应用单克隆抗体及时间分辨荧光免疫检测抗HBc、HBsAg的研究

时间分辨荧光免疫测定技术(TrFlATime-Resolved Fluoroimmunoassay),是一种新型非放射配基结合的超微量免疫测定技术。采用稀土元素铕(Eu~(3+))标记乙型肝炎病毒抗原或抗体,与被检物生成免疫复合物,在专用时间分辨荧光比色计上进行测定。完全排除了短衰变时间的非特异荧光物质的干扰,又加之使用增强液,最大限度地提高了检测的灵敏度。标记物制备简便,稳定性好,无放射性污染,便于组装试剂盒。1983年Siitari H等首次报告此项技术用于检测HBsAg。结合国内条件,本文采用传染病     

癌胚抗原时间分辨荧光免疫测定法

本文报告了癌胚抗原时间分辨荧光免疫测定法。采用McAb包被微量滴定条,以双功能基团螯合剂标记Eu~(3+)为示踪物。本法最小检出值为0.7ng/ml,标准曲线范围为2.5～500ng/ml,批内和批间CV平均为7.09％和10.56％,回收率为106.00％。测定方法简便快速,每天一个技术人员可完成300份样品。     

时间分辨荧光免疫分析方法建立中有关问题的探讨

本文报告了时间分辨荧光免疫分析技术建立中有关问题的实验研究结果。包括对Eu~(3+)标记技术的改进,增强液的配制和检定,抗体包被固相材料的选择及操作中必须注意的防止稀土元素污染的措施。     

固相时间分辨荧光免疫分析的标记技术及标记过程中的蛋白质测定

利用新型双功能螯合剂-4,7-二氯磺酰基苯基-1,10-菲啰啉-2,9-二羧酸（BCPDA）进行固相时间分辨荧光免疫分析（TRFIA）标记技术研究,结合标记牛血清白蛋白（BSA）实验,研究测定蛋白质方法,监测标记过程,计算回收率、标记比。确定考马斯亮蓝染色法为该体系标记研究中测定蛋白质含量方法。     

时间分辨荧光分析测定细胞活性和破膜法实验研究

采用Eu3+-DTPA标记L-929株靶细胞，经时间分辨荧光仪测定，建立一种崭新的时间分辨荧光分析法来测定细胞活性，此法具有灵敏度高，有效期长，不污染环境等优点。     

β2微球蛋白时间分辨荧光免疫检测法的建立及初步应用

为了建立β2微球蛋白(β2-m)时间分辨荧光免疫分析。以羊抗兔抗体包被板条,双功能螯合剂异氰酸苄基二乙烯三胺五乙酸络合Eu3+及标记β2-m,发光增强系统为以β二酮体为主的增强液。采用竞争法建立β2-m时间分辨荧光免疫分析,数据采用双对数法函数和四参数logistic函数数据处理程序处理。结果此法的批内和批间CV分别为2.25%和2.91%,平均回收率为101.06%,灵敏度为0.06mg/L,可测范围为0.06～12mg/L。本方法与白蛋白、肌红蛋白均无交叉反应。溶血对本法无影响。临床初步应用所检测结果与放免法吻合。试验结果表明,β2-m-TRFIA法的敏感度、特异性、准确度等指标符合临床诊断要求。     

Anytest-2000时间分辨荧光分析仪介绍

时间分辨荧光免疫分析方法(TRFIA)是一种先进的免疫检验新技术,TRFIA方法是20世纪80年代中期发展起来的一种新的荧光标记技术,它与传统的FIA、ELISA和RIA相比,具有很多优点：灵敏度高、特异性强、稳定性好、动态范围宽、试剂货架期长、无放射性危害等,迄今已被国际上公认为现代标记免疫最佳方法之一。本文介绍Anytest-2000时间分辨荧光分析仪(TRF仪)。     

血清胰岛素TRFIA的建立

目的 建立血清胰岛素( Ins)时间分辨荧光免疫分析法(time-resolved fluoroimmunoassay,TRFIA).方法 采用一株Ins-MAb包被于固相微孔板上,另一株Ins-MAb用DTTA- Eu3+标记作为示踪剂采用Victor 1420荧光仪测定,.并对本法进行评价.结果 本法的标准曲线范围...     

胃蛋白酶原Ⅰ时间分辨荧光免疫分析法的建立

目的　采用时间分辨荧光免疫分析技术建立高灵敏的胃蛋白酶原Ⅰ的快速全自动检测方法。方法　以PGⅠ单克隆抗体 80 0 3#包被板 ,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu3 及标记PGⅠ单克隆抗体 80 16 # ,发光增强系统为以 β 二酮体为主的增强液。采用平衡饱和法建立PGⅠ时间分辨荧光免疫分析 ,数据采用Log Logit法函数和四参数Logitc函数数据处理程序处理。结果　方法的批内和批间CV分别为 1.9%和 4 .7% ,平均回收率为 10 2 .6 5 % ,灵敏度为 0 .0 5 μg L ,可测范围为 3.5～ 32 8μg L ,ED2 0 、ED50 和ED80 分别为 11.34μg L、38.73μg L和 132 .3μg L。Eu3 标记抗体 2 0℃保存 8个月免疫反应性基本无损失 ,同批试剂连续 8个月应用分析结果稳定 ,临床结果与免疫放射分析法的结果相符。结论　胃蛋白酶原Ⅰ时间分辨荧光免疫分析法是目前胃蛋白酶原Ⅰ检测中最灵敏的方法 ,该分析法稳定性好 ,具有很好的应用前景。     

基于磁性微球AFP时间分辨荧光免疫分析法的建立

目的 建立基于磁性微球(磁珠)AFP时间分辨荧光免疫分析法.方法 用磁珠偶联标记抗AFP单克隆抗体(301M),Eu3标记抗AFP单克隆抗体(304M),采用双抗体夹心法建立基于磁性微球法的AFP-TRFIA,并对106名我院健康体检人员和住院患者进行该方法血清学检测.结果 其检测灵敏度为0.02 IU/mL,线性范围2.12～ 1210 IU/mL;癌胚抗原(CEA)、CA199、CAl25对AFP-TRFIA均无交叉反应.磁珠偶联301M 4℃保存3个月免疫反应性基本无损,而Eu3+标记304M于-20℃下保存6个月免疫反应性也基本无损;同批试剂连续3个月应用分析结果稳定,样品的平均添加回收率为98.75％,低、中、高质控的批内和CV分别为20.46IU/mL和4.75％,50.58 IU/mL和4.25％,120.51 IU/mL和2.89％.批间和CCV分别为19.54IU/mL和9.55％.46.32 IU/mL和7.38％,125.10 IU /mL和3.69％.血清AFP检测结果与传统时间分辨荧光法检测结果对比,结果高度相关,相关系数为0.976.结论 基于磁性微球建立的AFP时间分辨荧光免疫分析法敏感度、特异性、准确度等均符合临床应用要求,可用于临床血清标本中AFP含量的免疫测定.     

Time-resolved fluorescence using a europium chelate of 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labeling procedures and applications in immunoassays

We describe optimal conditions for protein labeling with a new fluorescent probe, 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The labeled proteins are suitable for time-resolved fluorometric applications because BCPDA forms fluorescent complexes with Eu3+ which exhibit very long fluorescence lifetimes. Labeling parameters such as the organic solvent used, pH, protein concentration, BCPDA excess and incubation times, were optimized accordingly. Excess BCPDA was removed by gel filtration and labeled proteins were characterized by absorbance and fluorescence measurements. The effect of labeling on the biological (binding) activity of the proteins streptavidin, avidin, monoclonal and polyclonal antibodies was also studied. It is shown that the labeled antibodies can be used for time-resolved fluoroimmunoassay applications.     

Determination of cytotoxic T-lymphocyte precursor frequencies using europium labeling as a nonradioactive alternative to labeling with chromium-51.

We report on the use of europium (Eu) as a suitable nonradioactive alternative for target cell labeling in limiting dilution analysis (LDA) assays set up to determine cytotoxic T-lymphocyte precursor (CTLp) frequencies. A nonradioactive alternative to the commonly used chromium-51 (51Cr) release assay seems desirable because working with radioisotopes has some major disadvantages concerning possible health risks, environmental load, costs of facilities necessary for working with radioisotopes, and shelf life. Some groups have successfully applied the Eu release assay based on detection by time-resolved fluorometry, to tests in which NK- or LAK-cell activity or cytotoxic T-lymphocyte reactions were measured. This led to the investigation whether this method could also be applicable to the more specific determination of CTLp frequencies in LDA assays. After optimal labeling conditions had been established, the sensitivity of the Eu release assay was determined by performing several LDA assays in which the target cells were labeled with either Eu or radioactive 51Cr. When CTLp frequencies were compared, it was shown that the Eu release assay is at least as sensitive and specific as the 51Cr release assay. Moreover, although the labeling procedure takes longer, sample processing is much faster: only 1 second per sample. The fact that the Eu release assay is not radioactive enables the assay to be performed at any laboratory and even--because the frequency of CTLps may have implications for organ graft survival and for donor selection in bone marrow transplantation--to do so on a routine basis.     

Preparation of europium-streptavidin in a time-resolved fluoroimmunoassay for interleukin-3

The preparation and immunoassay performance of europium-labeled streptavidin is described. The Eu-streptavidin conjugate can serve as a general detection reagent in time-resolved fluoroimmunoassays (TRFIA). The usefulness of such a strategy has previously been demonstrated with the Eu3+ chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (Diamandis et al. 1988). In this report the conjugation of streptavidin was accomplished with the Eu3+ chelate of N1-(p-isothiocyanatobenzyl)diethylenetriamine-N1,N3,N3(+)-tetraace tic acid, using a 50 M excess of the label. The conjugation ratio of Eu3+/streptavidin was 16. The use of the Eu-streptavidin reagent in a two-site immunometric assay to measure human recombinant interleukin-3 in human plasma, showed that the useful range of the assay was 20-25,000 pg/ml.     

Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay.

The polymerase chain reaction (PCR) has many potential applications in the field of nucleic acid diagnostics. In particular, it has been successfully applied to the detection of pathogens present in low copy numbers such as the human immunodeficiency virus type 1. Here we describe a time-resolved fluorescence-based hybridization assay which, combined with the PCR, offers an extremely sensitive method for the detection of nucleic acids. In this assay format, the PCR is run by standard procedures and the subsequent hybridization reaction is carried out in solution by using two oligonucleotide probes, one biotinylated and one labeled with europium (Eu3+). The sandwich hybrids are then collected onto a streptavidin-coated microtitration well, and the bound Eu3+ is measured in a time-resolved fluorometer. This assay is rapid, user friendly, and quantitative and lends itself to automation. The application of this assay to the detection of human immunodeficiency virus type 1 is described.     

新型铕络合物用于HBsAg的时间分辨荧光免疫检测

利用稳定的新型铕荧光络合物BHHCT与Eu3+标记羊抗人HBsAb和Eu3+标记BSA-SA,建立定量测定血清HBsAb的Eu3+-BHHCT-HBsAb-TRFIA法和Eu3+-BHHCT-BSA-SA-TRFIA法.结果表明,这两种方法最低检出值分别为0.2ng/mL和0.05ng/mL,标准曲线范围均为0-100ng/mL,批内变异系数CV均小于10%,后者回收率为85%-115%.用Eu3+-BHHCT-BSA-SA-TRFIA法与ELISA法同时检测118份血清样品,结果表明前者的检出率比后者高.     

Detection of hepatitis B virus PreS1 antigen using a time-resolved fluoroimmunoassay

The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu(3+) labeling of antibodies was performed with respective labeling kits, and Eu(3+) fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01?ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >10(3)?IU/mL (χ(2)?=?25.04, p?相似文献   

癌胚抗原的时间分辨免疫荧光分析及其诊断试剂的研制

目的利用时间分辨免疫荧光分析(TRFIA)技术,建立一种人血清癌胚抗原(CEA)的快速全自动检测方法并研制其诊断试剂。方法采用双抗体夹心法(用1株抗CEA的mAb M86160M用于包被微孔板,另1株抗CEA的mAbM86110M用于标记铕)建立CEA-TRFIA,增强液采用β-二酮体为主的发光增强系统。结果CEA-TRFIA的线性测量范围为(1~560)μg/L,分析的灵敏度为0.28μg/L,批内和批间的变异系数(CV)分别为7.2%~8.6%和8.9%~13.2%。与AFP、CA12-5、CA19-9和白蛋白均无交叉反应,与CA15-3的交叉反应值为1.62μg/L。将1000份血清标本用本法与国外罗氏化学发光试剂盒同时检测,其相关系数(r)为0.946。结论CEA-TRFIA的各项指标均达到临床检测的要求,可替代国外同类检测试剂盒,用于临床血清CEA水平的测定。     

TR-FIA与RIA法检测HBV标志物的比较分析

铕(Eu)标记时间分辨荧光免疫技术(TR-FIA)是新近推出的一种非放射性标记微量检测技术, 将其与RIA比较分析, 结果表明: TR-FIA法较RIA法灵敏度高, 测量范围宽, 准确度相近; 在乙型肝炎病毒标志物(HBV M)常见的8种组合模式中, 有7种完全符合(符合率为100%),只有HBSAg 抗-Hbe 抗-HBc组合模式符合率为73.3%,而HBeAg值差异较大.     

Cytotoxicity and cell imaging potentials of submicron color-tunable yttria particles

Increased demand of environment protection encouraged scientists to design products and processes that minimize the use and generation of hazardous substances. This work presents comprehensive result of large-scale fabrication and investigation of red-to-green tunable submicron spherical yttria particles codoped with low concentrations of Eu(+3) and Tb(+3). The color emission of synthesized particles can be precisely tuned from red to green by simple variation of Tb/Eu ratio and excitation wavelength. The Tb/Eu-codoped Y(2)O(3) particles did not adversely affect the viability of L-929 fibroblastic cells at concentrations less than 62.5 ppm. Through internalization and wide distribution inside the cells, Tb/Eu codoped Y(2)O(3) particles with intense bright green or red fluorescence rendered cell imaging to be possible. The high brightness, excellent stability, low-toxicity, and imaging capability along with fine color-tunability of synthesized particles enable to find promising application in various areas.