Fless G M, Snyder M L, Scanu A M
Department of Medicine, Pritzker School of Medicine, University of Chicago, IL 60637.
J Lipid Res. 1989 May;30(5):651-62.
Based on our findings that rabbit antisera raised against human Lp[a] or apo[a] have the potential to cross-react with plasminogen, and in some cases have nearly equal affinities for plasminogen and Lp[a], we have developed an assay for plasma Lp[a] based on a "sandwich" ELISA that is insensitive to the presence of plasminogen. This was accomplished through the use of anti-apo[a] as a capture antibody and quantitation of the bound Lp[a], i.e., the apoB-100-apo[a] complex, with an anti-apoB antibody. Although apo[a] is heterogeneous in size, all Lp[a] particles tested, either in pure form or contained in whole plasma, gave parallel dose-response curves and were immunologically equivalent. However, when purified Lp[a] particles with different apo[a] isoforms were studied, those having larger isoforms were, on a weight basis, less reactive than those having a smaller size. Nearly equivalent reactivity was observed when protein concentration was expressed on a molar basis. The distribution of Lp[a] in a population of 84 subjects was skewed with one-third of the individuals having less than 1 mg/dl Lp[a] protein. All subjects tested had measurable concentrations of Lp[a] with a lower limit of detection of 0.030 mg/dl Lp[a] protein. The mean level was 3.2 mg/dl with a range of 0.045 to 13.3 mg/dl. These studies demonstrate the successful development of an ELISA for Lp[a] protein that is insensitive to the presence of plasminogen; that heterogeneity of Lp[a] and apo[a] are an important source of variation in the assay; and the need for an appropriate Lp[a] standard in order to minimize this variation.
基于我们的研究发现，即针对人Lp[a]或载脂蛋白[a]产生的兔抗血清有可能与纤溶酶原发生交叉反应，并且在某些情况下对纤溶酶原和Lp[a]具有几乎相等的亲和力，我们开发了一种基于“夹心”ELISA的血浆Lp[a]检测方法，该方法对纤溶酶原的存在不敏感。这是通过使用抗载脂蛋白[a]作为捕获抗体，并使用抗载脂蛋白B抗体对结合的Lp[a]（即载脂蛋白B-100-载脂蛋白[a]复合物）进行定量来实现的。尽管载脂蛋白[a]在大小上是异质的，但所有测试的Lp[a]颗粒，无论是纯形式还是存在于全血中，都给出了平行的剂量反应曲线，并且在免疫上是等效的。然而，当研究具有不同载脂蛋白[a]异构体的纯化Lp[a]颗粒时，基于重量，具有较大异构体的颗粒比具有较小尺寸的颗粒反应性更低。当以摩尔为基础表示蛋白质浓度时，观察到几乎相等的反应性。在84名受试者群体中，Lp[a]的分布呈偏态，三分之一的个体Lp[a]蛋白含量低于1mg/dl。所有测试的受试者Lp[a]浓度均可测，检测下限为0.030mg/dl Lp[a]蛋白。平均水平为3.2mg/dl，范围为0.045至13.3mg/dl。这些研究表明成功开发了一种对纤溶酶原存在不敏感的Lp[a]蛋白ELISA检测方法；Lp[a]和载脂蛋白[a]的异质性是该检测中变异的重要来源；以及需要合适的Lp[a]标准品以尽量减少这种变异。
