The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases

Autoantibodies directed against intracellular antigens can be detected by i mmunoblotting (IB). Due to its high sensitivity this technique has many adv antages, but it can give misleading results when the specific bands are wea k or blurred against the background staining. To decrease background staini ng, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are general ly used as blocking agents. Moreover, these agents appear to have a renatur ating 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 au toantibody detection by IB, we rested the sera of 162 patients with connect ive 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 significan tly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa a nd C, Sm B/B' and D. Moreover, it increased the sensitivity for the detecti on of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantl y decreased the binding of autoantibodies specific for ribosomal P proteins , La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the me thod. We conclude that the addition of Tween 20 to standard IB is advantage ous for anti-nuclear antigen antibody detection and improves the sensitivit y of the method in revealing anti-Sm-positive sera in SLE. However, Tween 2 0 is not recommended for the detection of anti-cytoplasmic antibodies. (C) 2000 Elsevier Science B.V. All rights reserved.