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      A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients

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          Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibodies are lacking. The objective of this study was to design a simple set of steps to validate a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. An interleukin-24 (IL-24) sandwich ELISA system was prepared with a monoclonal mouse capture antibody and a polyclonal goat detection antibody and tested for interference by rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies. Plasma samples from 23 patients with RA and SpA were used. No differences were found between plasma samples measured in wells coated with anti-IL-24 specific antibody and in wells coated with isotype control antibody (false positive results), and recombinant human IL-24 was not recovered in spiked samples (false negative results). This interference was removed after preincubating the plasma samples from patients with arthritis with goat or bovine IgG, suggesting that anti-animal IgG antibodies found in the plasma of the arthritis patients caused the false results. Additional testing showed that the signal-to-noise ratio could be increased by titration of the capture and detection antibodies and by using the ELAST amplification system. Finally, the calculated concentration of IL-24 was increased in ethylenediaminetetraacetic acid (EDTA) plasma compared to heparin plasma and serum and decreased with repetitive freeze/thaw cycles of the samples illustrating how sample handling could additionally contribute to the variations reported by different laboratories in measurement of the same analyte. This study proposes a simple set of validation steps to evaluate and optimize a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. Anti-animal IgG antibodies are also present in healthy individuals, suggesting that validation of ELISA systems for measuring non-arthritis samples could also be improved by this simple set of validation steps.

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          Most cited references 41

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          The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.

          The revised criteria for the classification of rheumatoid arthritis (RA) were formulated from a computerized analysis of 262 contemporary, consecutively studied patients with RA and 262 control subjects with rheumatic diseases other than RA (non-RA). The new criteria are as follows: 1) morning stiffness in and around joints lasting at least 1 hour before maximal improvement; 2) soft tissue swelling (arthritis) of 3 or more joint areas observed by a physician; 3) swelling (arthritis) of the proximal interphalangeal, metacarpophalangeal, or wrist joints; 4) symmetric swelling (arthritis); 5) rheumatoid nodules; 6) the presence of rheumatoid factor; and 7) radiographic erosions and/or periarticular osteopenia in hand and/or wrist joints. Criteria 1 through 4 must have been present for at least 6 weeks. Rheumatoid arthritis is defined by the presence of 4 or more criteria, and no further qualifications (classic, definite, or probable) or list of exclusions are required. In addition, a "classification tree" schema is presented which performs equally as well as the traditional (4 of 7) format. The new criteria demonstrated 91-94% sensitivity and 89% specificity for RA when compared with non-RA rheumatic disease control subjects.
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              Regulation and functions of the IL-10 family of cytokines in inflammation and disease.

              The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29. Evolutionarily, IL-10 family cytokines emerged before the adaptive immune response. These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation. Finally, IL-10 itself can repress proinflammatory responses and limit unnecessary tissue disruptions caused by inflammation. Thus, IL-10 family cytokines have indispensable functions in many infectious and inflammatory diseases.

                Author and article information

                Springer International Publishing AG (Cham )
                15 June 2013
                15 June 2013
                : 2
                [ ]Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, Aarhus C, DK-8000 Denmark
                [ ]Department of Clinical Medicine, Aarhus University, Brendstrupgårdsvej 100, Aarhus N, DK-8200 Denmark
                [ ]Department of Rheumatology, Aarhus University Hospital, Nørrebrogade 44, Aarhus C, DK-8000 Denmark
                © Kragstrup et al.; licensee Springer. 2013

                This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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