Kimura H.^*, Mukaida M.^**, Yuan J.^***, Wang G.^***, Matsumoto K.^***
* Department of Forensic Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, Japan
^** Department of Forensic Medicine, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama, Japan
^*** Department of Chemistry, Waseda University, Okubo 3-4-1, Shinjuku-ku, Tokyo, Japan

Methods. Newly synthesized europium complex (BHHCT-Eu) was used as labeling material and MA was quantitated by one step and two step assay variations of competitive time-resolved fluoroimmunoassay. Microtiter plates coated with anti-MA rabbit IgG were used and BHHCT was bound to bovine serum albumin (BSA) and N-aminobutyl-MA complex, in the one step assay. In the two step assay, biotinylated MA-BSA and BHHCT conjugated streptavidin were used. Time-resolved fluorometric measurement was performed with an Arcus 1232 fluorometer (excitation at 340nm, fluorescence measurement at 615 nm).

Results and Discussion. The minimum detection limits of MA were 1 ng/ml (25 pg/assay) and 1 pg/ml (25 fg/assay), respectively. These are ten to one thousand times superior than any other immunoassay method and have enough detectability to measure a small quantity of MA in a segment of hair. We assayed 34 urine samples and there was a good correlation between this method and previously established gas chromatography (r=0.965). Intra-assay CV is about 3-6% at eight different concentrations (n=4). Time-resolved fluoroimmunoassay is characterized by its highly sensitive detection and a low background level. In our assay, the use of new fluorescent labeling makes highly sensitive and selective detection possible without interfering on the specificity of the anti-MA.