title: 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.
authors.affiliation: Chondrex Inc., 2607 151st Place NE, Redmond, WA 98052, United States. Electronic address: terato@chondrex.com.
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Terato
authors.affiliation: Chondrex Inc., 2607 151st Place NE, Redmond, WA 98052, United States. Electronic address: cdo@chondrex.com.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Do
authors.affiliation: Chondrex Inc., 2607 151st Place NE, Redmond, WA 98052, United States. Electronic address: dcutler@chondrex.com.
authors.forename: Dawn
authors.initials: D
authors.lastname: Cutler
authors.affiliation: Chondrex Inc., 2607 151st Place NE, Redmond, WA 98052, United States. Electronic address: twaritani@chondrex.com.
authors.forename: Takaki
authors.initials: T
authors.lastname: Waritani
authors.affiliation: Asama Chemical Co. Ltd, Chuo-ku, Tokyo, Japan. Electronic address: h.shionoya@asama-chemical.co.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shionoya
mesh_terms: D000906:Antibodies / Q000097:blood*; D000907:Antibodies, Bacterial / Q000097:blood; D001172:Arthritis, Rheumatoid / Q000175:diagnosis / Q000276:immunology; D001327:Autoimmune Diseases / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology; D005188:False Negative Reactions; D005189:False Positive Reactions; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis / Q000276:immunology; D011485:Protein Binding
chemical_list: D000906:Antibodies; D000907:Antibodies, Bacterial; D002021:Buffers; D007070:Immunoglobulin A; D007074:Immunoglobulin G
keywords: Autoimmune diseases; CCP antibody; ELISA; Environmental agents; Non-specific reactions; Rheumatoid arthritis