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。     

固相时间分辨荧光免疫螯合剂 BCPDA 的制备及其标记技术

报道了固相时间分辨荧光免疫螫合剂4,7-二氯磺基苯-1,10-菲罗啉-2,9-二羧酸(BCPDA)的制备方法,BCPDA 标记蛋白质及螫合 Eu³⁺方法,BCPDA-Eu³⁺标记物荧光光谱研究以及固相时间分辨荧光免疫分析法检测甲胎蛋白异质体(AFP-R-LCA)方法的建立.结果表明 BCPDA 能在温合条件下与蛋白质氨基结合并与 Eu³⁺螯合,BCPDA-Eu³⁺蛋白质标记物荧光特性、标记比度、生物结合活性与国外同类产品一致,所建立的检测 AFP-R-LCA 免疫分析法最小检测值0.6ng/ml,为提高我国非放射性同位素标记技术水平奠定了基础.     

流式微球一步法快速免疫检测马铃薯A病毒

[目的]建立流式微球一步法快速免疫检测马铃薯A病毒(PVA)的新方法.[方法]以荧光微球为反应载体,通过在微球表面进行双抗夹心免疫反应形成微球-捕获抗体-PVA-标记FITC检测抗体的复合物,利用流式细胞仪荧光检测系统收集荧光信号.[结果]通过实验优化检测条件,最佳捕获抗体工作浓度为4μg/mL、最佳检测抗体工作浓度为1:25倍稀释、最佳反应时间为2h;与马铃薯Y病毒、莴苣花叶病毒、番茄环斑病毒等均未出现交叉反应;阳性样品经64倍稀释后依然可检出,检测灵敏度是传统微孔板ELISA的4倍.[结论]流式微球一步法能灵敏、快速、简便的检测马铃薯A病毒.     

CA19-9时间分辨荧光免疫层析检测方法的建立

本研究旨在建立一种定量检测血清中CA19-9含量的时间分辨荧光免疫层析检测方法。采用双抗体夹心法与荧光免疫层析技术,以羧基荧光微球和NC膜为载体将CA19-9配对抗体进行标记和包被,制备CA19-9检测试纸条。通过标记、包被抗体量对工艺进行优化,并通过线性范围、最低检出限、精密性等性能指标对CA19-9时间分辨荧光层析检测方法进行评价。最终确定20μL荧光微球的标记抗体量为80μg,检测线包被抗体浓度为1.5 mg/mL时,检测时间为15 min,线性范围为12.5–800 U/mL,最低检出限为6.32 U/mL,批内精密性与批间精密性均小于15%,平均回收率为101%,与罗氏电化学发光检测试剂盒平行检测50份临床样本,两者相关系数为0.980 6。初步建立了定量检测血清中CA19-9的荧光免疫层析检测方法,有较好的临床应用前景。     

重组杆状病毒滴度免疫荧光法的建立及验证

目的 建立重组杆状病毒滴度间接免疫荧光法的检测方法,并对其进行验证。方法 构建含有重组质粒pGEX-6p-1-GST-GP64大肠埃希菌BL21(DE3),进行IPTG(isopropyl-β-D-thiogalactoside, IPTG)诱导,表达并纯化杆状病毒截短GP64糖蛋白。配伍佐剂后采用背部多点注射的方式免疫日本大耳白兔,制备兔抗杆状病毒GP64多抗作为免疫荧光结合抗体,在96孔板里贴壁培养Sf9细胞,接种10倍系列稀释的重组杆状病毒共孵育一定时间,80%冷丙酮固定、透化,一抗稀释比例为1∶200、荧光二抗稀释比例为1∶500,荧光显微镜下显色,计数荧光灶数目计算病毒滴度。并对建立的方法进行验证。结果 成功制备了GP64兔多抗;检测时Sf9细胞与重组杆状病毒共孵育时间4 d;重组杆状病毒感染细胞可与GP64抗体发生特异的荧光灶反应,未感染细胞对照组未出现特异性荧光灶;组内和组间测定结果的RSD均<5%;同一样品不同人员检测差异无统计学意义(F=1.86,F表);同一样品梯度稀释后,稀释倍数的对数与病毒滴度呈线性相关(R²     

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。     

小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法的建立

本研究旨在建立小反刍兽疫病毒H蛋白抗体的化学发光免疫分析检测方法。以H蛋白4个反应原性较好的B细胞表位串联后为检测抗原,在确定抗原包被量和血清稀释度,优化血清和酶标二抗反应时间的基础上,用ROC曲线分析确定检测临界值,建立了小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法,然后对该方法进行敏感性、特异性和重复性评价。结果显示,建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法最佳抗原包被浓度为1.5×10^-6μg/孔,待检血清最佳稀释度为1∶100;血清及酶标二抗孵育时间均为10 min,检测方法的临界值为S/P=9.77%,批内及批间变异系数均小于10%;与口蹄疫、羊痘、蓝舌病及山羊传染性胸膜肺炎阳性血清不发生交叉反应;用建立的化学发光法和市售小反刍兽疫抗体cELISA试剂盒平行检测247份田间血清,两者相对符合率为94.33%,但化学发光法敏感性更高。研究结果表明,建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析方法灵敏、特异、稳定,可用于田间血清样品小反刍兽疫抗体检测。     

碱性磷酸酶标记链霉亲和素

碱性磷酸酶标记链霉亲和素（AP-SA）是酶放大时间分辨荧光免疫分析（EATRFIA）通用的、最关键的试剂.报告了AP-SA的戊二醛二步标记方法.用自行研制的荧光发展溶液和AP的底物5-氟水杨酸磷酸酯测定了标记物AP-SA的特性,AP的标记回收率为38.7%;AP-SA稀释200～12 800倍时,稀释倍数和Tb的信/噪比呈线性关系;至少在两个月内,AP-SA的酶活性是稳定的.     

静注人免疫球蛋白中IgG含量检测方法的探讨

为了更好地进行生产质量控制,快速、准确的测定静注人免疫球蛋白中的IgG含量。实验中对2001~2008年的静注人免疫球蛋白中的IgG含量检测结果进行了抽样统计分析。分析结果表明:样品40倍稀释后测得的吸光值与标准曲线第三点的吸光值无显著性差异;样品40倍稀释后测得的IgG含量与样品进行20、30、40倍三个稀释度测得的IgG含量的平均值无显著性差异。从而证明,样品40倍稀释后测得的IgG含量可以代表该批样品的IgG含量,无需进行20、30倍的稀释。检测方法会更省时、省力、省材料。     

细胞骨架蛋白4荧光免疫层析检测方法的建立

采用基于免疫层析技术与荧光微球标记技术相结合的方式,建立一种快速、简单的定量检测肝癌肿瘤标记物的方法。依据双抗体夹心原理,将细胞骨架蛋白4(CKAP4)配对抗体分别作为标记与包被抗体,羊抗兔多抗作为质控线包被抗体,制备CKAP4荧光免疫层析试纸条,并对试纸条的线性、精密性、稳定性等各项性能指标进行评价。结果表明,采用时间分辨荧光微球所制备的CKAP4免疫层析试纸条灵敏度高,特异性好,精密性在15%以内,回收率在85%–115%之间,线性范围为25–1 000 pg/mL,可在37℃稳定保存20 d,与商业化的ELISA试剂盒的相关性良好。结论:初步建立了CKAP4荧光免疫层析方法,能够定量检测血清中CKAP4的含量,且具有快速、灵敏、简便、经济、可单人份操作等优点,有望成为肝癌辅助诊疗的新方法。     

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。     

Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix)

The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu³⁺-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 μg/mL for standard SSa sample with a detection limit of 0.006 μg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.     

Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels

Based on a novel cocoating strategy and dissociation enhancement lanthanide fluorescence immunoassay technique, a sensitive time-resolved fluoroimmunoassay (TRFIA) has been developed for simultaneous quantification of human serum thyroid-stimulating hormone (TSH) and thyroxin (T4) in a one-and-the-same assay procedure. The new cocoating strategy for preparing highly active surface anti-TSH and anti-T4 monoclonal antibodies (McAbs) was performed by a three-step protocol. Namely, anti-TSH McAb at high concentration (10 micro g/ml) and extensively biotinylated bovine serum albumin (BSA) at low concentration (0.5 micro g/ml) were coated on microwells by passive adsorption, then streptavidin was captured by the surface BSA-biotin, and finally biotinylated anti-T4 McAb was immobilized by the remnant binding sites of the bound streptavidin. In the present TSH/T4 TRFIA, both sandwich- and competitive-type configurations were involved, and Eu(3+) and Sm(3+) were used as labels for TSH and T4 detection, respectively. The method showed rapid kinetics; the equilibrium was reached within 30min at 37 degrees C due to the use of high concentrations of reaction reagents, rapid agitation, and small reaction volume. The lower limits of detection of the method were 0.028 mIU/L for TSH and 4.1 nmol/L for T4 with 20 micro L of sample volume. The assay ranges for TSH and T4 were 0.21-80.00 mIU/L and 20-300 nmol/L, respectively. The correlation between the TSH/T4 values obtained by the present TSH/T4 TRFIA and those obtained by commercial chemiluminescence immunoassay was satisfactory.     

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。     

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.     

现代荧光免疫分析技术应用及其新发展

免疫分析作为一类特殊的试剂分析技术,已经被广泛应用于多个不同领域。作为免疫分析方法家族中的一员,现代荧光免疫分析技术在相关领域里也扮演了重要角色。现代荧光免疫分析技术主要包括荧光偏振免疫分析(FPIA)和时间分辩荧光免疫分析(TRFIA)两种技术。本文从原理角度出发,综述了FPIA和TRFIA近年来在分析领域中的应用,并且阐述了二者的一些最新发展动态。     

Detection of Escherichia coli in blood using flow cytometry

A rapid method for the detection of Escherichia coli in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coli/ml and from in vivo rabbit model studies at less than 100 E. coli/ml.     

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.     

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.