Abstract
BackgroundThe 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. MethodsWe 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. ResultsWe 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). ConclusionWith 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.
Highlights
Immunoassays were first reported in the 1960
Purity of the antigen was validated by means of SDS PAGE and Western Blot using antibodies directed against either human chitinase-3-like protein 1 (CHI3L1) or 6 x His-Tag. 96 well plates were purchased from Thermo Scientific and display either a hydrophobic surface
Since there is no enzyme-linked immunosorbent assay (ELISA) available for the detection of antibodies against CHI3L1, we have developed a corresponding assay
Summary
Immunoassays were first reported in the 1960. This so-called radioimmunoassay (RIA) was used to determine the insulin concentration in human plasma [1]. The ELISA technology was employed for the verification of candidate biomarkers to improve the diagnostic and control of various diseases as well as the monitoring of drug response [3, 4]. 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. Methods: 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 1⁄4 23, respectively). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p 1⁄4 0.0005). 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
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