Enzyme-linked Immunosorbent Assay for Human Plasma Apolipoprotein B
Enzyme-linked Immunosorbent Assay for Human Plasma Apolipoprotein B
复制标题
人血浆载脂蛋白 B 的酶联免疫吸附测定
DOI：
--
复制
发表时间：
--
期刊：
影响因子：
--
通讯作者：
E. J. Schaefer
中科院分区：
文献类型：
--
作者：
J. Ordovás;J. P. Peterson;P. Santaniello;J. S. Cohn;P. W. Wilson;E. J. Schaefer;J. P. Peterson;P. Santaniello;J. S. Cohn;P. W. Wilson;E. J. Schaefer
已开发出一种非竞争性酶联免疫吸附测定 (ELISA)，用于使用亲和纯化的多克隆和单克隆抗体测量总血浆载脂蛋白 (apo) B。测试了不同制造商的微量滴定板的 IgG 结合特性；只有一块板产生小于 5% 的一致变异系数。该测定中的最佳血浆稀释度为 1:3000。与碱性磷酸酶缀合的 IgG 抗 apoB 抗血清用作第二抗体。使用磷酸对硝基苯酯作为显色底物，并使用与微计算机连接的 ELISA 读数器读取吸光度（410 nm）以进行数据处理。 Framingham 后代研究中测定了 1115 名男性和女性参与者的血浆 apoB 水平。平均 (f SD) 血浆浓度为 89 f 28 mg/dl。注意到 apoB 水平的显着年龄和性别相关差异。-Ordovas, J. 人血浆载脂蛋白 B 的酶联免疫吸附测定。补充关键词径向免疫扩散冠状动脉疾病脂蛋白从密度区域 1.019-1.063 g/ml 的人血浆中分离出的低密度脂蛋白 (LDL) 是含有（重量百分比）约 25% 蛋白质、40% 胆固醇、 25% 磷脂和 10% 甘油三酯 (1)。载脂蛋白 (apo) B-100 是 LDL 的主要蛋白质成分，占 LDL 蛋白质质量的 95% 以上。 ApoB 还占极低密度脂蛋白 (VLDL) 中蛋白质的约 30%。血浆 LDL 胆固醇水平升高与早发冠状动脉疾病 (CAD) 的风险增加相关 (2, 3)。有人建议，apoB 可能比 LDL 胆固醇更能预测早发 CAD (4)。通过 apoB 定量，人们可以识别出那些尽管 LDL 胆固醇水平正常（高脂β脂蛋白血症）但 apoB 水平升高的个体 (5)。后者患早发 CAD 的风险增加 (5)。许多方法已用于 apoB 的定量：径向免疫扩散 (RID)、电免疫测定 (EIA)、放射免疫测定 (RIA) 和浊度测量 (6-12)。所有这些方法都存在技术问题，这使得它们在某种程度上不适合用作临床测定。最近，出现了一些出版物，描述了基于酶联免疫吸附测定 (ELISA) 的 apoB 测量 (13-16)。我们本研究的目的是利用免疫纯化的多克隆抗体，通过 ELISA 方法开发一种灵敏、可重复且易于执行的血浆 apoB 水平测定方法。与之前报道的测定不同，我们的测定要求包括一致的运行间变异系数小于 5%。我们的检测结果是……
A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for measuring total plasma apolipoprotein (apo) B using affinity purified polyclonal and monoclonal antibodies. Microtiter plates from different manufacturers were tested with regard to their IgG binding characteristics ; only one plate yielded consistent coefficients of variation of less than 5%. The optimal plasma dilution in this assay was 1:3000. IgG anti-apoB antisera conjugated to alkaline phosphatase was used as a second antibody. p-Nitrophenyl phosphate was utilized as substrate for color development, and the absorbance (410 nm) was read utilizing an ELISA reader interfaced with a microcomputer for data processing. Plasma apoB levels in plasma have been determined in 1115 male and female participants in the Framingham Offspring Study. Mean (f SD) plasma concentrations were 89 f 28 mg/dl. Significant age and sex related differences in apoB levels were noted.-Ordovas, J. Enzyme-linked inmunosorbent assay for human plasma apolipoprotein B. Supplementary key words radial immunodiffusion coronary artery disease lipoproteins Low density lipoproteins (LDL) as isolated from human plasma in the density region 1.019-1.063 g/ml are particles that contain (weight percent) approximately 25% protein, 40% cholesterol, 25% phospholipid, and 10% triglyceride (1). Apolipoprotein (apo) B-100 is the major protein constituent of LDL comprising over 95% of LDL protein mass. ApoB also accounts for about 30% of the protein present in very low density lipoproteins (VLDL). Increased levels of plasma LDL cholesterol have been associated with an enhanced risk for premature coronary artery disease (CAD) (2, 3). It has been suggested that apoB may be a better predictor for premature CAD than LDL cholesterol (4). The quantitation of apoB allows one to identify those individuals who have elevated apoB levels despite having normal LDL cholesterol levels (hyperapobetalipoproteinemia) (5). These latter subjects are at increased risk for premature CAD (5). A number of methods have been utilized for quantita-tion of apoB: radial immunodiffusion (RID), electroim-munoassay (EIA), radioimmunoassay (RIA), and neph-elometry (6-12). All of these methods have technical problems that make them somewhat undesirable for use as clinical assays. More recently, several publications have appeared describing apoB measurements based on the use of enzyme-linked immunosorbent assays (ELISA) (13-16). Our purpose in this study was to develop a sensitive , reproducible, and easily performed assay for plasma apoB levels with ELISA methodology utilizing immunopurified polyclonal antibodies. Unlike previously reported assays, our assay requirements included having consistent between-run coefficients of variation of less than 5%. Our assay was …