Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers.

Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlying non-specific immunoglobulin deposition, we here analyse the phenomenon in detail and we propose means of reducing false positive test results caused by non-specific binding. The level of non-specific binding, in sera with suspected autoreactivity, was analysed in non-coated and autoantigen-coated ELISA wells and 4-32% of sera showed a high level of non-specific binding depending on the assay conditions and serum properties. Non-specifically binding sera were found to contain increased concentrations of IgG and other inflammatory mediators. Moreover, non-specific binding could be induced in serum by increasing the concentration of IgG and incubating the serum at 40 °C. This suggests that non-specific binding immunoglobulins can be formed during inflammation with high immunoglobulin levels and elevated temperature. We show that the level of non-specific binding correlates with the IgG concentration and therefore propose that non-specific binding may be interpreted as an informative finding indicative of elevated IgG and inflammation.

[1]  A Voller,et al.  Enzyme immunoassays in diagnostic medicine. Theory and practice. , 1976, Bulletin of the World Health Organization.

[2]  H. Moutsopoulos,et al.  Anti-Ro (SSA)/La (SSB) antibodies and Sjögren's syndrome , 1990, Clinical Rheumatology.

[3]  L J Kricka,et al.  Human anti-animal antibody interferences in immunological assays. , 1999, Clinical chemistry.

[4]  R A Kyle,et al.  Retrospective cohort study of 148 patients with polyclonal gammopathy. , 2001, Mayo Clinic proceedings.

[5]  P. Plagemann,et al.  Immune complexes that bind to ELISA plates not coated with antigen in mice infected with lactate dehydrogenase-elevating virus: relationship to IgG2a- and IgG2b-specific polyclonal activation of B cells. , 1992, Viral immunology.

[6]  Y. Shoenfeld,et al.  Novel Insights Into Associations of Antibodies Against Cardiolipin and beta2-glycoprotein I with Clinical Features of Antiphospholipid Syndrome , 2007, Clinical reviews in allergy & immunology.

[7]  I. Kaplan,et al.  When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. , 1999, Clinical chemistry.

[8]  K. Skjødt,et al.  High Prevalence of Human Anti‐bovine IgG Antibodies as the Major Cause of False Positive Reactions in Two‐Site Immunoassays Based on Monoclonal Antibodies , 2004, Journal of immunoassay & immunochemistry.

[9]  J. C. Cohen Tervaert,et al.  Antineutrophil Cytoplasmic Autoantibodies: How Are They Detected and What Is Their Use for Diagnosis, Classification and Follow-up? , 2012, Clinical Reviews in Allergy & Immunology.

[10]  S. S. Levinson Antibody multispecificity in immunoassay interference. , 1992, Clinical biochemistry.

[11]  E. Engvall,et al.  Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. , 1971, Immunochemistry.

[12]  B. Kocsis,et al.  Comparison of Blocking Agents for an Elisa for Lps , 2000, Journal of immunoassay.

[13]  D. Vergani,et al.  Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? , 1990, The Lancet.

[14]  W. Maddrey,et al.  False positive ELISA tests for anticardiolipin antibodies in sera from patients with repeated abortion, rheumatologic disorders and primary biliary cirrhosis: correlation with elevated polyclonal IgM and implications for patients with repeated abortion. , 1988, Clinical and experimental immunology.

[15]  G. Hernández-Molina,et al.  The meaning of anti-Ro and anti-La antibodies in primary Sjögren's syndrome. , 2011, Autoimmunity reviews.

[16]  T. Jaskowski,et al.  Heterophile Antibodies to Bovine and Caprine Proteins Causing False-Positive Human Immunodeficiency Virus Type 1 and Other Enzyme-Linked Immunosorbent Assay Results , 1999, Clinical Diagnostic Laboratory Immunology.

[17]  Bo Nilsson,et al.  C3 Adsorbed to a Polymer Surface Can Form an Initiating Alternative Pathway Convertase1 , 2002, The Journal of Immunology.

[18]  A. Wiik,et al.  Absence of high‐affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease , 2005, Scandinavian journal of clinical and laboratory investigation.

[19]  S. S. Levinson,et al.  Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. , 2002, Clinica chimica acta; international journal of clinical chemistry.

[20]  B. Nilsson,et al.  Conformational epitopes of C3 reflecting its mode of binding to an artificial polymer surface. , 1993, Molecular immunology.

[21]  R. Falk,et al.  False‐positive myeloperoxidase binding activity due to DNA/anti‐DNA antibody complexes: a source for analytical error in serologic evaluation of anti‐neutrophil cytoplasmic autoantibodies , 2000, Clinical and experimental immunology.

[22]  P. Elder,et al.  Rheumatoid factor and false positive sex-hormone binding globulin. , 2003, Clinica chimica acta; international journal of clinical chemistry.

[23]  D. Schultz,et al.  Antineutrophil cytoplasmic antibodies: major autoantigens, pathophysiology, and disease associations. , 1995, Seminars in arthritis and rheumatism.

[24]  N. Després,et al.  Antibody interference in thyroid assays: a potential for clinical misinformation. , 1998, Clinical chemistry.

[25]  B. van Weemen,et al.  Immunoassay using antigen—enzyme conjugates , 1971, FEBS letters.

[26]  Yuhong Xiao,et al.  Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)--not all BSAs are alike. , 2012, Journal of immunological methods.

[27]  P. Meroni,et al.  Pathogenesis of antiphospholipid syndrome: understanding the antibodies , 2011, Nature Reviews Rheumatology.

[28]  L. Cole,et al.  False-positive hCG assay results leading to unnecessary surgery and chemotherapy and needless occurrences of diabetes and coma. , 1999, Clinical chemistry.