化学发光酶联免疫分析测定血清中抗DNA抗体

建立起适于临床应用的抗ＤＮＡ抗体化学发光酶联免疫分析法。该法精密度良好，相对标准偏差为２．４％。比ＥＬＩＳＡ法更加简便、经济、省时，同时提高了灵敏度（８倍）和血清的阳性检出率。探讨了检测系统中对碘苯酚增强鲁米诺－过氧化氢－辣根过氧化物酶化学发光反应机理。     

化学发光单孵多层免疫技术检测粪样中轮状病毒

以酶联免疫吸附分析（ＥＬＩＳＡ）夹心测定粪样中轮状病毒（ＲＶ）实验为模型，将传统又孵式免疫操作变成单孵式，即在包被ＲＶ抗体Ａｂ的微孔中同时加入含ＲＶ的粪样上清液和根过氧化物酶（ＨＲＰ）标记抗ＲＶ抗原共同孵育，其结果在载体表面形成多层酶免疫复合物，以ＨＲＰ催化鲁米诺－对碘苯酚－过氧化氢化学体系作为最终信号检测系统，从而建立起化学发光单孵多层免疫技术（ＣＬ－ＳＩＭＩＴ）。其灵敏度是ＥＬＩＳＡ的１６倍，     

麻疹病毒抗体的酶免疫电化学分析法研究

建立了测定麻疹病毒抗体的酶免疫电化学分析法。其原理是在异相酶联免疫之后用极谱法检测酶促产物。本法采用小型三电极系统“ＤＭＥ－Ｐｔ－Ａｇ／ＡｇＣｌ”直接与商品酶联板配套使用，简化了操作步骤。对测定麻疹抗体的合适条件以及灵敏度，特异性等作了考察。结果表明，本法特异性强，灵敏度高，检测范围宽，不受样品颜色、浊度、杂散光及酶联板自身光吸收不均一的影响；应用于测定三批共１１８份血清样品，阳性率分别为１５％（６／４０），６２．５％（２５／４０）和３１．５８％（１２／３８），用４６份样品对本法和ＥＬＩＳＡ作相关性检验，其相关性良好（γ＝０．７７．ｔ＿ｒ＝１０．８４，Ｐ＜０．００１，相关显著）。     

化学发光磁酶免疫法检测血清游离hCGβ

采用高灵敏度、信号稳定的化学发光分析法对血清中游离的人绒毛膜促性腺激素β亚单位 （Free hCGβ）进行检测, 并对免疫分析进行条件优化和改进, 建立化学发光磁酶免疫检测方法. 利用3-（2′-螺旋金刚烷）-4-甲氧基-4-（3″-邻氧酰苯基）-1, 2-二氧杂环丁烷（AMPPD）-碱性磷酸酶（ALP）化学发光体系, 用磁珠酶联免疫法对Free hCGβ进行检测, 其检测灵敏度较光度法提高了10倍, 线性范围为0.45～185.2 mIU/mL, 批间和批内相对标准偏差（RSD）均小于12%, 回收率为84%～120%之间, 临床标本的检测值与分光光度法的相关系数为0.955. 因此, 化学发光磁酶免疫分析法较分光光度法的精密度和灵敏度高, 可以作为检测血清中的Free hCGβ水平的一种可靠方法.     

ELISA法和化学发光法对血清中HIV-1HIV-2抗体、梅毒抗体和丙肝抗体的检测意义

目的探讨分析ELLSA法(酶联免疫吸附法)和化学发光法对血清中HIV-1/HIV-2抗体、梅毒抗体和丙抗体的临床检测意义。方法将382份脐血作为研究分析对象,分别采用酶联免疫吸附和化学发光法对所选标本进行检测,包含丙肝抗体、梅毒抗体、HIV-1/HIV-2抗体检测。结果采用ELLSA检测后HIV-1/HIV-2抗体、梅毒抗体、丙肝抗体阳性率分别为0.52%、0.79%、1.05%。采用化学发光法检测HIV-1/HIV-2抗体、梅毒抗体、丙肝抗体3种检测的阳性率分别为0.79%、1.05%、1.31%。标准抗体品梯度稀释后再进行检测,将此3种抗体稀释程度为10 pg/m L,三组抗体采用化学发光法进行检测,其结果均为阳性,采用酶联免疫吸附方式进行检测的只有梅毒抗体呈阳性,其余两项均为阴性。结论化学发光法作为临床进行血清中HIV-1/HIV-2抗体、梅毒抗体、丙肝抗体检测的首选方案,此检测方式具有更高的灵敏性。     

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…     

神经生长因子的化学发光标记与检测

以辣根过氧化物酶（ＨＲＰ）和吖啶酯（ＡＥ）为化学发光标记试剂分别标记神经生长因子（ＮＧＦ）单克隆抗体，经分离纯化制成标记抗体（ＨＲＰ－Ａｂ，ＡＥ－Ａｂ），采用化学发光免疫分析法（ＣＬＩＡ）对ＮＧＦ进行检测，其检出限为０．５ｎｇ／ｍＬ，线性范围为２～１２８ｎｇ／ｍＬ．１０例样本分别用ＣＬＩＡ和ＲＩＡ进行检测，其结果无显著性差异．     

4-(1H-三氮唑)苯酚增强化学发光酶联免疫分析法检测甲胎蛋白

增强化学发光酶联免疫分析法除具有化学发光信号持续稳定时间长、发光强度高、灵敏度高等优点外,而且简便、快速、安全、应用面广,克服了直接化学发光用于免疫分析的缺点,提高了化学发光免疫分析的灵敏度~([1]).     

红细胞标记抗体的化学发光免疫分析

用红细胞代替辣根过氧化物酶作为双抗体夹心免疫分析中第二抗体的标记物, 建立了一种红细胞标记抗体的免疫化学发光测定乙型肝炎病毒表面抗原的新方法. 在免疫反应完成后, 结合了抗原-抗体免疫复合物的致敏红细胞在低渗溶液中溶血, 释放出血红蛋白. 基于血红蛋白对鲁米诺-H2O2体系化学发光具有催化作用的原理, 采用化学发光法测定血红蛋白含量. 测得的血红蛋白发光强度与待测抗原浓度呈线性关系. 采用这种方法可检测出0.5 ng/mL的乙型肝炎病毒表面抗原. 将该方法与酶联免疫吸附分析(ELISA)结合起来对乙型肝炎患者血清乙肝病毒表面抗原(HBsAg)进行检测, 两者符合率均为97%, 表明本法具有良好的灵敏度和特异性, 可用于临床标本测试.     

农药环境内分泌干扰物的免疫分析

综述了环境内分泌干扰物中农药抗原、抗体制备技术、放射免疫、酶免疫、荧光免疫、化学发光免疫技术和免疫传感器现状及其免疫分析研究发展趋势.     

Development of an Indirect Chemiluminescent Competitive ELISA to Detect Danofloxacin Residues in Milk

In this study, a sensitive and specific indirect chemiluminescent enzyme-linked immunosorbance assay method (CL-ELISA) was developed to detect danofloxacin residue in milk. The influence of experimental factors on the results of the method was investigated, including the concentration of coating antigen and primary antibody, the dilution factor of the IgG-HRP antibody, as well as the type of microtiter plate. Under the optimized condition, the linear working range and IC₅₀ value were determined to be 0.025–0.5 ng · mL^?1 and 0.1 ng · mL^?1, respectively. Compared with traditional colorimetric ELISA, the CL-ELISA developed in this research demonstrated significant improvement in terms of sensitivity (20 times improvement) and linear range (25 times wider). The developed method was applied in detection of danofloxacin residue in milk and satisfied recovery rates of 92.6–105.2% and coefficient variations of 2.4–9.5% were obtained, demonstrating that the proposed method was accurate and reliable. Moreover, there was almost no any meaningful cross-reactivity with nine other (fluoro)quinolones, indicating its high specificity.     

Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection

To identify the explosive used in a terrorist attack, or to obtain an early sign of environmental pollution it is important to use simple and rapid assays able to detect analytes at low levels, possibly on-site. This is particularly true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because of its toxicity, a well known pollutant. In this work we describe the development of an indirect competitive ELISA with chemiluminescent detection (CL-ELISA) and of a lateral-flow immunoassay (LFIA) based on colloidal gold nanoparticle labels. A commercially available monoclonal antibody was used and 13 specially synthesized conjugates were tested. We optimized the assay by determining the optimal concentration of monoclonal antibody and conjugates and the influence of various non-specific factors such as: tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The sensitivity and reproducibility of the CL-ELISA were good (LOD and IC₅₀ values in the ng mL⁻¹ range, and CV value about 7%). It has been applied to real samples of various materials involved in a controlled explosion of an “improvised explosive device”. Three extraction procedures were tested on these samples, all employing methanol as the solvent. The lateral flow immunoassay (LFIA), developed by using the same immunoreagents, reached a detection limit of 1 μg mL⁻¹ when tested on the same samples analysed by CL-ELISA.      

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.     

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.     

Determination of aculeatisides based on immunoassay using a polyclonal antibody against aculeatiside A

An enzyme-linked immunosorbent assay (ELISA) was developed for determination of aculeatisides. Aculeatiside A was conjugated with bovine serum albumin (BSA) for immunization. The ratio of hapten in an antigen conjugate was determined by matrix-assisted laser desorption/ionization TOF mass spectrometry. Polyclonal antibody was developed in rabbits against an aculeatiside A-BSA conjugate. The antibody was specific for aculeatiside A and aculeatiside B. The range of the immunoassay extended from 100 ng ml(-1) to 5 pg ml(-1) of aculeatisides. Good correlation between ELISA and HPLC methods was obtained when crude extracts of plant samples were analyzed. The optimized ELISA was found to be applicable to the determination of total aculeatisides in various plant 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.     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate: application to enzyme-linked immunosorbent assay for urinary and serum cortisol.

Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype gamma1, kappa) showed an affinity constant (K(a)) for cortisol of 1 x 10(9) M(-1) and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17alpha-hydroxyprogesterone (12%), 6beta-hydroxycortisol (8.4%), and tetrahydrocortisol (< 0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 +/- 17.0 microg/day (n = 7) and 80.8 +/- 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.