Preventing intense false positive and negative reactions attributed to the principle of ELISA to re-investigate antibody studies in autoimmune diseases

Abstract

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™" 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.

Antigen-free control wells in an ELISA set-up for the determination of autoantibodies against G protein-coupled receptors—a requisite for correct data evaluation

“…Terato et al [6] also described this phenomenon, while especially focusing on sera from autoimmune diseases and developing a special buffer, which was able to reduce this existing problem. These authors demand that in order 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 (background) noise reactions of individual samples”.…”

“…As already done by Güven et al [5] and Terato et al [6], we would also, therefore, like to invite the autoantibody research community to enlarge the panel of control experiments about the non-specific non-coated plate or a proper control plate, if sophisticated membranes for detection of autoantibodies to membrane receptors are used and to see and report what happens with the results. Just check it out and test it at your set-up.…”

“…We are looking forward to receive not only discussion contributions to this question and problem but also results and experiences from different labs about similar constellations when using solid-phase ELISA technology (including marketed ELISA) for the detection of autoantibodies against G protein-coupled receptors. Such essential controls “are frequently omitted in plates made by investigators conducting basic research, and are not even included in commercially prepared plates” [6]. …”

Opportunistic Microorganisms in Rheumatic Diseases

“…Возможность участия УПМ в патогенезе РЗ подтвер-ждает не только выделение микроорганизмов, но и обна-ружение антибактериальных антител [22,23]. При РА су-щественно чаще, чем у больных ОА и здоровых лиц, опре-деляются антитела против O-и H-антигенов бактерий ро-да Proteus и O-антигена -E. coli [24].…”

The immunomodulation of dynorphin 1-17 and its biotransformation fragments on inflammatory signalling pathways

“…These antibody-based approaches, however, have several limitations including high levels of inter-assay variability, and importantly, the lack of available specific antibodies for different target proteins of interest (222,412,413). A minor modification in the protein structure serving to distinguish endogenous and exogenous variants (414), may not be detected by ELISA thereby, potentially resulting in false positive or negative findings (415). The development of specific antibodies is additionally challenging; it is an exhaustive and laborious process in addition to the requirement in designing various species-specific antibody epitopes (416).…”