title: Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers.
abstract: 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.
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Esin
authors.initials: E
authors.lastname: Güven
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Karen
authors.initials: K
authors.lastname: Duus
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Magnus Christian
authors.initials: MC
authors.lastname: Lydolph
authors.affiliation: Department of Microbiological Diagnostics & Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Charlotte Sværke
authors.initials: CS
authors.lastname: Jørgensen
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Inga
authors.initials: I
authors.lastname: Laursen
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark. Electronic address: gh@ssi.dk.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
mesh_terms: D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens; D001666:Binding Sites, Antibody; D015415:Biomarkers / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D018836:Inflammation Mediators / Q000097:blood*; D011237:Predictive Value of Tests; D011489:Protein Denaturation; D055550:Protein Stability; D013696:Temperature
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D015415:Biomarkers; D007074:Immunoglobulin G; D018836:Inflammation Mediators
keywords: Autoantibodies; ELISA; Elevated immunoglobulin concentration; Inflammation; Non-specific binding