Wilkinson Ryan J, Elliott Phillip, Hohmann Art, Francis Geoffrey, Carragher John
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia.
Comp Biochem Physiol B Biochem Mol Biol. 2004 Oct;139(2):193-201. doi: 10.1016/j.cbpc.2004.06.016.
This paper describes the development and validation of a competitive, polyclonal antibody enzyme-immunoassay (EIA) for the measurement of salmon and trout insulin-like growth factor-II (IGF-II). A polyclonal antiserum was raised against a synthetic peptide epitope, corresponding to amino acid residues 1-9 of the N-terminus of mature Atlantic salmon (Salmo salar) IGF-II. The antiserum was purified by hydrophobic charge induction chromatography (HCIC). The partially purified immunoglobulins were used in an enzyme-immunoassay system (EIA) resulting in a highly specific assay for salmon IGF-II with cross-reactivity of less than 0.01% for recombinant salmon IGF-I and recombinant salmon growth hormone (GH), and 5.57% for salmon insulin (sIns). The recombinant salmon IGF-II (rsIGF-II) standard curve limit of detection was 1.37 ng/ml with an EC(50) of 44.97+/-0.82 ng/ml. Intra- and interassay coefficients of variation were determined at 7.47% (n=15) and 7.42% (n=15), respectively. Added rsIGF-II was adequately recovered from acid-treated Atlantic salmon and rainbow trout (Oncorhynchus mykiss) plasma samples. Parallel dose-response inhibition curves were demonstrated for the plasma of both fish species tested. Circulating IGF-II levels of 22.26+/-2.66 and 18.24+/-1.43 ng/ml were determined for acid-treated plasma of normal adult Atlantic salmon and rainbow trout, respectively. This EIA should prove to be useful in the study of factors which influence circulating plasma levels of IGF-II in these fish species.
本文描述了一种用于测量鲑鱼和鳟鱼胰岛素样生长因子-II（IGF-II）的竞争性多克隆抗体酶免疫测定法（EIA）的开发与验证。针对与成熟大西洋鲑（Salmo salar）IGF-II N端氨基酸残基1-9对应的合成肽表位制备了多克隆抗血清。通过疏水电荷诱导色谱法（HCIC）纯化抗血清。将部分纯化的免疫球蛋白用于酶免疫测定系统（EIA），从而建立了一种对鲑鱼IGF-II具有高度特异性的测定方法，对重组鲑鱼IGF-I和重组鲑鱼生长激素（GH）的交叉反应率小于0.01%，对鲑鱼胰岛素（sIns）的交叉反应率为5.57%。重组鲑鱼IGF-II（rsIGF-II）标准曲线的检测限为1.37 ng/ml，半数有效浓度（EC50）为44.97±0.82 ng/ml。批内和批间变异系数分别测定为7.47%（n = 15）和7.42%（n = 15）。从酸处理的大西洋鲑和虹鳟（Oncorhynchus mykiss）血浆样本中能充分回收添加的rsIGF-II。对所测试的两种鱼类的血浆均显示出平行的剂量反应抑制曲线。正常成年大西洋鲑和虹鳟酸处理血浆中循环IGF-II水平分别测定为22.26±2.66 ng/ml和18.24±1.43 ng/ml。这种EIA在研究影响这些鱼类循环血浆中IGF-II水平的因素方面应会被证明是有用的。
