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.     

高效液相色谱法与酶免疫法快速测定鲫鱼中残留显/隐色孔雀石绿的比较

采用高效液相色谱(HPLC)法与酶免疫法(EIA)两种方法检测鲫鱼中残留显/隐色孔雀石绿的含量,对这两种分析方法所得的回收率进行了比较,当添加浓度在5.0～50.0 μg·kg-1时,其中 HPLC 法所测得的回收率为 68.2%～90.0%,相对标准偏差小于7.0%,检出限为 2 μg·kg-1;EIA 法所测得的回收率为 72.4%～92.3%,相对标准偏差小于 6.8%,检出限为1 μg·kg-1;两种方法均具有高效、快速、稳定的特点,能满足日常检验工作的需要.     

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。     

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…     

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%。     

Novel and Sensitive Noncompetitive Enzyme Immunoassay (Hetero-Two-Site Enzyme Immunoassay) for Arginine Vasopressin

Abstract

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for arginine vasopressin in plasma is described. Plasma (0.3 ml) was diluted 1.3-fold with an appropriate buffer and filtered by centrifugation in a micro-concentrator with polysaccharide membrane to eliminate plasma proteins. Arginine vasopressin in plasma filtrates was biotinylated and trapped onto anti-arginine vasopressin IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated arginine vasopressin was eluted from the polystyrene balls with HCl and was reacted with anti-arginine vasopressin Fab′-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The detection limit of arginine vasopressin was 11 fg (10 amol)/tube. This was 45-fold lower than that by competitive enzyme immunoassay using the same antiserum as used in this study and 9 to 400-fold lower than those previously reported by competitive radioimmunoassays. The assay range of arginine vasopressin in plasma was 0.14–140 ng /l using 100 μl of plasma filtrates corresponding to 75 u1 o f plasma. Plasma levels of arginine vasopressin i n 8 healthy subjects aged 25–41 yr with, ad libitum water in take and normal activity approximately 4 h after breakfast were 0.72 ± 0.22 (SD) ng /l (range, 0.42–1.04 ng /l).     

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。     

Amperometric Enzyme Sensor-Based Enzyme Immunoassay for Factor VIII-Related Antigen

Abstract

The coupling of an enzyme immunoassay for factor VIII-related antigen with a commercial glucose oxidase based amperometric sensor permits the determination of 1.6 to 16 ng of factor XIII-related antigen in human plasma. Further pure amperometric sensors or amperometric enzyme sensors for determination of the main marker-enzymes of enzyme immunoassays are described.     

化学发光酶免疫法检测猪肉中氯丙嗪残留

采用棋盘滴定法确定包被抗原浓度和抗体稀释倍数,通过单因素实验优化了竞争反应时间、磷酸盐缓冲液浓度、甲醇含量、pH值等参数,建立了氯丙嗪的间接竞争化学发光酶免疫检测方法,并考察了方法的特异性、灵敏度和稳定性.结果表明,最佳反应条件为包被抗原浓度为0.05 μg/L;氯丙嗪抗体稀释32000倍;体系缓冲液为含10％甲醇、0.1 mol/L磷酸盐缓冲液(pH 7.0).本方法的IC50为0.12 μg/L;检出限为0.02 μg/L;线性范围0.02～24.78 μg/L;批内和批间相对标准偏差均小于10％.与其它结构类似物没有明显交叉反应.猪肉检测的平均回收率为87.4％～105.6％,与高效液相色谱方法的相关性良好.本方法具有较高的灵敏度和较好的稳定性.     

Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich Transfer Enzyme Immunoassay) for Antigens

Abstract

A novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens is described. Antigens were reacted with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates. The complex formed of antigens with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates was trapped onto affinity-purified rabbit (antidinitrophenyl bovine serum a1bumin) IgG-coated polystyrene balls. After eliminating excess of the conjugates, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. ß-D-Galactosidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed by fluorimetry. Nonspecifically bound ß-D-galactosidase activity considerably decreased with less decrease in specifically bound ß-D-galactosidase activity. As a result, the detection limits of human thyroid-stimulating hormone (0.01 nu, 0.02 amol) and human growth hormone (10 fg, 0.5 amol) by the present enzyme immunoassay were 30-fold lower than those by the conventional enzyme immunoassay, in which antigens were incubated with monoclonal mouse antibody IgG₁-coated polystyrene balls and rabbit antibody Fab′-ß-D-galactosidase conjugates.     

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。     

化学发光酶免疫分析测定鱼肉中呋喃它酮代谢物方法研究

建立了呋喃它酮代谢物5-吗啉甲基-3-氨基-2-6唑烷基酮(AMOZ)间接竞争化学发光酶免疫分析(icCLEIA)检测方法,通过单因素实验优化了包被原浓度、抗体稀释倍数、反应缓冲体系及浓度、竞争反应时间等参数,结果表明icCLEIA最佳反应条件为:包被抗原浓度为10 ng/mL,抗体稀释60000倍,最佳竞争时间为50 min,体系缓冲液0.01 mol/L PBS(pH 7.4).在优化的条件下,本方法的线性检测范围为0.026 ～3.52 μg/L,IC50为0.29 μg/L,检出限(LOD,IC10)为0.012 μg/L.对鱼肉样品的平均添加回收率在101.4％～115.5％之间.建立的icCLEIA方法可用于实际样品中AMOZ残留检测.     

Novel and Sensitive Noncompetitive Enzyme Immunoassay to Measure Peptides by Biotinylation

Abstract

A novel and sensitive noncompetitive enzyme immunoassay for angiotensin I as a peptide model is described. Angiotensin I in buffer containing bovine serum albumin or in plasma was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. the biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. the biotinylated angiotensin I eluted was reacted with anti-angiotensin I Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. the detection limit of angiotensin I was 13 fg (10 amol)/tube and 6.5 ng/1 of plasma, which was 10 to 480-fold lower than that previously reported by competitive radioimmunoassay and competitive enzyme immunoassay. and other peptides could also be measured more sensitively by the present method than by competitive radioimmunoassay.     

化学发光酶免疫分析法检测水产品中残留的麻醉剂丁香酚

将丁香酚(Eul)与4-溴丁酸叔丁酯进行衍生化反应,合成了半抗原4-(4-烯丙基-2-甲氧基-苯氧基)-丁酸(Eul-4-Tbl).通过偶联载体蛋白并免疫动物后获取丁香酚多克隆抗体,建立了丁香酚的间接竞争化学发光酶免疫分析方法(ic-CLEIA).水产样品经乙腈提取,Oasis PRiME HLB固相萃取小柱净化后用于...     

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…     

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.     

微板式磁化学发光酶免疫分析法对人绒毛膜促性腺激素(HCG)的灵敏快速测定

以磁性酶免疫测量分析为模型,建立了微板式磁化学发光酶免疫分析法.利用3-(2′-螺旋金刚烷)-4-甲氧基-4-(3″-磷酰氧基)苯-1,2-二氧杂环丁烷(AMPPD)-碱性磷酸酶(ALP)化学发光体系对人绒毛膜促性腺激素(HCG)进行测定,测量的灵敏度较分光光度法提高了13倍.测定的线性范围为0.15～500 mIU/mL;批内变异系数(C.V.%)和批间变异系数(C.V.%) 均在18%之内;回收率在80%～116%之间;利用本法对血清样品进行了测定,并与其它化学发光免疫分析方法进行了比较,其相关系数为0.965.首次将酶免疫分析方法用于唾液中人绒毛膜促性腺激素(HCG)的测定,为建立非侵入式样品测定方法打下了基础.     

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。     

增强化学发光体系酶联免疫分析法测定人绒毛膜促性腺激素

应用活化鲁米诺,用优化的增强化学发光酶联免疫分析体系测定人绒毛膜促性腺激素,检测限为0.2mIU/ml。线性范围0～200mIU/ml,与放射免分析测定结果比较,相关性良好。进而又发展了一种半定量的照相测定法,通过实际血清样品测定,效果良好。     

荧光偏振免疫分析方法分析磺胺二甲基嘧啶

建立了检测磺胺二甲基嘧啶的荧光偏振免疫分析方法。合成了3种结构不同的荧光标记物,并用薄层色谱法提纯。研究了不同结构的荧光标记物对FPIA方法灵敏度的影响。该FPIA方法在缓冲液中的检出限为1.6μg/L,半数抑制量(IC50)为41μg/L,检测范围为5~458μg/L,可以达到食品中SMZ最低残留限量的要求。研究了FPIA的动力学过程及对其它16种磺胺类药物的交叉反应,结果显示,SMZ、磺胺甲基嘧啶和磺胺二甲基恶唑的交叉反应率分别为100%、8.7%和2.4%,其它磺胺类药物的交叉反应率均低于1%。