title: The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases.
abstract: Autoantibodies directed against intracellular antigens can be detected by immunoblotting (IB). Due to its high sensitivity this technique has many advantages, but it can give misleading results when the specific bands are weak or blurred against the background staining. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Moreover, these agents appear to have a renaturating action towards proteins and antigens. Tween 20 has a more pronounced renaturating effect on proteins than other detergents and thereby improves antigen-antibody binding. To evaluate the effect of Tween 20 on specific autoantibody detection by IB, we tested the sera of 162 patients with connective tissue diseases (CTDs) by adding this detergent at certain steps of the IB assay. We found that the use of Tween 20 in the IB procedure significantly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa and C, Sm B/B' and D. Moreover, it increased the sensitivity for the detection of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantly decreased the binding of autoantibodies specific for ribosomal P proteins, La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the method. We conclude that the addition of Tween 20 to standard IB is advantageous for anti-nuclear antigen antibody detection and improves the sensitivity of the method in revealing anti-Sm-positive sera in SLE. However, Tween 20 is not recommended for the detection of anti-cytoplasmic antibodies.
authors.affiliation: Cattedra e Divisione di Reumatologia, Dipartimento di Scienze Mediche e Chirurgiche, Universit¿a di Padova, Padova, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Zampieri
authors.forename: A
authors.initials: A
authors.lastname: Ghirardello
authors.forename: A
authors.initials: A
authors.lastname: Doria
authors.forename: M
authors.initials: M
authors.lastname: Tonello
authors.forename: R
authors.initials: R
authors.lastname: Bendo
authors.forename: K
authors.initials: K
authors.lastname: Rossini
authors.forename: P F
authors.initials: PF
authors.lastname: Gambari
mesh_terms: D001323:Autoantibodies / Q000097:blood*; D003240:Connective Tissue Diseases / Q000097:blood / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D011136:Polysorbates; D014407:Tumor Cells, Cultured
chemical_list: D001323:Autoantibodies; D011136:Polysorbates