Ebinuma Hiroyuki, Miyazaki Osamu, Yago Hirokazu, Hara Kazuo, Yamauchi Toshimasa, Kadowaki Takashi
Diagnostics Research Laboratories, Daiichi Pure Chemicals Co. Ltd., Ryugasaki, Ibaraki 301-0852, Japan.
Clin Chim Acta. 2006 Oct;372(1-2):47-53. doi: 10.1016/j.cca.2006.03.014. Epub 2006 May 12.
Adiponectin, an antiatherogenic adipocyte-derived protein exists in human blood as multiple isoforms--trimeric low molecular weight (LMW), albumin-binding LMW (Alb-LMW), hexameric middle molecular weight (MMW), and high molecular weight (HMW) forms. We developed a novel ELISA system to detect total human adiponectin and the selective level of each adiponectin multimer for investigating the distribution of these levels in human blood.
Two monoclonal antibodies that were raised against human adiponectin were used to construct a sandwich ELISA to measure adiponectin levels. Adiponectin multimers were selectively measured after sample pretreatment with two proteases that specifically digested the trimeric forms or both the hexameric and trimeric forms.
The ELISA had a dynamic range of 0.075-4.8 ng/ml. Intraassay variations (CV) were 5.3% (total adiponectin), 4.1% (MMW+HMW), and 3.3% (HMW). Comparison of the results of ELISA and quantitative western blot analysis of multimeric adiponectin in serum samples revealed good correlation (LMW+Alb-LMW, r=0.873; MMW, r=0.907; HMW, r=0.950). Each of the three forms of adiponectin multimer levels closely correlated with total adiponectin levels in healthy subjects.
This ELISA system can be used to further investigate the physiological roles of human adiponectin multimers.
脂联素是一种具有抗动脉粥样硬化作用的脂肪细胞衍生蛋白，以多种异构体形式存在于人体血液中，即三聚体低分子量（LMW）、白蛋白结合LMW（Alb-LMW）、六聚体中分子量（MMW）和高分子量（HMW）形式。我们开发了一种新型酶联免疫吸附测定（ELISA）系统，用于检测人总脂联素以及每种脂联素多聚体的选择性水平，以研究这些水平在人体血液中的分布情况。
使用两种针对人脂联素产生的单克隆抗体构建夹心ELISA，以测量脂联素水平。在用两种特异性消化三聚体形式或六聚体和三聚体形式的蛋白酶对样品进行预处理后，选择性测量脂联素多聚体。
该ELISA的动态范围为0.075 - 4.8 ng/ml。批内变异系数（CV）分别为5.3%（总脂联素）、4.1%（MMW + HMW）和3.3%（HMW）。血清样品中脂联素多聚体的ELISA结果与定量蛋白质印迹分析结果比较显示出良好的相关性（LMW + Alb-LMW，r = 0.873；MMW，r = 0.907；HMW，r = 0.950）。在健康受试者中，脂联素多聚体的三种形式中的每一种水平都与总脂联素水平密切相关。
该ELISA系统可用于进一步研究人脂联素多聚体的生理作用。
