Development of a bead-based immunoassay to routinely measure vimentin autoantibodies in the clinical setting

Abstract

IntroductionVimentin is an intermediate filament protein generally expressed in the cytosol of many adult cell types, including leukocytes, fibroblasts and endothelial cells. Several tissue and/or injury-specific isoforms of vimentin are known to exist that may trigger autoimmune responses due to aberrant structural or conformational variations. Such scenarios include allograft rejection and certain autoimmune diseases, such as rheumatoid arthritis. The primary objective for this study was to develop a Luminex immunobead assay to quantitate circulating levels of vimentin antibodies and, secondarily, to appraise the feasibility of these autoantibodies as a biomarker for clinical diagnosis. MethodsRecombinant human vimentin was conjugated to MagPlex® beads using standard carbodiimide/NHS chemistry and coupling efficiency tested and assay parameters determined using a commercial anti-vimentin polyclonal antibody. A limited number of serum samples (n=71) were then tested to evaluate the diagnostic value for future biomarker development efforts. ResultsFindings from repeated testing of three distinct batches of assays provide assay range parameters of 0.18–15μg/mL, median inter-assay recovery parameter within 1% of completion, and inter-assay variation (%CV) at 7%. The assay was found to be stable at several conditions with less than 5% loss in a month. Preliminary evaluation of the assay demonstrates significantly (p=0.022) higher circulating levels of anti-vimentin antibodies in 51 cases of renal allograft rejection relative to 20 cases of age-matched controls. ConclusionA direct capture assay for vimentin autoantibodies was developed and analytically validated. Preliminary evaluation of this assay against patient materials was promising and justifies additional testing with larger cohorts in future studies.