在时间分辨荧光免疫分析中铕标记技术的初步探讨

本文介绍了稀土元素标记白蛋白和时间分辨荧光测定。采用二步标记法,借助二乙基三胺五醋酸酐将Eu~(3+)标记到白蛋白抗原上的简便方法。用LKB的时间分辨荧光计和紫外分光光度计测得标记率为10Eu~(3+)/白蛋白分子,为建立白蛋白的时间分辨荧光免疫分析法打下基础。     

时间分辨荧光分析中的铕和钐

时间分辨荧光分析中的铕和钐ＴｏｍＳｔａｈｌｂｅｒｇＥｅｖａＭａｒｋｅｉａＨｅｉｋｋｉＭｉｋｏｌａＰｈｉｌｉｐＭｏｔｔｒａｍＩｌｋｋａＨｅｍｍｉｌａ（ＷａｌｌａｃＯｙ，Ｔｕｘｋｕ，Ｆｉｎｌａｎｄ）时间分辨荧光在常规免疫诊断工作中的实力，比如ＤＥＬＦＩＡ...     

CEA时间分辨荧光免疫分析与放射免疫分析

ＣＥＡ时间分辨荧光免疫分析与放射免疫分析黄建荣，尤海清，邵鹤生癌胚抗原（ＣＥＡ）放射免疫分析（ＲＩＡ）已在临床广泛开展，用于多种肿瘤的检测，而ＣＥＡ时间分辨荧光免疫分析（Ｔｉｍｅ－ｒｅｓｏｌｖｅｄＦｌｕｏｒｏｉｍｍｕｎｏａｓｓａｙＴｒＦＩＡ）在临床的...     

时间分辨荧光免疫分析技术研究的新进展

时间分辨荧光免疫分析技术研究的新进展高平，李振甲时间分辨荧光免疫分析（Ｔｉｍｅ－ｒｅｓｏｌｖｅｄＦｌｕｏｒｏｉｍｍｕｎｏａｓｓａｙ，ＴｒＦＩＡ）是当代最灵敏的标记免疫分析技术。自８０年代初问世以来，该技术在临床医学和基础研究中的应用日益广泛，其自身的...     

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     

用同一产前筛查软件分析不同检测系统测定数据比较

本文采用不同检测系统即时间分辨荧光免疫分析（Tr-FIA）和电化学发光分析（ECLIA）,测定数据用同一产前筛查软件进行分析,现将结果报道如下。     

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...     

周时间分辨荧光免疫法测定孕早期妇女血清甲胎蛋白的初步研究

本文用时间分辨荧光免疫法测定了孕６－１６周妇女的血清甲胎蛋白水平。共７４２份血清本。其中无流产史组３００分标本／３００例，有习惯性流产史组２３８份／６１例，为了天筛查，前者用毛细管微量采血与静脉血测定ＭＳＡＦＰ浓度对照为２０４份。     

BAS—时间分辨荧光免疫测定中的一些实验条件

本文综述ＢＡＳ－时间分辨荧光免疫测定技术的实验条件。包括链亲合系的Ｅｕ＾３＾＋标记，抗体或抗原生物素化方法，因相抗体或抗原包被等最佳实验条件。并观察了Ｅｕ＾３＾＋链亲合素的稳定性以及影响测定结果的主要因素。     

BAS─时间分辨荧光免疫测定中的一些实验条件

本文综述ＢＡＳ─时间分辨荧光免疫测定技术的实验条件。包括链亲合素的Ｅｕ（３＋）标记，抗体或抗原生物素化方法，固相抗体或抗原包被等最佳实验条件。并观察了Ｅｕ（３＋）链亲合素的稳定性以及影响测定结果的主要因素。     

基于时间分辨透射光谱的血液血红蛋白无创检测方法

目的 基于时间分辨结合遮蔽光谱(TRACOS)技术进行血红蛋白在体无创测量方法的理论研究.方法 在人体手指部位及人工血流动力学条件下,检测和分析动态双波长时间分辨透射.通过对时域数据进行拉普拉斯变换,提高血红蛋白的检测灵敏度.使用质量浓度范围为6～16 g/dl的血红蛋白对时间分辨检测法进行验证.结果 模拟结果表明,与连续波法相比,当使用拉普拉斯变换参数p=5×1010 s-1时,时间分辨检测法可以提供更高的检测灵敏度.结论 采用小正参数对时间分辨透射光强进行拉普拉斯变换可以强调早到达光子效应,从而增强血红蛋白探测的灵敏度.     

Evaluation of a new human immunodeficiency virus (HIV) antibody detection method based on time-resolved fluoroimmunoassay

We have developed a time-resolved fluoroimmunoassay for the detection of anti-HIV antibody using purified virus, recombinant gp120 or a synthetic peptide derived from gp41. The results of time-resolved fluoroimmunoassay correlated well with those obtained by enzyme immunoassay and with the results of immunoblotting. This time-resolved fluoroimmunoassay has sufficient sensitivity and specificity for clinical use.     

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.     

Imaging of in vitro chicken leg using time-resolved near-infrared optical tomography

Near-infrared optical imaging gains much attention because of its noninvasiveness and deep penetration depths into tissue. Here, we report near-infrared optical tomographic imaging of an in vitro chicken leg from time-resolved measurements. The in vitro chicken leg, dipped in a cylindrical container filled with diluted Intralipid-10% solution, was imaged with a multichannel time-resolved imaging system. A two-dimensional reconstruction algorithm based on a modified generalized pulse spectrum technique has been developed to reconstruct the images of both the absorption and reduced scattering coefficients simultaneously and quickly. The results demonstrate that a simultaneous reconstruction of absorption and reduced scattering coefficients from time-resolved measurement has a potential to reveal the changes in the optical properties associated with not only the physiological information but also the anatomical structure of the organ.     

Time-resolved study of the mechanical response of tissue phantoms to nanosecond laser pulses

We present a time-resolved study of the interaction of nanosecond laser pulses with tissue phantoms. When a laser pulse interacts with a material, optical energy is absorbed by a combination of linear (heat generation and thermoelastic expansion) and nonlinear absorption (expanding plasma), according to both the laser light irradiance and material properties. The objective is to elucidate the contribution of linear and nonlinear optical absorption to bubble formation. Depending on the local temperatures and pressures reached, both interactions may lead to the formation of bubbles. We discuss three experimental approaches: piezoelectric sensors, time-resolved shadowgraphy, and time-resolved interferometry, to follow the formation of bubbles and measure the pressure originated by 6 ns laser pulses interacting with tissue phantoms. We studied the bubble formation and pressure transients for varying linear optical absorption and for radiant exposures above and below threshold for bubble formation. We report a rapid decay (of 2 orders of magnitude) of the laser-induced mechanical pressure measured (by time-resolved shadowgraphy) very close to the irradiation spot and beyond 1 mm from the irradiation site (by the piezoelectric sensor). Through time-resolved interferometry measurements, we determined that bubble formation can occur at marginal temperature increments as low as 3°C.     

Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand

A new 96-well microtiter plate, time-resolved fluorometric assay was developed to measure leukocyte adhesion in vitro. The assay is based on loading leukocytes with a fluorescence enhancing ligand 2,2':6', 2"-terpyridine-6,6"-dicarboxylic acid (TDA), which in its acetoxymethyl ester form readily diffuses through the cell membrane. After hydrolysis by nonspecific intracellular esterases, the impermeable TDA accumulates inside the cells. When the TDA-labeled adherent leukocytes are lysed, the ligand is released and reacts with europium present in the lysis solution to produce a highly fluorescent and stable chelate. The fluorescence signal can be measured by time-resolved fluorometry and correlates directly with the number of adherent cells. In this study, we have optimized both the TDA-labeling and adhesion assay conditions in isolated human neutrophils. Furthermore, we have compared the assay with a traditional microscopic counting method. This time-resolved fluorometric assay provides a rapid, reproducible and convenient method for the routine analysis of leukocyte adhesion.     

Assessment of inflow and washout of indocyanine green in the adult human brain by monitoring of diffuse reflectance at large source-detector separation

Recently, it was shown in measurements carried out on humans that time-resolved near-infrared reflectometry and fluorescence spectroscopy may allow for discrimination of information originating directly from the brain avoiding influence of contaminating signals related to the perfusion of extracerebral tissues. We report on continuation of these studies, showing that the near-infrared light can be detected noninvasively on the surface of the tissue at large interoptode distance. A multichannel time-resolved optical monitoring system was constructed for measurements of diffuse reflectance in optically turbid medium at very large source-detector separation up to 9 cm. The instrument was applied during intravenous injection of indocyanine green and the distributions of times of flight of photons were successfully acquired showing inflow and washout of the dye in the tissue. Time courses of the statistical moments of distributions of times of flight of photons are presented and compared to the results obtained simultaneously at shorter source-detector separations (3, 4, and 5 cm). We show in a series of experiments carried out on physical phantom and healthy volunteers that the time-resolved data acquisition in combination with very large source-detector separation may allow one to improve depth selectivity of perfusion assessment in the brain.     

Noncontact backscatter-mode near-infrared time-resolved imaging system: Preliminary study for functional brain mapping

To improve the spatial resolution and to obtain the depth information of absorbers buried in highly scattering material, we developed a noncontact backscatter-mode near-infrared time-resolved imaging system (noncontact B-TRIS) that is intended for functional human brain mapping. It consists of mode-locked Ti-sapphire lasers as light sources and a charge-coupled device camera equipped with a time-resolved intensifier as a detector. The system was tested with a white polyacetal phantom as a light-scattering medium and black polyacetal particles as absorbers. Illumination and detection of light through an objective lens system (phi = 150 mm) enabled us to capture images from an area whose diameter is about 70 mm without coming into contact with it. The scattering and absorption coefficients of the white phantom obtained by B-TRIS were similar to those obtained by a conventional time-resolved spectroscopy. Although the imaged diameter of an absorber buried within a phantom was considerably larger than the actual diameter, the center position of the absorber coincided with the actual position with accuracy <2 mm. Furthermore, the depth information can be also detected by the noncontact B-TRIS. These results suggest a potential of noncontact B-TRIS for imaging cognitive human brain function.     

Time-resolved fluoroimmunoassay of pancreatic phospholipase A2

Summary We have developed an immunoassay for human pancreatic secretory phospholipase A₂ based on time-resolved fluorescence. The labeled antibody technique in combination with the time-resolved fluorometric detection of the Europium label, which essentially eliminates all background fluorescence, resulted in a high sensitivity and a wide linear range. Our assay is a one-incubation, multi-site, solid-phase assay on polystyrene micro-titer strips, even though a polyclonal antibody was used. Increased levels of immunoreactive phospholipase A₂ are found by this assay in sera of patients suffering from acute pancreatitis.     

Simultaneous quantification of two plant viruses by double-label time-resolved immunofluorometric assay

For simultaneous and sensitive detection of two antigens in one sample, monoclonal antibody (MAb) to potato virus M (PVM) was labelled with a lanthanide Eu3+ and MAb to potato virus X (PVX) with another lanthanide Sm3+. A mixture of the labelled MAbs was used as a tracer. After performing the immunoreactions, the fluorescence of the dissociated lanthanides was measured at different wave-lengths in a time-resolved fluorometer to quantificate the PVX and PVM amount in a sample. Double-label time-resolved fluoroimmunoassay (TR-FIA) detected 1 ng/ml of each virus and was therefore more sensitive for simultaneous detection of PVX and PVM than reported for a single virus detection with double antibody sandwich ELISA (DAS-ELISA).