Flow Injection Fluoroimmunoassay for Human Transferrin Using a Protein a Immunoreactor

Abstract

A protein A immunoreactor (immobilized protein A) incorporating flow injection technique was used for on-line fluoroimmunoassay of human transferrin. Antibody immobilized on protein A and antibody-antigen complex formation took place in phosphate buffered saline (PBS, PH 7.4). After washing off excess lucifer yellow VS labelled human transferrin, the antibody-antigen complex was eluted with acid buffer and detected. Experimental variables have been studied and the method has been used to determine the transferrin contents in human serum.     

茶（叶）碱的流动注射免疫试验法研究

本研究建立了一种多相抗哮喘麻醉药茶碱的酶免疫电化学检测法。此法使用含有Ａ蛋白免疫反应体的流动注射分析装置来分离与抗体结合和与抗体不结合的茶碱磷酸酯酶。抗体、标示剂和试样相继附着在Ａ蛋白免疫反应体上。当ｐＨ值约为中性时，抗体Ａ蛋白和抗体Ａ蛋白标示剂或试样发生反应，合成物则在ｐＨ为酸性情况下洗提。曾用ｐ－磷酸胺基苯（ＰＡＰＰ）和ｐ－胺基酚（ＰＡＰ）作为检测剂。在诸如不同的流动速率和不同的反应物浓度条件下进行了研究，并已用该法测定了血清茶碱，较好地得到了ｒ、ｓ、ｄ值和萃取率，其结果符合要求。     

Magnetic Separation Immunoassay for Digoxin in Plasma with Flow Injection Fluorescence Detection

Immunoassay (IA) is a sensitive and selective approach for low level quantitation of drugs. Magnetic separation immunoassays use magnetic beads to facilitate the separation of bound labeled antigens from free antigens in solution. Digoxin was chosen for this study because low level analysis (ngmL^–1) in biological samples isrequired, antibodies to digoxin were commercially available and derivatization procedures for fluorescence labeling were well established. A competitive immunoassay format was used in this study. Streptavidin coated magnetic beads were attached to biotinylated anti-digoxin antibodies for the separation. The inhibition curve for off-line magnetic separation immunoassay of digoxin in spiked plasma was characterized and the dynamic range of the curve was 0.25–2.5ngmL^–1. A power fit weighted by the inverse of concentration was found to provide the best fit to the data (r=0.9934). The percent RSDs for the two controls, 0.8 and 2.2ngmL^–1, were 9.95% and 20.62% (n=6) and the percent errors were 11.75% and 22.63% (n=6), respectively. The limit of detection (LOD) in plasma is 0.14ngmL^–1. The dynamic range of the inhibition curve for on-line magnetic separation immunoassay of digoxin was 0.5–15ngmL^–1 of digoxin. A quadratic fit was found to provide the best fit to the data (r=0.9937). The percent RSDs for the two controls, 4.0 and 12ngmL^–1, were 14.1% and 10.7% (n=6) and the percent errors were 5.8% and 3.3% (n=6) from the spiked value, respectively. The LOD was estimated to be 0.44ngmL^–1 (determined as two times the standard deviation of the blank, n=6). The on-line method has the advantages of being relatively easy to automate in the continuous flow mode and is adaptable for use in conjunction with HPLC separations.     

顺序注射可更新表面固相荧光免疫法测定人血清中免疫球蛋白G

建立了采用芯片式微型流通池的测定人血清中免疫球蛋白G(IgG)的顺序注射可更新表面非均相荧光免疫分析法。将羊抗人IgG抗体固定于包被有蛋白A的Sephamse CL4B凝胶微珠,然后制备成固相抗体。用标记FITC的抗人IgG抗体作为第二抗体。固相抗体、血清试样和荧光标记第二抗体由顺序注射系统注入芯片式微型流通池,并在其中进行免疫反应生成夹心式抗体．抗原荧光复合物。荧光分光光度计通过光纤与流通池耦合测定截留于流通池中的抗体一抗原复合物荧光强度。一次测定完成后,微珠即被排出流通池。流通池经缓冲液清洗后即可进行下一次测定。体系经优化后,检出限为0．1mg／L IgG,分析速率达到11次／h。3．9mg／L IgG的日内和日间精密度(RSD)分别为1．7％和5．2％;校正曲线的线性范围为0．3—7．0mg／L IgG。所建立的方法已成功地应用于人血清中IgG的测定。     

流动注射在线氧化荧光分析法测定柳氮磺吡啶

研究了硫酸介质中,高锰酸钾氧化柳氮磺吡啶（SASP）体系的荧光特性,结合流动注射进样技术,建立了流动注射在线氧化荧光分析测定SASP的新方法。SASP本身不发荧光,但在硫酸介质中与高锰酸钾反应后可产生1种有强荧光的产物（λex=301 nm,λem=366 nm）。在优化条件下,SASP的质量浓度在1.2×10-5~2.0×10-3 g/L范围内与相对荧光强度呈良好线性,相关系数为0.999 6。方法的检出限为3.0×10-6 g/L,对1.2×10-4 g/L的SASP标准溶液平行测定11次的相对标准偏差为0.6%。该法简单、快速、灵敏度高、选择性好,可用于药物制剂中SASP含量的直接分析。     

Flow Injection Chemiluminescence for the Determination of Estriol via a Noncompetitive Enzyme Immunoassay

A novel approach to the detection of estriol using a flow injection system coupled to enhanced chemiluminescent immunoassay was developed based on noncompetitive immunoassay formats. A conjugated estriol-ovalbumin immobilized immunoaffinity column was inserted into the flow system to trap the unbound horseradish peroxidase (HRP)-labeled antibody after an off-line incubation of estriol and HRP-labeled anti-estriol antibody. The trapped enzyme conjugate was detected by the injection of chemiluminescent substrates to produce enhanced chemiluminescence. The linear range for the determination of estriol is 10.0 to 400 ng · mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 5.0 ng · mL⁻¹. The total time for sampling and chemiluminescent detection of one sample is 400 seconds after 30 min of pre-incubation. The results for pregnancy serum samples obtained by this method are in good agreement with those obtained using ELISA.     

芯片式流通池用于顺序注射可更新表面荧光免疫法测定人血清中的IgG

采用芯片式流通池作为非均相免疫反应和原位固相荧光检测的场所,用双岔光纤将芯片式流通池与荧光光度计耦联,以双抗夹心式非均相免疫反应的模式,研究建立了测定人血清中IgG的顺序注射可更新表面非均相免疫分析新方法.     

海水中痕量锌的流动注射在线预富集和电感耦合等离子体原子荧光检测

一种较新的螯合树脂CPPI,被用来从海水中富集痕量锌。在线预富集由流动注射分析仪控制,经富集浓缩后的锌试液亦由流动注射分析仪送往电感耦合等离子体原子荧光光谱仪检测。检出限在0.1μg/L以下。     

流动注射协同增强化学发光免疫分析法的建立与评价

以兔IgG为模型 ,NaTPB与PPP协同增强的Luminol H2 O2 HRP混合液为发光体系 ,HRP标记抗体 ,建立了灵敏度高、特异性强、重现性好的流动注射协同增强化学发光免疫分析法。结果表明 :在 2～ 6 0 μg L范围内兔IgG量与发光强度有良好的线性关系 ,相关系数r=0 .994 1 (P <0 .0 1 ) ;绝对检测限为 0 .6 5fmol;方法精密度为 4 .72 %～ 9.31 % ;回收率为 92 .5 0 %～ 99.4 0 %。免疫柱可反复使用 2 0 0次以上。本法测定不同浓度兔IgG标准溶液的结果与RIA法的测定结果基本一致     

流动注射在线分离富集光度法测定铜

本文提出痕量铜的在线流动注射离子交换树脂分离富集的吸光光度测定方法，以显色液作为淋洗液，直接洗脱Ｄ４０１螯合树脂上富集的铜，与此同时显色剂与铜（Ⅱ）生成有色络合物，在６３２ｎｍ处测定铜。     

毛细滤管在线浓缩结合流动注射测定二氧化氯

提出了一种用毛细滤管浓缩结合流动注射分析测定ClO2的新方法。在ClO2存在下,4-氨基安替比林和苯酚显色反应,生成的有色络合物与ClO2的量成正比且能被有效地富集在聚四氟乙烯(Teflon)毛细滤管内,然后被洗脱进入流通池检测。经富集后,方法的灵敏度提高了近2个数量级,可测定痕量水平(μg/L级)的ClO2,是目前测定ClO2最灵敏的方法之一。方法用于测定ClO2的线性范围为5—250μg／L;检出限为0．5μg／L。其它氯化物和金属离子的干扰可通过聚四氟乙烯滤膜处理而得以除去。     

吩噻嗪类药物的流动注射在线氧化荧光分析法测定

结合流动注射进样技术,借助时间扫描荧光方式,建立了流动注射在线氧化荧光分析法测定吩噻嗪类药物的新方法。在优化的实验条件下,盐酸氯丙嗪、奋乃静、盐酸异丙嗪的质量浓度分别在0.12~36、0.4~20、0.4~36 mg/L范围内与荧光强度呈良好的线性关系,方法检出限分别为0.003 6、0.02、0.03 mg/L。该方法简便、快速、准确、灵敏度高,用于药物制剂、尿样中吩噻嗪类药物的直接测定,结果满意。     

痕量铜的流动注射-固相萃取-光度法测定

根据在酸性介质中,Cu²⁺对溴酸钾氧化藏红T的反应具有催化作用,利用自制的二苯卡巴腙功能键合硅胶做固相萃取固定相,联用流动注射样品处理技术,建立了在线选择性固相萃取催化动力学流动注射光度法测定痕量Cu²⁺的新方法。 研究了富集时间、反应管长、富集和洗脱流速、反应介质及洗脱液、试剂浓度、反应温度和共存物质的影响。 在最佳实验条件下,对铜测定的线性范围为0.0010~0.60 mg/L,检出限为7.9×10^-4 mg/L。 在线分离富集后分析方法的灵敏度提高20多倍,允许干扰物质存在的倍率提高了近10倍。 11次重复测定的相对标准偏差小于2.6%。 一份样品溶液从分离富集处理到测定,仅需6 min和6 mL试剂。 方法已用于中药和茶叶中痕量铜的测定。     

Catalytic Determination of Trace Formaldehyde with a Flow Injection System Using the Indicator Reaction between Crystal Violet and Bromate

A sensitive flow injection method is proposed for the determination of formaldehyde based on its catalytic action upon the redox reaction between crystal violet and potassium bromate in phosphoric acid medium. The reaction is monitored spectrophotometrically by measuring the decrease in absorbance of crystal violet injected at the maximum absorption wavelength of 600nm. A calibration graph from 0.018 to 1.400µgmL^–1 is obtained, and the detection limit is 0.007µgmL^–1 in a sample volume of 30µL. Up to 15 samples can be analyzed per hour with a relative standard deviation of 1.2 and 2.3%, respectively, for the determination of 1.0 and 0.1µgmL^–1 of formaldehyde. The method is free from most interference. This method has been applied to the determination of formaldehyde in air, decoration boards and paints, and the results compare well with those provided by iodimetry.     

流动注射在线萃取荧光法测定痕量阴离子表面活性剂

报道了痕量阴离子表面活性剂的流动注射在线萃取荧光分析方法．该方法测定的线性范围为 ０．０１～０． ２０ ｍｇ／Ｌ；进样频率为 ２０样／ｈ．应用本法测定了水样中痕量阴离子表面活性剂，得到了满意的结果．     

夹心法免疫层析试条的数学模型与仿真研究

基于夹心法免疫层析试条检测原理,结合对流扩散方程和流体动力学方程,建立了夹心法免疫层析试条动态反应过程的数学模型,并通过COMSOL软件对试条动态反应过程进行仿真.分别探究了目标待测物A浓度在0 ～ 20 mol/L,标记物P浓度在1×10-2～1×103 mol/L以及硝酸纤维素膜的孔隙率在0～1范围内变化时,检测线上夹心复合物浓度关于位置和时间的浓度变化情况,并分析了各物质初始浓度以及试条结构对于检测结果和检测时间的影响.结果表明,在一定浓度范围内,目标待测物A以及标记物P浓度的增加将提高试条的定量检测性能,而孔隙率通过影响混合液流速和混合液中各物质反应接触情况来影响检测结果.     

Flow injection immunoassays: A review

This review covers the application of flow injection analysis to immunoassays (FIA-IA). The advantages and limitations of this approach are discussed. Future directions, particularly those involving microfluidic systems are highlighted.     

在线氧化还原流动注射分光光度法测定多贝斯

建立了在线氧化还原流动注射分光光度法测定多贝斯的新方法。研究了多贝斯与Fe3 + 的反应摩尔比和各种影响因素 ,发现Fe3 + 可以定量氧化多贝斯。产生的Fe2 + 与邻菲咯啉在弱酸性介质中生成稳定的红色络合物 ,该络合物在 5 12nm处的最大吸收与多贝斯浓度在 4 .2 4× 10 -6～ 4 .6 6× 10 -5mol/L范围内呈线性关系。回归系数为 0 .9987;方法的检出限为 5 .3× 10 -7mol/L ;相对标准偏差为 0 .5 % (n =8) ;采样率为 30次/h。     

Spectrophotometric Determination of Bromate in Water Using Multisyringe Flow Injection Analysis

A multisyringe flow injection system for the spectrophotometric determination of bromate in water is proposed, based on the oxidation of phenothiazine compounds by bromate in acidic medium. Several phenothiazines were tested, including chlorpromazine, trifluoperazine, and thioridazine. Higher sensitivity and lower LOD were attained for chlorpromazine. Interference from nitrite, hypochlorite, and chlorite was eliminated in-line, without any changes in the manifold. The automatic methodology using chlorpromazine allowed the determination of bromate between 25 and 750 µg L^?1, with LOD of 6 µg L^?1, good precision (RSD < 1.6%, n = 10), and determination frequency of 35 h^?1.     

基于可控相转变温度热敏高分子的人IgG酶联荧光免疫分析

以温度敏感高分子聚N－异丙基丙烯酰胺－丙烯酰胺[P(NIP-AA)]作为载体,建立了酶联荧光免疫分析人IgG的新方法。AA摩尔含量为8％的高分子P(NIP-AA)其临界溶解温度为37 °C。竞争型免疫测定中,被固定的IgG和标准溶液（或样品）在33 °C均相条件下竞争性地与辣根过氧化物酶标记抗体反应,升高温度分离出高分子免疫复合物,沉淀重新溶解后通过偶联过氧化氢与对羟基苯乙酸的荧光反应进行定量,线性范围为100-1000 ng/mL,检测限为2.0 ng/mL。方法灵敏、快速操作简便,且提高了免疫反应效率。此外,灵敏度与用传统微孔板做载体相似,但测定时间更快（从100-120分钟减少到30分钟）。该法用于测定人血清中IgG的含量,结果满意。