复合材料的结构出发,介绍了化学合成POSS基聚氨酯及物理共混POSS/聚氨酯复合材料,总结分析了悬垂型、星型、串珠型以及物理混合POSS基聚氨酯复合材料的结构特点,以及POSS结构差异对其复合材料性能的影响与影响机理。     

氨基甲酸酯类杀虫剂速灭威酶联免疫吸附分析方法研究

利用间-甲酚和β-丙氨酸合成了速灭威半抗原β-(间-甲基苯氧基羰基)氨基丙酸(HOM)。通过活泼酯法将HOM交联于牛血清白蛋白(BSA)和卵清蛋白(OVA)上;HOM-BSA为免疫原制备了速灭威的兔抗血清,ELISA法测得其效价高达1.28×106,交叉反应测定表明抗体方法特异性识别速灭威。对离子强度等影响因素进行了研究,确定了速灭威酶联免疫吸附分析方法(ELISA)的最佳工作条件,建立了定量测定速灭威的间接竞争ELISA方法,结果表明,该方法检测线性范围为1~10000μg/L;IC50为40.74μg/L;检出限为0.08~0.10μg/L;批内相对标准偏差为2.9%;批间相对标准偏差4.6%。土壤、稻谷和水中的平均添加回收率分别为80%、93.4%和107%。本研究建立了一种快速检测环境和农产品中速灭威残留的有效方法。     

疏水化水溶性两性纤维素接枝共聚物与粘土的相互作用

运用紫外光谱法研究了疏水化水溶性两性纤维素接枝共聚物(羧甲基纤维素接枝丙烯酰胺及N,N 二甲基辛基(2 甲基丙烯酰氧乙基)溴化铵的共聚物, CGAO)在粘土上的吸附,考察了聚合物浓度、无机盐浓度、温度、 pH、 表面活性剂和粘土浓度等因素对CGAO在粘土上吸附量的影响,以及通过X射线衍射分析了CGAO在粘土上的吸附位置.结果表明， CGAO在粘土上的吸附规律与一般聚合物有很大差别,而且CGAO未深入到粘土晶层间,只在其表面吸附. 粘土与CGAO作用前后的粒度分析表明CGAO对粘土粒子有很好的桥接聚集作用. 扫描电镜分析显示粘土与CGAO作用后,其颗粒形态发生了显著变化.     

Development of an analytical protocol for a fast,sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) analysis of proteins offers a particularly promising approach for investigations in cultural heritage on account of its appreciated properties of being highly specific, sensitive, relatively fast, and cost-affordable with respect to other conventional techniques. In spite of that, it has never been fully exploited for routine analyses of painting materials in consideration of several analytical issues that inhibited its diffusion in conservation science: limited sample dimensions, decrease of binder solubility and reduced availability of antibody bonding sites occurring with protein degradation. In this study, an ELISA analytical protocol suited for the identification of aged denatured proteins in ancient painting micro-samples has been developed. We focused on the detection of bovine β-casein and chicken ovalbumin as markers of bovine milk (or casein) and chicken albumen, respectively. A systematic experimentation of the ELISA protocol has been carried out on mock-ups of mural and easel painting prepared with 13 different pigments to assess limits and strengths of the method when applied for the identification of proteins in presence of a predominant inorganic matrix. The analytical procedure has been optimized with respect to protein extraction, antibodies’ concentrations, incubation time and temperature; it allows the detection of the investigated proteins with sensitivity down to nanograms. The optimized protocol was then tested on artificially aged painting models. Analytical results were very encouraging and demonstrated that ELISA allows for protein analysis also in degraded painting samples. To address the feasibility of the developed ELISA methodology, we positively investigated real painting samples and results have been cross-validated by gas chromatography–mass spectrometry.     

流动注射化学发光免疫分析研究I. 血清中乙型肝炎表面抗原的测定

我们首次以键合有抗体的多孔玻璃作为固相免疫分析的免疫反应器, 以化学发光作为最终检测手段, 建立了一种新的、高效率的免疫分析技术-流动注射化学发光免疫分析技术。实验表明: 采用该技术可使单次测定时间从ELISA(Enzyme-linked Immunosorbent Assay)法的二十多小时降至二十分钟, 且所有操作均可在微机控制下自动完成。用该方法对人血清中乙型肝炎表面抗原的测定结果与ELISA法所得结果一致, 对同一样品连续九次测定的相对标准偏差为7.2%。因此, 该方法具有自动化程度高、分析速度快、稳定性好的优点。     

Fluorescence immunoassay based on alkaline phosphatase-induced in situ generation of fluorescent non-conjugated polymer dots

Alkaline phosphatase (ALP) activity assay is not only significant to the clinical diagnosis of some related disease, but also momentous to the construction of ALP-based enzyme-linked immunosorbent assay (ELISA). Herein, for the first time, we have discovered that ascorbic acid (AA) can specially react with N-methylethylenediamine (N-MEDA) to generate fluorescent non-conjugated polymer dots (NCPDs) under mild conditions. On the basis of the AA-responsive emission and ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to AA, we have exploited a fluorometric ALP activity assay with high sensitivity and selectivity. Furthermore, by means of conventional ALP-based ELISA platform, a conceptual fluorescent ELISA has been constructed and applied in the potential clinical diagnosis, during which cardiac troponin I (cTnI), a well-established biomarker of acute myocardial infarction, has been chosen as the model target. We envision that such original fluorescent NCPDs generation-enabled ELISA could become a versatile tool in biochemical sensing and medical diagnosis in the future.     

Polarographic Enzyme-Immunoassay for Trace Hepatitis B Surface Antigen

Abstract

A polarographic enzyme-immunoassay for Hepatitis B Surface Antigen(HBsAg) has been established, in which horseradish peroxidase(HRP) is used as the labeled enzyme, o-phenylenediamine(OPD) as the substrate, and the enzyme-generated product,2,2′-diaminoazobenzene (DAA). is detected by linear-potential scan polarography. Under optimal conditions, the second derivative current of DAA is linear with the concentration of HBsAg from 0.1 to 5 ng/mL. The correlation coefficient(r) is 0.9994. The detection limit is 0.05ng/mL and the relative standard deviation is 6.7%(8 replicates). The sensitivity of the assay is about 20-fold higher than that of ELISA. The assay has been successfully applied for minute determination of HBsAg in both human serum and the negative control serum from ELISA kits.     

Reliable enzyme immunoassay detection for chlorothalonil: fundamental evaluation for residue analysis and validation with gas chromatography

This work describes the analytical performance of the enzyme-linked immunosorbent assay (ELISA) for the fungicide chlorothalonil to effectively exploit as a simple and rapid detection system for pesticide residue on the scenes of the agricultural production and distribution. This ELISA represents the satisfactory analytical characteristics (I50 value, 0.34 ng/g; limit of detection, 0.052 ng/g) to detect chlorothalonil at the regulatory values or thereabout in a sample. Noticeable cross-reactivities were shown with two fungicides, fthalide (58.8%) and pentachloronitrobenzene (quintozene) (20.0%), and some non-agrochemicals such as tetrachloroterephthalonitrile (96.8%) and tetrachlorophthalonitrile (68.3%). The influence of three organic solvents (methanol, acetone, and acetonitrile) used as extractants for chlorothalonil residue was evaluated, with the result that methanol was the most suitable solvent for the ELISA, and the final concentration in the well could be up to 5% (v/v) without any negative influence on the ELISA. It has been possible to directly analyze chlorothalonil residue only by giving dilution of each sample extract with water prior to the ELISA analysis. The average recovery values from the spiked samples by the ELISA were between 101.7 and 113.6% with the average coefficients of variation between 2.6 and 5.9%. Although the results obtained from the ELISA correlated well with those from the reference GC/MS methods for all agricultural samples (r>0.98), the linear function inclined to the ELISA results because of loss during complex sample preparations for GC/MS analysis. Nevertheless, the results demonstrated that the proposed ELISA is a reliable, cost-effective, and rapid quantitative method for chlorothalonil residue.     

Development of a solid-phase extraction—enzyme-linked immunosorbent assay method for the determination of estrone in water

Estrone has been identified as a potential endocrine-disrupting chemical (EDC). To facilitate its analysis, a highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) method with a simple solid-phase extraction (SPE) for analysis of estrone in aquatic environments has been developed. The specific polyclonal antibody was produced against a conjugate of estrone-3-hemisuccinate and keyhole limpet hemocyanin (KLH). The obtained ELISA showed specific recognition of estrone, without cross-reactions for three other major estrogenic compounds (17β-estradiol, estriol, and 17α-ethynylestradiol) commonly found in water. The ELISA had a limit of detection of 0.14 μg/l estrone in water. Combining a SPE method to extract and pre-concentrate estrone from water samples and ELISA to specifically quantify estrone content, the SPE-ELISA can detect estrone down to 1.25 ng/l level in water. Good recovery with spiked river water was obtained with this SPE-ELISA method. The developed SPE-ELISA system was applied to analyze the real influent and effluent samples of sewage treatment plant in Penrith (Australia) and the results correlated well with those obtained using GC and HPLC methods. The developed SPE-ELISA method is capable of being applied for the specific detection and routine monitoring of estrone in environmental water samples.     

Monoclonal antibody to polycyclic aromatic hydrocarbons based on a new benzo[a]pyrene immunogen

Benzo[a]pyrenebutyric acid (B[a]PBA) has been synthesized and covalently coupled to bovine serum albumin to generate monoclonal antibodies (Mab). A competitive indirect enzyme-linked immunosorbent assay (ELISA) for polycyclic aromatic hydrocarbons (PAH) has been developed with Mab B[a]P-13. It was shown by testing with 21 parent PAH and 10 compounds carrying methyl, hydroxy, or butyric acid functions that the antibody had broad specificity. Highest affinity was found for four- to six-ring PAH. Different organic co-solvents were tested. No loss in sensitivity, compared with controls in PBS, were found with methanol, dimethyl sulfoxide, and glycerol at final concentrations of 5 to 10%. Further, an observation was made that a modification (fine-tuning) of the affinity and specificity of the antibodies was possible by changing the type of the added organic co-solvent. The high susceptibility of the ELISA with regard to inorganic ions might be an indication of a more hydrophilic binding pocket e.g. involving a pi-cation interaction. Investigation of the effect of pH revealed that for pH between 6 to 9 there was no noticeable impairment. With an LOD as low as 30 pg per well for B[a]P the sensitivity of the ELISA is sufficient for analyses of solvent extracts of many environmental samples. As an example, the determination of a PAH sum parameter, given as B[a]P-equivalents, in crude aerosol extracts by both ELISA and HPLC revealed good correlation (r2=0.717) but approximately five-fold overestimation by the immunochemical method, obviously as a result of cross-reacting analytes.     

Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin

Plumbagin (PL; 5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural compound mainly isolated from Plumbago zeylanica. This plant is distributed in Southeast Asia, and well known as Ayurvedic medicine in India for its medicinal properties. PL has been shown to have various pharmacological activities. We have successfully prepared monoclonal antibodies against PL, and developed an enzyme-linked immunosorbent assay (ELISA) system for determination of PL. 3-(5-Hydroxy-2-methyl-1,4-naphthoquinone-3-yl) propanoic acid was synthesized and purified to prepare PL-bovine serum albumin conjugate (PL-BSA), which was used as an immunogen. PL-BSA conjugate was administered into BALB/c male mice for production of monoclonal antibodies against PL. The monoclonal antibody against PL which is secreted from established hybridoma cell line 3A3 (MAb 3A3) has been proven to have highly-specific to PL resulting from cross-reactivities test. The range for calibration of PL by ELISA was 0.2-25 μg mL⁻¹. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of PL in plant.     

Quantitative Assessment of Protein Adsorption by Combination of the Enzyme-Linked Immunosorbent Assay with Radioisotope-Based Studies

Protein adsorption at polymer surfaces has been investigated by means of both ELISA and radiolabeling techniques. Most of the data obtained are linearly related to each other for protein concentrations between 0.01 and 1 μg/ml, i.e., the concentration range in which the maximum amount of adsorbed active protein (ELISA) is achieved. The correlation of ELISA data with radioisotope-based measurements allows quantification of the former. Specific correlation factors are described. Adsorption is shown to be strongly dependent on the polymer/protein system.     

增强化学发光酶免疫分析法测定血清中的铁蛋白

本文采用对碘苯酚增强的Ｌｕｍｉｎｏｌ－Ｈ２Ｏ２－ＨＲＰ化学发光反应体系作为免疫分析的最终检测手段，建立了一种新的铁蛋白的免疫分析方法，与酶联免疫分析法相比，该方法具有灵敏度高，线性范围宽等特点。方法的相对标准偏差３．７％，标准曲线线性范围为０．１２－２０００ｎｇ／ｍｌ，用该方法对血清中的铁蛋白进行了测定，其结果与ＥＬＩＳＡ法所得结果一致。