Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder, Verasper moseri
Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder, Verasper moseri
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DOI：
10.1006/gcen.2001.7760
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发表时间：
2002-02-15
影响因子：
2.7
通讯作者：
Nagasawa, H
中科院分区：
文献类型：
--
作者：
Andoh, T;Nagasawa, H
建立了一种时间分辨荧光免疫分析（TR-FIA）系统，用于定量测定巴芬比目鱼体内的胰岛素水平。该TR-FIA系统是一种固相测定法，基于未标记胰岛素和生物素化的叉鳍比目鱼胰岛素-II与抗叉鳍比目鱼胰岛素-II抗体的竞争。在该TR-FIA中，大鳍比目鱼胰岛素-I和胰岛素-II的最低可检测水平为10 pg/孔，相当于1.0 ng/ml，胰岛素-II显示出比胰岛素-I略高的交叉反应性。通过专属性试验、验证试验和使用血浆添加胰岛素-II的回收率试验，确保了该TR-FIA的准确度。结果表明，该检测系统具有较高的特异性和足够的准确度，用于胰岛素水平的测量。应用该系统测定了饥饿和饥饿条件下长鳍比目鱼的血浆胰岛素水平。在实验过程中，进食比目鱼的血浆胰岛素水平（平均值+/- SEM）在2小时达到最大值（9.3 +/- 1.7 ng/ml），此后逐渐下降，而禁食比目鱼的血浆胰岛素水平保持在低水平（1.1 +/- 0.1-2.0 +/- 0.2 ng/ml）。在通过酸化和随后的凝胶过滤去除蛋白质后，通过反相HPLC分别对喂食后2 h从喂食和禁食的比目鱼中采集的血浆样品进行分级分离。在美联储比目鱼，胰岛素免疫反应性检测到的馏分对应的胰岛素-I或-II。各峰的积分胰岛素免疫反应性比值为0.378 +/- 0.044（平均值+/- SD）。该值与反相HPLC上来自巴芬比目鱼Brockmann体提取物的每种胰岛素的吸光度面积（0.355 +/- 0.019）非常一致。在禁食比目鱼，非常弱的胰岛素免疫反应性观察到的保留时间对应的胰岛素-I和-II。这些结果表明，胰岛素-I和胰岛素-II分泌到血液中的喂养刺激诱导的比例大致相同的数量窝藏在布罗克曼体。(C)2002 Elsevier Science（美国）。
A time-resolved fluoroimmunoassay (TR-FIA) system was developed to quantify insulin levels in the barfin flounder. This TR-FIA system is a solid-phase assay based on competition of unlabeled insulins and biotinylated barfin flounder insulin-II against an anti-barfin flounder insulin-II antibody. The minimum detectable level of barfin flounder insulin-I and -II in this TR-FIA was 10 pg/well which corresponded to 1.0 ng/ml, and insulin-II showed slightly higher crossreactivity than insulin-I. The accuracy of this TR-FIA was assured by specificity test, validation test, and recovery test using plasma added insulin-II. The results indicated the high specificity and sufficient accuracy of this assay system for insulin level measurement. This system was applied to the measurement of plasma insulin levels of fed and fasted barfin flounders. Plasma insulin levels (average +/- SEM) in fed flounders reached a maximum 2 h (9.3 +/- 1.7 ng/ml) and decreased gradually thereafter, while those in fasted flounders remained at low levels (1.1 +/- 0.1-2.0 +/- 0.2 ng/ml) during the experiment. After removing proteins by acidification and subsequent gel filtration, plasma samples taken from fed and fasted flounders at 2 h after feeding were fractionated separately by reversed-phase HPLC. In fed flounders, insulin immunoreactivity was detected in fractions corresponding to those of insulin-I or -II. The ratio of integrated insulin immunoreactivities of each peak was 0.378 +/- 0.044 (average +/- SD). This value was in good agreement with those (0.355 +/- 0.019) of absorbance areas of each insulin from Brockmann body extracts of the barfin flounder on reversed-phase HPLC. In fasted flounders, very weak insulin immunoreactivities were observed at retention times corresponding to those of insulin-I and -II. These results indicated that both insulin-I and -II were secreted into the blood being induced by feeding stimulation with approximately the same ratio as that of the quantities harbored in the Brockmann body. (C) 2002 Elsevier Science (USA).