DESIGN OF A SPECIFIC, SENSITIVE ASSAY FOR THE DETECTION OF ANTI- VIMENTIN ANTIBODIES USING LUMINEX BEAD-BASED TECHNOLOGY: CHARACTERIZATION AND DISCUSSION OF ANTI-VIMENTIN ANTIBODY IN THE CARDIAC ALLOGRAFT TRANSPLANT POPULATION By

Background: The intermediate filament vimentin is not normally exposed to the immune system, however when exposed, anti-vimentin antibodies (AVA) can be provoked. AVA have been implicated in the development of cardiac allograft vasculopathy (CAV), limiting the long-term benefits of cardiac transplantation. Our aim was to develop a more specific and sensitive assay for AVA than currently available and determine if low levels of AVA are associated with CAV. Methods: A multiplexed assay for AVA was designed and validated by parallel testing with a commercially available ELISA for AVA. Sera titration and competitive inhibition with soluble vimentin were used to assess sensitivity and specificity. Preand post-transplant sera from forty-six patients were tested under IRB approval. Post-transplant sera were obtained within 12 months after transplantation. Results: In parallel titration studies, the bead-based assay was found to be twice as sensitive as the commercially available ELISA. Competitive inhibition studies of five sera resulted in a mean of 60%±28% reduction of antibody binding, confirming the Luminex assay specificity. The incidence of AVA in preii transplant sera was 39.1%. Conclusions: A Luminex bead based assay for AVA was developed that is both specific and twice as sensitive as a commercially available ELISA. Although a high incidence of AVA among cardiac transplant candidates was observed pretransplant, longer term studies will be needed to confirm any association with CAV post-transplant. Thesis Readers: Mary S. Leffell, Ph.D., Mentor Andrea A. Zachary, Ph.D., Mentor Christov Roberson, Ph.D., Reader Bob Lessick, Lecturer Patrick Cummings, Ph.D., Academic Advisor