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

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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