Development of a time-resolved fluoroimmunoassay for salmon insulin-like growth factor binding protein-1b
Development of a time-resolved fluoroimmunoassay for salmon insulin-like growth factor binding protein-1b
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DOI：
10.1016/j.cbpa.2015.04.016
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发表时间：
2015-09-01
影响因子：
2.3
通讯作者：
Shimizu, Munetaka
中科院分区：
文献类型：
--
作者：
Fukuda, Miki;Kaneko, Nobuto;Shimizu, Munetaka
在鲑鱼血浆/血清中，三种主要的胰岛素样生长因子结合蛋白（IGFBPs）始终在22-，28-和41-kDa检测。22-kDa形式已被鉴定为IGFBP-1b，并显示在分解代谢条件下增加。我们开发了鲑鱼IGFBP-1b的竞争性时间分辨荧光免疫分析法（TR-FIA）。纯化的鲑鱼IGFBP-1b用于生物素标记、测定标准品和抗血清生产。TR-FIA与41-kDa形式（IGFBP-2b）没有交叉反应，但与28-kDa形式（IGFBP-1a）显示出3%的交叉反应性。它测量IGFBP-1b水平低至0.4 ng/ml，ED 80和ED 20分别为0.9和24.6 ng/ml。IGF-I的干扰似乎很小。使用TR-FIA，血清IGFBP-1b水平测定个别标记的未成年马苏鲑鱼喂养或禁食5周，或禁食3周，然后再喂养2周。禁食3周显著增加了循环IGFBP-1b水平，而在延长禁食2周后恢复到基础水平。血清IGFBP-1b水平与体重、体质指数、特定生长率、血清IGF-I水平呈负相关。在parr-smolt转换的马苏鲑鱼，平均循环IGFBP-1b水平是最高的五月。血清IGFBP-1b与IGF-I呈正相关，与禁食/喂养实验结果相反。IGFBP-1b与鳃Na+，K+-ATPase活性呈正相关。这些结果表明，循环中IGFBP-1b和IGF-I之间的关系在银化过程中不同于在禁食期间，IGFBP-1b可能在低血糖调节能力的发展中发挥作用。(C)2015 Elsevier Inc. All rights reserved.
In salmon plasma/serum, three major insulin-like growth factor binding proteins (IGFBPs) are consistently detected at 22-, 28- and 41-kDa. The 22-kDa form has been identified as IGFBP-1b and shown to increase under catabolic conditions. We developed a competitive time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1b. Purified salmon IGFBP-1b was used for biotin-labeling, assay standard and antiserum production. The TR-FIA did not cross-react with the 41-kDa form (IGFBP-2b) but showed 3% cross-reactivity with the 28-kDa form (IGFBP-1a). It measured IGFBP-1b levels as low as 0.4 ng/ml, and ED80 and ED20 were 0.9 and 24.6 ng/ml, respectively. There appears to be little interference by IGF-I. Using the TR-FIA, serum IGFBP-1b levels were measured in individually-tagged underyearling masu salmon fed or fasted for 5 weeks, or fasted for 3 weeks followed by refeeding for 2 weeks. Fasting for 3 weeks significantly increased circulating IGFBP-1b levels, while it returned to the basal levels after prolonged fasting for additional 2 weeks. Serum IGFBP-1b level negatively correlated with body weight, condition factor, specific growth rate and serum IGF-I level. During parr-smolt transformation of masu salmon, average circulating IGFBP-1b levels were the highest in May. There was a positive correlation between serum IGFBP-1b and IGF-I, which is in contrast to that in the fasting/feeding experiment. IGFBP-1b also showed a positive relationship with gill Na+, K+-ATPase activity. These results suggest that the relationship between circulating IGFBP-1b and IGF-I during smoltification differs from that during fasting and IGFBP-1b may play a role in the development of hypoosmoregulatory ability. (C) 2015 Elsevier Inc. All rights reserved.