Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers
Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers
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DOI：
10.1016/j.jim.2013.11.014
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发表时间：
2014-01-31
影响因子：
2.2
通讯作者：
Houen, Gunnar
中科院分区：
文献类型：
--
作者：
Guven, Esin;Duus, Karen;Houen, Gunnar
酶联免疫吸附试验（ELISA）是一种经过验证的、灵敏的检测人类自身抗体的方法，但可能存在特异性问题。非特异性结合是一个众所周知的问题，经常在自身抗体测试中观察到，当血清在塑料表面孵育时，例如ELISA板。为了了解非特异性免疫球蛋白沉积的机制，我们在这里详细分析了这一现象，并提出了减少非特异性结合引起的假阳性检测结果的方法。在未包被和自身抗原包被的ELISA孔中分析了怀疑具有自身反应性的血清中的非特异性结合水平，根据检测条件和血清性质，4-32%的血清显示出高水平的非特异性结合。发现非特异性结合血清中IgG和其他炎症介质的浓度增加。此外，通过提高IgG浓度并在40℃下培养血清，血清中可诱导非特异性结合。这表明在免疫球蛋白水平高、温度升高的炎症过程中可形成非特异性结合免疫球蛋白。我们发现非特异性结合水平与IgG浓度相关，因此提出非特异性结合可能被解释为IgG升高和炎症的信息发现。(C) 2013 Elsevier B.V.版权所有
Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlying non-specific immunoglobulin deposition, we here analyse the phenomenon in detail and we propose means of reducing false positive test results caused by non-specific binding.The level of non-specific binding, in sera with suspected autoreactivity, was analysed in non-coated and autoantigen-coated ELISA wells and 4-32% of sera showed a high level of non-specific binding depending on the assay conditions and serum properties. Non-specifically binding sera were found to contain increased concentrations of IgG and other inflammatory mediators. Moreover, non-specific binding could be induced in serum by increasing the concentration of IgG and incubating the serum at 40 degrees C. This suggests that non-specific binding immunoglobulins can be formed during inflammation with high immunoglobulin levels and elevated temperature. We show that the level of non-specific binding correlates with the IgG concentration and therefore propose that non-specific binding may be interpreted as an informative finding indicative of elevated IgG and inflammation. (C) 2013 Elsevier B.V. All rights reserved.