微板式化学发光酶免疫分析法测定人血清中癌胚抗原

采用辣根过氧化物酶(HRP)催化鲁米诺(luminol)-H2O2化学发光体系,建立了一种测定人血清中癌胚抗原(CEA)的高灵敏度、高特异性、简便快速的微板式化学发光酶免疫分析方法。对免疫反应条件、酶结合物稀释度、发光反应时间、封闭液等进行了考察和优化。采用双抗体夹心法,室温静置1h,洗涤后加入100μL发光底物液,10min后检测。该方法的线性相关系数为0.9998;最低检出限为0.57μg/L;批内和批间变异均在10%之内;低、中、高3个不同浓度值样品的平均回收率分别为107.4%、93.3%和104.5%。使用本方法与进口发光试剂盒对40份人血清样品进行测定,结果表明,本方法显示了良好的相关性,其相关系数为0.9115。表明本分析体系稳定可靠,可用于商品化诊断试剂盒的开发和应用。     

Homogeneous Amperometric Immunoassay for Digoxin

Abstract

A homogeneous spectrophotometric EMIT immunoassay for the measurement of digoxin has been converted to an amperometric immunoassay format capable of analysing digoxin in diluted serum. NADH produced by glucose-6-phosphate dehydrogenase (G6PDH) labelled digoxin was detected amperometrically by its rapid, efficient oxidation at platinized activated carbon electrodes (PACE) poised at +150 mV vs Ag/AgCl. The new amperometric procedure resulted in a simplified assay protocol with a decreased overall assay time, enhanced NADH detection and minimal sample pretreatment.     

微孔板化学发光酶免疫分析法分析人血清中游离前列腺特异性抗原

建立了一种定量分析人血清中游离前列腺特异性抗原(f-PSA)的高灵敏度微孔板化学发光酶免疫分析方法,以碱性磷酸酶(alkaline phosphatase,ALP)为标记酶,4-甲氧基-4-(3″-磷酰氧基苯)-螺旋-(1,2-二氧杂环丁烷-3,2′-金刚烷)(4-methoxy-4-(3″-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane),AMPPD)-ALP高灵敏的化学发光反应为检测体系,通过检测发光强度对人血清中f-PSA进行定量。对几种物理化学参数如温育时间、免疫反应步骤以及检测时间等进行了优化,采用了一步双抗体夹心法,37℃恒温静置温育2h,洗涤后加入50μL AMPPD,30~80min内检测。该方法线性范围为0.15~20μg/L,相关系数大于0.998;检出限可达0.01μg/L,批内和批间相对标准偏差(C.V.)均小于7%;回收率在88%~108%之间。对人体血清中共存的6种常见肿瘤标志物CA50、CA125、CA15-3、CA242、CEA和AFP的偶联反应进行考察,特异性良好。为了验证该方法用于商业试剂盒的可行性,在4℃和37℃条件下分别进行了3d,5d,7d的稳定性考察,其线性相关系数仍均大于0.998,相对标准偏差小于6%。对98例实际血样进行测定,并与进口试剂盒(Monobind.USA)作临床比对,相关性良好。这些结果均表明,该分析体系稳定,可靠,可以用于商业化试剂盒的开发,在临床分析人血清中f-PSA的辅助诊断前列腺癌方面具有很高的应用价值。     

Fast Amperometric Immunoassay Utilizing Highly Dispersed Electrode Material

Abstract

An immunoassay technique based on a high surface area carbon immunoelectrode is described. Dispersed ULTI carbon serves as a carrier for immobilized antibodies and also as an electrode material. ‘Sandwich’ scheme of immunoassay of rabbit IgG (as a model analyte) has been used. Iodine formed as a result of the enzymatic oxidation of iodide by peroxidase-label has been detected amperometrically. Using dispersed carbon material as a solid support for immobilization of immunoagents and as an electrode material improves the efficiency of immuno-interaction and the sensitivity of electrochemical assay due to the high area-to-volume ratio of solid to liquid phases.

The method based on ‘sandwich’ immunoassay is conducted at 21°C. The time of each stage of incubation (with analyte and with peroxidase-antibody conjugate) is 10 min. Electrochemical detection of peroxidase-labeled immuno-complex does not exceed several minutes. The technique allows fast determination of rabbit IgG with a low detection limit, in the nanomolar range. By comparison, the conventional technique based on ELISA, being performed under the same conditions (temperature and incubation time) does not allow any quantitative determination of IgG for concentration lower then 100 nM.     

Glutathione Amperometric Enzyme Microsensor

Abstract

Glutathione micro-enzyme sensors were developed based on the immobilization of glutathione oxidase at the tip of a 25 μm Pt wire sealed in glass. An inner membrane constructed from cellulose acetate (CA) was deposited onto the tip of the Pt microelectrode prior to enzyme immobilization with glutaraldehyde. The final outer diameter of the microelectrodes is approximately 30 μm. The analytical characteristics of the microelectrodes, including calibration curves, apparent K_M′, pH response curves, stability and selectivity over enzymatic interferences were determined. The response was linear in the concentration range 1.0 × 10^?5 M - 1.9 × 10^?4 M glutathione, reaching 95% steady-state current in 15–20 seconds. The microelectrodes were useful for more than two months.     

氯化血红素作为模拟酶荧光免疫分析乙肝表面抗原

乙肝表面抗原的测定在临床诊断上是一项很重要的指标．现在一般采用酶联吸附免疫分析技术测定，但是酶本身性质不稳定且价格昂贵、操作繁琐；更重要的是大分子的酶作为标记物，由于空间位阻效应而阻碍抗原-抗体的免疫反应．所以，用小分子催化剂代替大分子酶的研究显得日益重要[1，2]．近年来有关模拟酶在免疫分析中的应用已有报道[3，4]．本文提出了以氯化血红素作为辣根过氧化物酶（HRP）的模拟酶来标记抗体，以盐酸硫胺素（维生素B1）作为供氢体，成功地实现了乙肝表面抗原（HBsAg）的夹心法荧光免疫分析．测定范围是2．5～500ng／wel…     

An Enzyme Immunoassay for the Determination of Methabenzthiazuron

Abstract

A sensitive enzyme immunoassay is described for the determination of the urea herbicide methabenzthiazuron. The assay is carried out with polyclonal antibodies, which were raised in rabbits by immunization with a methabenzthiazuron-BSA conjugate containing five methabenzthiazuron residues per molecule. The ELISA was optimized on microtiter plates with a peroxidase-methabenzthiazuron tracer. The middle of the test (50% B/B₀) was found at 1.0 μg/l. The lower detection limit of methabenzthiazuron is c. 0.05 μg/l. Samples can be measured up to 10 μg/l methabenzthiazuron (upper detection limit). The assay does not require concentration or clean-up steps for drinking or ground water samples. Validation experiments showed a good accuracy and precision. Work with monoclonal antibodies is in progress.     

An Enzyme Immunoassay of Catalytically Active Proteases

Abstract

A method is proposed to assay catalytically active protease. The method is based on the subsequent inter action of the enzyme with a protein inhibitor immobi lized on polysterene and with antibodies labelled with a high-activity enzyme. Optimal conditions have been chosen for assaying alkaline protease from Bacillus subtilis (subtilisin) by ELISA with peroxidase as label, employing two protein serine protease inhibitors isolated from duck egg white and maize seeds. The proposed method enables the detection of the catalyti cally active enzyme at concentrations as low as 1 ng/ml for 1.5 hr. The c. v. of the assay is less than 5%. The method can be used to determine the equilibrium con stant of the enzyme-inhibitor reaction in solution.     

化学发光酶免疫分析研究进展

叙述了化学发光免疫分析的分类及特点,从发光底物、增强剂、过氧化物酶、过氧化物及其他方面,对鲁米诺类化合物化学发光酶免疫分析的进展进行了综述,引用文献34篇。     

非竞争性毛细管电泳免疫分析乙肝表面抗原

用硫脲标记乙肝抗体(抗-HBs),基于抗原．抗体高选择性识别,建立了非竞争性毛细管电泳免疫分析乙肝表面抗原的新方法。研究了混合温育时间、缓冲溶液酸度和浓度、进样时间的影响。在5mmol／L Tris-20mmol／L H3BO3-3mmol／L EDTA(pH7．0)的运行缓冲溶液中,游离的标记抗体和抗原抗体复合物在15min内完全分离。HBsAg在4．0—50mg／L范围内与复合物的峰面积呈良好的线性关系;相对标准偏差小于6％;检出限为3．0mg／L。该方法用于乙肝病人血清和正常人血清中HBsAg的测定,结果令人满意。     

化学发光酶免疫法测定游离三碘甲腺原氨酸

以辣根过氧化物酶为三碘甲腺原氨酸类似物的标记物,鲁米诺为化学发光反应底物,建立了一种高灵敏度化学发光免疫检测游离三碘甲腺原氨酸的方法。该法线性范围为0.90~80 ng/L,批内变异小于12.1%,批间相对标准偏差均小于15%,其余各项指标均良好。利用本法对血清样品进行了测定,关于甲状腺机能亢进组病人的临床诊断符合率达到83.3%。与其它化学发光免疫分析方法比较所得相关系数为0.9005。在方法条件选择上,以尽可能保持游离激素和蛋白结合激素之间的平衡为原则,提高游离三碘甲腺原氨酸浓度测定的准确性。     

Determination of Thyroxine with Capillary Electrophoretic Enzyme Immunoassay

Capillary electrophoretic immunoassay (CEIA) is a new analytical technique1,2. In CEIA, the detection methods were UV absorbance detection1 and laser induced fluorescence detection (LIF)2. However, The UV detection is lack of sensitivity. The LIF is difficult to use due to its high cost, and furthermore, most biological fluids are strongly luminescent when excited by the laser in the blue or green region of the spectrum. In our present work, a capillary electrophoretic enzyme immuno…     

Electrochemical Determination of Cortisol by Capillary Electrophoretic Enzyme Immunoassay

In recent years, capillary electrophoretic immunoassay (CEIA) has become a primary analytical tool in clinical diagnostics. The primary detection methods in CEIA are UV absorbance detection and laser induced fluorescence detection (LIF)1. The major disadvantages of the UV detector are the lack of sensitivity and not available for many antigens which are small molecules without strong UV absorbance. LIF is a more general approach to improve sensitivity. However, it is difficult to u…     

Separation-free amperometric enzyme immunoassay

A new technique for conducting a separation-free amperometric enzyme immunoassay is described using DNP-aminocaproic acid as the analyte. The technique is based on the combined use of a recently described separation-free enzyme immunoassay (19) and an electrode system that senses H₂O₂. Oxidation of glucose to gluconate and H₂O₂ by the enzyme reconstituted from DNP-conjugate apoglucose oxidase (DPN-CAGO) and FAD was continuously measured amperometrically. The reconstitution was inhibited by preincubation with anti-DNP antibody before adding FAD. This antibody-induced inhibition of the reconstituting of the holoenzyme was reversed by adding DNP-amino caproic acid to DNP-CAGO before adding the antibody to DNP-CAGO. Based on (a) the antibody-induced inhibition of holoenzyme reconstitution, (b) a specific ligand-induced reversal of the inhibition, and (c) an electrochemical system that measures H₂O₂, we developed a separation-free (homogeneous) amperometric enzyme immunoassay.     

Disposable Amperometric Label-Free Immunosensor on Chitosan–Graphene-Modified Patterned ITO Electrodes for Prostate Specific Antigen

A facile and highly sensitive determination of prostate-specific antigen (PSA) is of great significance for the early diagnosis, monitoring and prognosis of prostate cancer. In this work, a disposable and label-free electrochemical immunosensing platform was demonstrated based on chitosan–graphene-modified indium tin oxide (ITO) electrode, which enables sensitive amperometric determination of PSA. Chitosan (CS) modified reduced graphene oxide (rGO) nanocomposite (CS–rGO) was easily synthesized by the chemical reduction of graphene oxide (GO) using CS as a dispersant and biofunctionalizing agent. When CS–rGO was modified on the patterned ITO, CS offered high biocompatibility and reactive groups for the immobilization of recognition antibodies and rGO acted as a transduction element and enhancer to improve the electronic conductivity and stability of the CS–rGO composite film. The affinity-based biosensing interface was constructed by covalent immobilization of a specific polyclonal anti-PSA antibody (Ab) on the amino-enriched electrode surface via a facile glutaraldehyde (GA) cross-linking method, which was followed by the use of bovine serum albumin to block the non-specific sites. The immunosensor allowed the detection of PSA in a wide range from 1 to 5 ng mL⁻¹ with a low limit of detection of 0.8 pg mL⁻¹. This sensor also exhibited high selectivity, reproducibility, and good storage stability. The application of the prepared immunosensor was successfully validated by measuring PSA in spiked human serum samples.     

细交链格孢菌酮酸间接竞争酶联免疫检测方法及配套试剂盒的研制

建立了间接竞争酶联免疫吸附法检测食品中细交链格孢酮酸(Tenuazonic Acid,TA)残留,并研制快速检测试剂盒。采用肟化法对TA进行衍生化,活泼酯法偶联半抗原和载体蛋白得到人工抗原TAO-BSA和TAO-KLH,以TAO-KLH作为免疫原免疫雌性Balb/c小鼠制备特异性抗体。对包被浓度、包被时间、封闭液类型、抗体工作浓度、二抗稀释度、底物显色时间等参数进行研究,建立了TA残留间接竞争酶联免疫检测方法并进行了配套试剂盒的研制。该试剂盒半抑制浓度Ic_(50)为1.48ng/mL,检测线性范围为0.06~35.95ng/mL(R~2=0.9941);检测限为0.02ng/mL;加标样品平均回收率大于88.50%;试剂盒的批间和批内平均变异系数分别为2.84%和9.49%,与食品中常见真菌毒素交叉反应率均小于1%。     

板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125

在传统的板式化学发光免疫分析法和管式磁颗粒化学发光免疫分析法基础上,建立了人血清中糖类抗原125(CA125)的板式磁颗粒化学发光免疫分析方法.该方法以磁性微粒子作为分离固相,96孔板为反应容器,辣根过氧化物酶(HRP)催化H2O2-luminol化学发光体系作为检测体系.本法测定CA125的检测灵敏度可达2.0U/mL,线性范围为0～400U/mL.与常用的包被板化学发光免疫分析方法对比,该方法检测范围宽.与管式磁颗粒化化学发光法比较,其分析灵敏度与精密度高、线性范围、分析通量以及分析成本方面均显示了很好的优越性.采用该方法对人血清中CA125进行测定并与罗氏全自动电化学发光系统的测值结果进行了比对,两者显示了良好的相关性.     

Improvement of an Enzyme Immunoassay for the Determination of Mercury(II)

Three systems were tested for the optimization of a heterogenous non-competitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the non optimized Hg-EIA with a detection limit of 2.1 μg/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 μg/L Hg (II)). A conventional competitive ElA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 μg/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 μg/L Hg (II).     

Development of an Ultra-sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Diethylstilbestrol in Seafood

An ultra-sensitive indirect competitive chemiluminescence enzyme immunoassay was developed for screening diethylstilbestrol in fish and shrimp samples. The concentration of diethylstilbestrol that caused 50% inhibition of the binding enzyme marker (IC₅₀) was 0.32 ng/mL and the limit of detection was 0.0068 ng/mL; the linear range was from 0.028 ng/mL to 3.60 ng/mL. The assay showed cross-reactivity of 7.1% and 2.8% with dienestrol and hexoestrol, respectively, but negligible cross-reactivity with estradiol, estrone, ethinyloestradiol, and progestin. The recovery from spiked fish and shrimp samples varied from 68.5% to 92.5%, and the mean coefficients of variation within groups and between groups were 6.2% and 8.0%, respectively. Our results indicated that the assay is a simple, sensitive, specific, and accurate method for screening fish and shrimp samples for diethylstilbestrol.     

增强化学发光酶免疫法对猪肉中盐酸克伦特罗的检测

建立了猪肉中克伦特罗(CLB)的直接竞争化学发光酶免疫检测(dc-CLEIA)方法。通过优化离子强度、pH值确立了化学发光酶免疫法的标准曲线,优化后dc-CLEIA的检出限可达0.02μg/L。猪肉中的盐酸克伦特罗用高氯酸提取、SPE净化后绘制基质曲线,基质曲线与标准曲线吻合,说明基质影响基本消除。测定0.10、1.0、5.0μg/kg 3个水平的添加回收率,平均回收率为83%~98%,批内与批间的相对标准偏差均小于15%。进一步研究了dc-CLEIA与HPLC两种方法测定猪肉样品的相关性,结果显示两者测定结果相关性良好,r=0.964 7,说明所建立的直接化学发光酶免疫方法可用于实际样品的检测,结果准确可靠。