Eriksson Susann, Halenius Heidi, Pulkki Kari, Hellman Jukka, Pettersson Kim
Department of Biotechnology, University of Turku, Turku, Finland.
Clin Chem. 2005 May;51(5):839-47. doi: 10.1373/clinchem.2004.040063. Epub 2005 Feb 17.
There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI.
We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery.
Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (approximately 50 kDa) and light chains (approximately 25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly higher in 10 samples with low recovery than in 10 samples with normal recovery of cTnI (P < or = 0.007).
Circulating autoantibodies to cTnI or other proteins of the troponin complex can be a source of negative interference in cTnI immunoassays.
免疫分析中有多种潜在的干扰源。我们的目的是确定在基于针对心肌肌钙蛋白I（cTnI）中心部分的抗体的cTnI免疫分析中导致负干扰的血液成分。
我们从添加的cTnI回收率低的样本中分离出一种干扰因子（IF），采用了几个连续的纯化步骤：辛酸沉淀、硫酸铵沉淀以及在Cibacron Blue凝胶和蛋白G柱上进行纯化。通过凝胶电泳和蛋白条带的质谱分析鉴定纯化后的IF。为了直接检测血清样本中针对心肌肌钙蛋白的人抗体，我们使用三种不同的抗人免疫球蛋白抗体开发了免疫分析方法，并测量了cTnI回收率低和正常的样本中的肌钙蛋白抗体。
用辛酸处理未使IF沉淀，但IF在40%硫酸铵饱和度时沉淀。IF与Cibacron Blue凝胶柱结合，用线性盐梯度从该柱上洗脱；它也与蛋白G结合。纯化后的IF的凝胶电泳显示两条主要条带，其分子量对应于免疫球蛋白的重链（约50 kDa）和轻链（约25 kDa），通过质谱法确认了它们的身份。通过三种不同的免疫分析方法在cTnI回收率低的样本中证实了肌钙蛋白特异性自身抗体的存在。cTnI回收率低的10个样本的中位信号明显高于cTnI回收率正常的10个样本（P≤0.007）。
针对cTnI或肌钙蛋白复合物其他蛋白的循环自身抗体可能是cTnI免疫分析中负干扰的一个来源。
