Giri T K, Hillarp A, Härdig Y, Zöller B, Dahlbäck B
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Thromb Haemost. 1998 Apr;79(4):767-72.
A new method to determine the concentration of free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1:20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specificity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies.
本文描述了一种测定血浆中游离蛋白S浓度的新方法。它是一种酶联配体吸附测定法（ELSA），利用天然配体C4b结合蛋白（C4BP）与蛋白S的结合能力，从血浆样本中捕获游离蛋白S。在该测定中使用C4BP作为配体是可行的，这是由于蛋白S与C4BP之间相互作用的高亲和力（Kd = 0.1 nM）以及复合物解离速率较慢。一种单克隆抗体（HPS 54）与辣根过氧化物酶偶联，并用作靶抗体。该抗体识别蛋白S第一个表皮生长因子样结构域中依赖Ca2+的表位，且不干扰蛋白S的C4BP结合位点。在测定中添加钙有助于防止C4BP - 蛋白S - HPS 54复合物解离。三个不同的实验证明该测定对游离蛋白S具有特异性。第一，血浆中的蛋白S和纯化的蛋白S获得了几乎相同的剂量反应曲线。第二，向正常血浆中添加纯化的C4BP导致游离蛋白S减少。第三，蛋白S缺乏的血浆检测值为零，添加到蛋白S缺乏血浆中的约80%纯化蛋白S以及添加到蛋白S缺陷血浆样本中的约70%蛋白S可通过该测定法回收。该测定快速（仅涉及30分钟的单次孵育步骤）、灵敏，当血浆稀释1:20代表100%时，测量范围为游离蛋白S的3%至200%。两个水平的批内和批间变异系数分别为2.3 - 4.3%和5.1 - 7.4%。在一个大型蛋白S缺陷家族中，该测定对致病突变显示出100%的敏感性和特异性。此外，抗凝的蛋白S缺乏患者的游离蛋白S水平与非抗凝对照完全分开。这种新的游离蛋白S测定法适用于自动化，为常规临床检测蛋白S缺乏提供了一种有用的手段。
