Preventing intense false positive and negative reactions attributed to the principle,of ELISA to re-investigate antibody studies in autoimmune diseases
Preventing intense false positive and negative reactions attributed to the principle,of ELISA to re-investigate antibody studies in autoimmune diseases
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DOI：
10.1016/j.jim.2014.03.013
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发表时间：
2014-05-01
影响因子：
2.2
通讯作者：
Shionoya, Hiroshi
中科院分区：
文献类型：
--
作者：
Terato, Kuniaki;Do, Christopher T.;Shionoya, Hiroshi
为了研究潜在的环境病原体在自身免疫性疾病发病机制中的可能作用，有必要研究对各种环境因子和自体成分的抗体反应。然而，由于其原理是利用蛋白质与塑料表面的高结合亲和力，传统的ELISA缓冲液都不能防止假阳性和假阴性反应。这项研究的目的是揭示与传统缓冲系统相关的所有类型的非特异反应，并使用ELISA最新开发的缓冲系统“ChonBlock(TM)”重新研究自身免疫性疾病患者对潜在环境致病和自体抗原的抗体反应。与传统缓冲系统相比，新型缓冲系统在减少样品中免疫球蛋白疏水结合到塑料表面所引起的最强烈的假阳性反应、“背景(BG)噪声反应”和其他非特异性反应方面非常有效，而不干扰抗原-抗体反应。应用此缓冲液，我们发现类风湿性关节炎(RA)和系统性红斑狼疮(SLE)患者对大肠杆菌O111：B4、大肠杆菌脂多糖(LPS)和多肽多糖(PG-PS)的抗体反应明显低于或有降低趋势，而对这些抗原的IgA抗体反应与正常对照组相当或有升高趋势。因此，在RA和SLE患者中，除克罗恩病外，针对这些抗体的IgA/Ig G抗体比率显著升高。要检测人血清中的抗体，必须使用适当的缓冲系统来消除假阳性和阴性反应，并包括抗原非包被空白井来确定个体样本的BG噪声反应。最后，基于我们在这项研究中的初步分析，我们认为，对潜在致病环境因素的低免疫球蛋白抗体应答可能是自身免疫性疾病的根本障碍。(C)2014 Chondrex，Inc.由Elsevier B.V.出版
To study the possible involvement of potential environmental pathogens in the pathogenesis of autoimmune diseases, it is essential to investigate antibody responses to a variety of environmental agents and autologous components. However, none of the conventional ELISA buffers can prevent the false positive and negative reactions attributed to its principal, which utilizes the high binding affinity of proteins to plastic surfaces. The aims of this study are to reveal all types of non-specific reactions associated with conventional buffer systems, and to re-investigate antibody responses to potential environmental pathogenic and autologous antigens in patients with autoimmune diseases using a newly developed buffer system "ChonBlock (TM)" by ELISA.Compared to conventional buffers, the new buffer was highly effective in reducing the most intense false positive reaction caused by hydrophobic binding of immunoglobulin in sample specimens to plastic surfaces, "background (BG) noise reaction", and other non-specific reactions without interfering with antigen-antibody reactions. Applying this buffer, we found that IgG antibody responses to Escherichia coli O111:B4, E. coli lipopolysaccharide (LPS) and peptidoglycan polysaccharide (PG-PS) were significantly lower or tended to be lower in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), whereas IgA antibody responses to these antigens were equal or tended to be higher compared to normal controls. As a consequence, the IgA/IgG antibody ratios against these agents were significantly higher in patients with RA and SLE, except for Crohn's disease, which showed significantly higher IgG responses to these antigens.To assay antibodies in human sera, it is indispensable to eliminate false positive and negative reactions by using an appropriate buffer system, and to include antigen non-coated blank wells to determine BG noise reactions of invidual samples. Finally, based on our preliminary analysis in this study, we propose that low IgG antibody responses to potential pathogenic environmental factors may be the fundamental disorder in autoimmune diseases. (C) 2014 Chondrex, Inc. Published by Elsevier B.V.