Deutschmann Claudia, Roggenbuck Dirk, Schierack Peter, Rödiger Stefan
Institute of Biotechnology, Faculty Environment and Natural Sciences, Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, 01968 Senftenberg, Germany.
Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus - Senftenberg, The Brandenburg Medical School Theodor Fontane, The University of Potsdam, Senftenberg, Germany.
Heliyon. 2020 Jan 18;6(1):e03270. doi: 10.1016/j.heliyon.2020.e03270. eCollection 2020 Jan.
The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios.
We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems.
We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005).
With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies.
酶联免疫吸附测定（ELISA）是临床诊断中不可或缺的工具，可用于识别或区分自身免疫性疾病等疾病，还可监测疾病进展或控制药物疗效。ELISA的一个应用案例是区分不同状态（如健康与患病）。另一个目标是对相关生物标志物进行定量评估，如自身抗体。因此，ELISA技术也用于新自身抗体的发现和验证。然而，关键的兴趣点在于开发免疫测定法，以便在疾病早期阶段灵敏且特异地检测此类生物标志物。因此，用户必须处理许多参数，如缓冲系统或抗原 - 自身抗体相互作用，才能成功建立ELISA。通常，需要进行微调，如测试几种封闭物质，以获得高信噪比。
我们开发了一种ELISA，用于检测对照组和疾病组（每组n = 23）血清中针对几丁质酶 - 3样蛋白1（CHI3L1）的IgA和IgG自身抗体，CHI3L1是炎症性肠病（IBD）中一种新发现的自身抗原。测试了具有不同表面修饰（PolySorp和MaxiSorp包被）的微孔板，以检测重现性问题。
我们发现微孔板的表面性质有显著影响。在MaxiSorp包被的板上测量时，IgA抗体反应性显著降低，因为其处于背景噪声范围内（p < 0.0001）。不同板上的IgG抗体反应性没有差异，但板表面对测试结果有显著影响（p = 0.0005）。
通过本报告，我们希望引起读者对固相性质及其对ELISA检测自身抗体的影响的关注。我们希望让读者意识到选择错误的板可能导致假阴性测试结果，这反过来对自身抗体的发现会产生严重后果。
