时间分辨荧光免疫分析在兽药残留检测中的应用

近年来,兽药残留引起食物中毒的报道日益增多,兽药残留检测的意义重大。传统的气相色谱法、液相色谱法存在前处理复杂、仪器成本昂贵等缺陷,酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)灵敏度也不高,而时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)操作简便、灵敏度高,已在兽药残留检测领域引起重视。介绍了TRFIA的原理和优势,综述了其在促生长繁殖类、瘦肉增产类和杀菌驱虫类兽药残留检测中的应用,并与传统方法进行了对比,TRFIA有望取代传统的检测方法成为兽药残留检测的常规方法。     

赭曲霉毒素 A 的高灵敏时间分辨荧光免疫分析

采用时间分辨荧光免疫分析 (TRFIA) 技术建立快速的高灵敏度的赭曲霉毒素 A (OTA) 全自动检测方法 . 将 OTA-BSA 作为免疫分子免疫 Balb/c 小鼠, OTA-KLH 为筛选用抗原包被板,用间接酶联免疫分析 (ELISA) 法筛选出专一针对 OTA 的高效价的阳性克隆 3G9 ,用杂交瘤细胞制备抗 OTA 单克隆抗体 . 用 OTA-BSA 包被 96 孔板为固相抗原,与游离 OTA 共同竞争有限的抗 OTA 单克隆抗体,以稀土离子 Eu3+ 标记的羊抗鼠抗体进行示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立 OTA-TRFIA. 该方法的灵敏度为 0.03 μg /L ,测量范围为 0.03 ~1 000 μg /L ,批内和批间变异分别为 3.7% 和 5.3% ,平均回收率为 94.2% ,与赭曲霉毒素 B 的平均交叉反应为 3.7% ,与黄曲霉毒素 B1 、 牛血清白蛋白和苯基丙氨酸无交叉反应,说明抗体的特异性很好 . 8 条不同时间进行的间接竞争 OTA-TRFIA 的效应点均值 ED80 、 ED50 、 ED20 分别为 (0.33±0.02) μg /L、 (1.44 ±0.08) μg /L 和 (5.22 ± 0.12) μg /L ,说明方法的稳定性好,样品经 TRFIA 和 ELISA 试剂盒同时检测 OTA ,两者的相关系数为 0.925 ,结果相符 . 研究表明, OTA-TRFIA 是目前报道的 OTA 检测中最灵敏的方法,该分析方法稳定性好,可测范围宽,具有很好的应用前景 .     

IgG-Eu-IDPA对抗原IOV的时间分辨荧光免疫检测

镧系螯合物已经被广泛应用于高灵敏度的时间分辨荧光免疫分析.但是,使用紫外光激发镧系螯合物会对生物分子和细胞产生很大的损害.合成了能够被可见光(最大波长565nm)激发的IgG-Eu-IDPA,并测量了IgG-Eu-IDPA的光谱属性,如荧光寿命、在不同pH值和不同浓度环境下的荧光强度.在自制的时间分辨荧光仪上使用IgG-Eu-IDPA作为荧光探针,检测IOV抗原.数据显示,它的灵敏度远远高于传统的荧光仪.结果表明,IgG-Eu-IDPA能够在高灵敏度的原位和活体分析中作为一种新的、有潜力的荧光探针.     

荧光偏振技术在生命科学中的研究进展

荧光偏振技术(FP)作为一种新的检测技术已经广泛应用于生命科学的各个方面.本文对荧光偏振技术在生命科学中的研究进展做了详细的评述.介绍了荧光偏振技术的原理,评述了荧光偏振技术在生命科学中的研究热点方向.     

时间分辨荧光免疫分析技术研究现状及进展

时间分辨荧光免疫分析是一种新型的超微量的免疫标记分析方法,集酶标记免疫分析和放射免疫分析等优点于一身,且无放射性污染等.本文主要介绍了时间分辨荧光免疫分析的原理、优点,螯合剂的种类,以及各种分析技术及其应用现状和进展.     

橙色荧光蛋白的研究进展及其应用

荧光蛋白(Fluorescent protein,FPs)可作为探针用以探究细胞内分子间相互作用,追踪特定代谢物的代谢途径,对活细胞内的各种代谢过程和细胞通路进行详细、准确的描述。目前已有的FPs几乎已经覆盖了从紫外光到远红外光的所有光谱波段,这些FPs借助高分辨率显微技术应用于生命科学的诸多领域,为生物学的发展作出巨大贡献。橙色FPs通常指光谱区间在540–570nm的FPs,近几年来关于橙色FPs的研究进展较快,并且其作为标记蛋白以及荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET)中的荧光受体在生物学及医学领域得到较多的应用。文中综述了近15年橙色FPs领域的相关研究,重点聚焦橙色FPs的发展和应用,为今后橙色FPs的研究提供依据。     

荧光纳米生物传感器研究进展

荧光纳米生物传感器检测物质具有灵敏度高、响应迅速、抗干扰性强、无需参比电极等特点而被广泛地运用于生物传感技术领域。本文综述了荧光纳米生物传感器种类和特点,介绍了国内外近期在荧光纳米生物传感器及在生物检测方面的一些研究成果及进展,并作了分析比较。着重讨论了纳米粒子荧光生物传感器和光纤纳米荧光生物传感器的特性及其在生物分析中的应用。     

解离增强镧系元素荧光免疫分析灵敏度的改进

为提高解离增强镧系荧光免疫分析(DELFIA)的灵敏度或信／噪比,进行了一些重要的方法学研究．观察到了对于不同的铕量,荧光响应和信／噪比都随着增强液体积而明显地变化．对于确定的铕量,存在一个最佳体积,且铕量越小,其最佳体积也越小．实验中选择最佳体积是重要的．研究了增强液制备技术,发展了微滴定板条有效的清洗和干燥方法,使本底荧光明显降低．     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

C肽与胰岛素双标记时间分辨荧光免疫分析法的建立及初步临床应用

建立钐(Sm3+)标记检测C肽(C-peptide)及铕(Eu3+)标记检测胰岛素(insulin)的双标记时间分辨荧光免疫分析法(TRFIA),并初步尝试检测人血C肽以及胰岛素含量.将抗C肽单克隆抗体(Biodesign No.E54094M)与抗insulin单克隆抗体(BiodesignNo.E86306M)混合包被96孔板,然后用Sm3+标记抗C肽单克隆抗体(Medix No.9103),Eu3+标记抗insulin单克隆抗体(Biodesign No.E86802M),运用双抗体夹心一步法建立C肽/胰岛素双标记时间分辨免疫荧光分析法.结果显示:C肽分析灵敏度为0.2μg/L,线性范围为0.5～22μg/L,平均回收率达到99.6%,分析内和分析间变异系数分别为4.6%～6.0%和5.1%～7.6%.胰岛素分析灵敏度为0.8 mU/L,线性范围为3.6～180 mU/L,平均回收率为99.4%,分析内和分析间变异系数分别为3.7%～6.0%和5.1%～8.0%.C肽/胰岛素双标记检测试剂与PerkinElmer公司对应的单标记进口试剂盒分别同时测定血清样本200份,检测结果高度相关,具有较好的一致性,相关系数分别为0.98与0.99.总之,自建C肽/胰岛素双标记时间分辨荧光免疫分析方法的性能均可以达到临床检测要求,有望替代现有国内外较为昂贵的单标记试剂,可用于胰岛素分泌不足导致的糖尿病诊断及糖尿病的大规模普查筛选.     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.     

时间分辨荧光免疫分析及其在临床检测中的应用

本文介绍了时间分辨荧光免疫分析法的检测原理、检测方法,分析了时间分辨荧光免疫分析仪的结构并介绍了其在临床检测方面的应用。     

时间分辨荧光免疫分析仪的临床实验方法和评价方案

时间分辨荧光免疫分析技术是一种利用稀土离子及其螯合物作为示踪剂的灵敏度高、线性范围宽、应用范围广的非放射性标记免疫分析技术。研制了一种性能稳定的时间分辨荧光免疫分析仪,为了进一步将这一先进超微量物质检验技术推广于临床应用,根据美国国家临床实验室标准化委员会（NCCLS）制订的临床评价方案,选择放射免疫分析法、化学发光免疫分析法以及Perkin Eimer Life Sciences公司的Auto DFLFIA-1235全自动时间分辨荧光免疫分析仪作为比较方法,提出了三套检验时间分辨荧光免疫分析仪性能的临床实验方法和评价方案。并利用其中的一套方案进行了实验,结果表明这些方案可操作性强,结果可信,经济适用,可作为同类医疗检验仪器进行临床实验的参考。     

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20–1000 pg mL⁻¹, and its limit of quantitation was 20 pg mL⁻¹. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.     

Fluorescence polarization immunoassay of ractopamine

A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopamine–fluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.3–50 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.     

Comparison of high-performance liquid chromatography with fluorescence polarization immunoassay for the analysis of vancomycin in patients with chronic renal failure

Eighty-two plasma samples from patients with chronic renal failure undergoing vancomycin treatment and hemodialysis (HD) were analyzed with fluorescence polarization immunoassay (FPIA) and high-performance liquid chromatography (HPLC). Vancomycin was infused once and the samples were collected during three subsequent HD sessions at 2 h, 3 days and 5 days post-infusion. The HPLC method, modified from an earlier assay, was simple. There was a wide variation in the estimated concentration between the two assay methods. The results obtained by HPLC were 69% lower than those obtained by FPIA. This difference in vancomycin concentration was independent of the sampling time after vancomycin infusion. HPLC analysis commenced approximately 1.5 year after that of FPIA. To study the effect of in vitro degradation, the vancomycin concentration in ten of the samples was redetermined with FPIA during HPLC analysis. The concentrations of those samples decreased to 78–98% (average 92%) of the original concentration. Because FPIA appears to lack specificity, there is a need of other methods such as HPLC for vancomycin measurements, particularly in samples from patients with end-stage renal failure.     

Development of a time‐resolved fluorescence immunoassay for herpes simplex virus type 1 and type 2 IgG antibodies

Enzyme‐linked immunosorbent assays (ELISA) specific for anti‐HSV glycoprotein G (gG) are most commonly used in the clinical diagnosis of HSV infection. But most of them are qualitative and with narrow detection ranges. A novel time‐resolved fluoroimmunoassay (TRFIA) methodology was developed for the quantitative determination of HSV IgG in human serum. The assay was based on an indirect immunoassay format, and performed in 96‐well microtiter plates. HSV‐1 and HSV‐2 were used as the coating antigens. Eu³⁺‐labeled goat anti‐(human IgG) polyclonal antibodies were used as tracers. The fluorescence intensity of each well was measured and serum HSV IgG levels quantified against a calibration curve. The detection range of the novel TRFIA was between 5 and 500 AU/mL. Assay sensitivity was 0.568 AU/mL. The intra‐ and inter‐assay coefficients of variation were 0.59–3.63% and 3.65–6.81%, respectively. Analytical recovery, dilution tests and serum panel tests were performed using TRFIA and the results proved satisfactory. There were no statistically significant differences in sensitivity and specificity between the TRFIA and commercial ELISAs. An effective, sensitive and accurate quantitative HSV type 1 and type 2 IgG TRFIA was successfully developed and provided diagnostic value in clinical use. Copyright © 2014 John Wiley & Sons, Ltd.     

快速同时检测玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇的研究

目的：利用稀土离子作为示踪剂,建立DON/ZEN双标记间接竞争时间分辨荧光免疫分析方法同时检测DON、ZEN。 方法：以DON BSA、ZEN-BSA共包被于固相微孔板,与DON/ZEN标准或样品中的DON、ZEN竞争结合抗DON多抗、抗ZEN单抗,然后分别用稀土离子Eu3+-羊抗兔IgG及Sm3+ 羊抗鼠IgG进行示踪检测,并对建立DON/ZEN-双标记TRFIA进行方法学的考核。结果：DON/ZEN-双标记TRFIA检测灵敏度,DON为0.2 ng/ml、ZEN为0.7 ng/ml,检测范围为：DON 0.2~100 ng/ml,ZEN 0.7~50 ng/ml,批内、批间变异率均小于10%。不同样品添加回收实验表明玉米、小麦样品中DON平均回收率分别为102.8%、98.8%,ZEN平均回收率分别为94.2%、95.7%。DON/ZEN-双标TRFIA检测时,DON与ZEN不相互干扰,该方法特异性好。玉米样品检测结果表明,DON/ZEN双标记TRFIA与单标记DON -TRFIA、ZEN-TRFIA试剂盒结果高度相关,具有较好的一致性,两者检测DON的结果相关系数为0.9760,检测ZEN结果的相关系数为0.9695,结论：DON/ZEN-双标记TRFIA灵敏度高,检测范围宽,重复性、稳定性好,一次检测可同时得到DON、ZEN两个结果,是一种简便、快速、经济、稳定、可进行大批量样品筛查的检测方法。     

A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 microl of reagents and sample, and is applicable to a wide range of analytes.     

Interference of 7-hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays

The accuracy of two clinical assays, the enzyme-multiplied immunoassay (EMIT) and fluorescence polarization immunoassay (FPIA2), universally employed for measurement of plasma levels of methotrexate (MTX) in children administered a high dose of this drug for treatment of acute lymphoblastic leukemia was evaluated here. Because of its superior specificity, sensitivity, and precision, high performance liquid chromatography (HPLC) was selected as the reference method with which the other two procedures were compared using approximately 420 different plasma samples for method comparison. 7-Hydroxymethotrexate (7-OHMTX), the major plasma metabolite of MTX, that can be detected in plasma at relatively high concentrations for long periods following infusion of a high dose of MTX, was also quantitated by HPLC. Forty-two and 66 h after infusion, the plasma level of MTX was overestimated in 2% and 3% of the samples by the FPIA2 procedure in 5% and 31% by the EMIT assay. The overall correlation coefficients (r2) for the values obtained by FPIA2 or EMIT versus those based on HPLC were 0.989 and 0.663, respectively. The presence of 7-OHMTX exerted a highly significant influence (p=0.0007 as determined by the unpaired t-test) on MTX measurement by the EMIT assay. We conclude that the rapid automated procedures routinely used at present and in particular EMIT, suffer from cross-reactivity with metabolites of MTX. Thus, the relatively high percentage of samples in which the level of MTX is overestimated at check-points by EMIT may result in longer periods of hospitalization, higher costs and prolonged administration of elevated doses of "rescue" leucovorin with an increased risk for relapse.