The development of time-resolved fluoroimmunoassay using new fluorescent chelates as labels and its forensic medical applications

新型荧光螯合物标记的时间分辨荧光免疫分析方法的发展及其法医学应用

• 批准号: 09670453
• 负责人: KIMURA Hiroko
• 金额: $ 1.09万
• 依托单位: Juntendo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670453/
• 关键词: Time-resolved fluoroimmunoassay; Europium; Samarium; Methamphetamine; Desipramine; Chlorpromazine; IgE; Bensulfuron-methyl; ルイス式血液型抗原; DNAハイブリダイゼーションアッセイ; デシプラミン; クロルプロマジン; α-フェトプロテイン; 癌胎児性抗原

A simple and highly sensitive time-resolved fluoroirnmunoassay (TR-FIA) using a new fluorescent europium chelate (BHHCT-Eu^<3+>) as a label was developed for forensic science analysis. First, drugs such as stimulants (methamphetamine, MA), psychopharmaceuticals (desipramine, DSP and chiorpromazine, CPZ) were assayed. MA analysis of 34 urine samples with the new method and the conventional gas chromatography showed a good correlation (r=0.954). The high detectability of this assay also enabled the segmental hair analysis with a few centimeters of a hair. The chelates of some lanthanides have their fluorescence peaks at different wavelengths. Combined use of europium and samarium enabled dual-label TR-FIA to assay two kinds of drugs (MA and DSP or MA and CPZ) contained in a sample simultaneously. The dual-label TR-FIA also enabled to assay two cancer markers, CEA and AEP, in a human serum. Development of another new chelates and combined use of these would enable multiple-label TR-FIA to … More assay several drugs contained in a sample. A new chelate has been synthesized and its application to the multiple-TR-FIA is now being developed.A paddy field herbicide bensulfuron-methyl was also assayed. The high detactability of the pesticide would be useful to the environmental analysis.Second, IgE in human sera was assayed. The detection limit was 1.5-3.O*10^<-3> IU/ml and this detectability was ten to one hundred times superior to other conventional immunoassays.Third, Lewis blood group antigens in the bloodstain and plasma samples were assayed. The past researchs on the postnatal change in the Lewis blood group were carried out by the traditional serological method. In order to know accurately the appearance time of Lewis^a and Lewis^b and the following changes in antigen expression, we assayed the Lewis antigen level quantitatively using the TR-FIA.The quantity of Lewis antigens in bloodstains and blood plasmas, from cord blood (40 samples), infant blood (39), and adult blood (33) were assayed. In the umbilical cord blood, Lewis^a antigen, almost equal in quantity to adult Le(a+b-) type, and a little amount of Lewis^b antigen already existed. And from the assay results of bloodstain and plasma samples, we considered that these antigens exist not on the red blood cells but in the plasma. And, in case of the newborn infants of Le(a-b+), the antigen level of Lewis^b in blood plasma began to rise about five days after birth, and it continued increasing for two or three months, and this antigen in the plasma was considered to adsorb to the blood cell surface. The detection limit of Lewis^a antigen by this method is 1 ng/ml, and detection limits of the anti-Lewis antibodies are I : 2* 10^6, and were some hundreds times more sensitive than the results obtained by the conventional ELISA method. Until now, Lewis blood group has been considered to change after the antigens appeared in blood plasma several months after birth. But our results revealed that the antigens appeared at a much earlier stage. The TR-FIA using a new label has revealed this fact. As to the Lewis^a, there is every likelihood that Lewis^a antigen in the cord blood is different in quality from adult Lewis^a antigen, because in spite of its abundant existence it does not adsorb to the cell membrane.Fourth, (BHHCT-Eu^<3+>) was found applicable to the label of DNA hybridization assay, and 4 pg/well of DNA was detected. Less

以新型铕螯合物（BHHCT-Eu^<3+>）为标记物，建立了一种简便、高灵敏度的时间分辨荧光免疫分析法（TR-FIA）。首先，分析了兴奋剂（甲基苯丙胺，MA）、精神药物（地昔帕明，DSP和氯丙嗪，CPZ）等药物。对34份尿样进行了MA分析，结果与常规气相色谱法测定结果有良好的相关性（r=0.954）。该测定的高检测能力也使得能够对几厘米的毛发进行分段毛发分析。某些镧系元素的螯合物在不同波长处有其荧光峰。铕和钐的联合使用使双标记TR-FIA能够同时测定样品中含有的两种药物（MA和DSP或MA和CPZ）。双标记TR-FIA还能够测定人血清中的两种癌症标志物CEA和AEP。开发另一种新的螯合物并联合使用这些螯合物将使多标记TR-FIA能够 ...更多信息 分析样品中含有的几种药物。合成了一种新的螯合物，并将其应用于多重TR-流动注射分析中，同时对稻田除草剂苄嘧磺隆进行了测定。该农药的高检出率为环境分析提供了依据。检测限为1.5 ~ 3.0 × 10 ~（-15）<-3>IU/ml，上级其它常规免疫分析方法的检测灵敏度高10 ~ 100倍。以往对刘易斯血型的生后变化研究多采用传统的血清学方法。为了准确地了解刘易斯^a和刘易斯^B出现的时间以及随后抗原表达的变化，我们使用TR-FIA定量测定了刘易斯抗原水平，并测定了来自脐带血（40份）、婴儿血（39份）和成人血（33份）的血痕和血浆中刘易斯抗原的量。脐带血中已存在刘易斯^a抗原，其数量与成人Le（a+b-）型相当，并有少量刘易斯^B抗原。从血斑和血浆样品的检测结果来看，这些抗原可能存在于血浆中，而不是红细胞上。并且，在Le（a-b+）新生儿的情况下，血浆中刘易斯^B的抗原水平在出生后约5天开始升高，并且持续升高2或3个月，并且血浆中的该抗原被认为吸附到血细胞表面。该方法对刘易斯α抗原的检测限为1 ng/ml，抗刘易斯抗体的检测限为1：2 × 10 ^6，比常规ELISA方法获得的结果灵敏几百倍。直到现在，刘易斯血型一直被认为是在出生后几个月血浆中出现抗原后发生变化的。但我们的结果表明，抗原出现在更早的阶段。使用新标签的TR-FIA揭示了这一事实。至于刘易斯^a，脐带血中的刘易斯^a抗原很可能在质量上不同于成人刘易斯^a抗原，因为尽管其大量存在，但它不吸附到细胞膜上。少

项目成果

Yuan.J., Wang, G., Kimura, H., Matsumoto, K.: "Highly sensitive time-resolved fluoroimmunoassay of human immunoglobulin E by using a new europium fluorescent chelate as a label." Anal Biochem.254. 283-287 (1997)

Yuan.J.、Wang, G.、Kimura, H.、Matsumoto, K.：“使用新型铕荧光螯合物作为标记，对人免疫球蛋白 E 进行高灵敏度时间分辨荧光免疫测定。”

Yuan, J., Wang, G., Kimura, H., Matumoto, K.: "Highly sensitive time-resolved fluoroimmunoassay of human immunogiobulin E by using a new europium fluorescent chelate as a label." Anal Biochem. 254. 283-287 (1997)

Yuan, J.、Wang, G.、Kimura, H.、Matumoto, K.：“使用新型铕荧光螯合物作为标记，对人免疫球蛋白 E 进行高灵敏度时间分辨荧光免疫测定。”

Yuan, J., Wang, J., Kimura, H., Matsumoto, K.: "Highly sensitive detection of bensulfuron-methyl by time-resolved fluoroimmunoassay using a tetradentate beta-diketonate europium chelate as a label." Anal Sci. 15. 125-128 (1999)

Yuan, J.、Wang, J.、Kimura, H.、Matsumoto, K.：“使用四齿β-二酮铕螯合物作为标记，通过时间分辨荧光免疫测定法对苯磺隆进行高度灵敏的检测。”

Kimura, H., Yuan, J., Wang, G., Matsumoto, K. and Mukaida, M: "Highly sensitive quantitation of methamphetamine by time-resolved fluoroimmunoassay using a new europium chelate as a label." J Anal Toxicol. 23. 11-16 (1999)

Kimura, H.、Yuan, J.、Wang, G.、Matsumoto, K. 和 Mukaida, M：“使用新的铕螯合物作为标签，通过时间分辨荧光免疫测定法对甲基苯丙胺进行高灵敏度定量。”

Kimura, H., Yuan, J., Wang, G., Matsumoto, K.and Mukaida, M: "Highly sensitive quantitation of methamphetamine by time-resolved fluoroimmunoassay using a new europium chelate as a label." J Anal Toxicol. 23. 11-16 (1999)

Kimura, H.、Yuan, J.、Wang, G.、Matsumoto, K. 和 Mukaida, M：“使用新的铕螯合物作为标签，通过时间分辨荧光免疫测定法对甲基苯丙胺进行高灵敏度定量。”