Time-resolved Fluoroimmunoassay for Secretin and Cholecystokinin using Europium chelate-labeled Streptavidin

DOI Web Site

Bibliographic Information

Other Title
  • ユウロピウムキレート標識ストレプトアビジンを用いるセクレチンおよびコレシストキニン-8の時間分解蛍光免疫測定法

Search this article

Description

We have developed a sensitive time-resolved fluoroimmunoassay for secretin and cholecystokinin (CCK). Europium chelate-labeled streptavidin (Eu-SA) was prepared by labeling Euchelate [N'-(p-isothiocyanatobenzyl)diethylenetriamine-N, N N ', N"-tetraacetato]europium (III)]. The assay procedure was as follows: A standard secretin or CCK-8 solution (100 ul), rabbit anti-human secretin or anti-human CCK anti-serum solution (50 pl) and biotinylated secretin or CCK-33 solution (50 ul) were added to the well of microtiter plate coated with coated with goat anti rabbit IgG antibody and incubated for 16 or 72 h at 4 °C. Then, the wells were washed with 0.05 mol/L Tris-HC1 buffered saline containing 0.05% Tween 20(pH 7.7). The solution of Eu-SA (100 pl) was added to the biotinylated secretin or CCK-33bound to the well. After incubation for 90 minutes, the well was washed and then enhancement solution (Pharmacia Co. Ltd. ) (200 AO was added to each well and incubated at room temperature for 10 minutes. The time-resolved fluorescence intensity was measured by Arcus 1230 fluorometer (Pharmacia Co. Ltd. ). A measurable range and fifty percent value of inhibition concentration (IC 50) for secretin and CCK-8 were from 25 to 1000 pg/m/ and 520 pg/m/ and from 62.5 to 32000 pg/m/ and 1450pg/m/, respectively, and the coefficients of variation for within-run was 1.7 -3.30% (n= 8)and 2.34-7.56% (n = 6). Further, the sensitivity of IC 50 value and detection limit for CCK-8 with ELISA amplification system (Daiichi kagakuyakuhin Co. ) was 18.5-fold and 31.3-fold better than that was obtained by without ELISA amplification system.539

Journal

  • NIPPON KAGAKU KAISHI

    NIPPON KAGAKU KAISHI 1993 (5), 534-538, 1993-05-10

    The Chemical Society of Japan

Keywords

Details 詳細情報について

Report a problem

Back to top