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Int J Clin Exp Pathol 2013;6(6):1202-1210

Original Article
Novel detection of pancreatic and duodenal homeobox 1 autoantibodies (PAA) in human
sera using luciferase immunoprecipitation systems (LIPS) assay

William Donelan, Hai Wang, Shi-Wu Li, David Pittman, Yi Li, Shuhong Han, Yu Sun, Christopher Carter, Mark Atkinson, Westley Reeves, William
E Winter, Li-Jun Yang

Department of Pathology, Immunology and Laboratory Medicine, Department of Medicine, University of Florida College of Medicine, Gainesville,
Florida 32610, USA; Tianjin University of Science & Technology, Tianjin, 300457, China

Received February 24, 2013; Accepted April 16, 2013; Epub May 15, 2013; Published June 1, 2013

Abstract: We have previously identified pancreatic and duodenal homeobox 1 (Pdx1) autoantibodies (PAA) in sera from both non-obese
diabetic (NOD) mice and human type 1 diabetic (T1D) patients. A suitable non-radioactive, sensitive and specific assay is needed for large-
scale testing to determine the clinical utility of PAA. Here we reported a liquid-phase luciferase immunoprecipitation system (LIPS) assay by
generating a renilla luciferase (Rluc)-Pdx1 fusion protein as a sensitive non-radioactive antigen from mammalian cells combined with
immunoprecipitation to detect PAA in human sera. Sera from healthy donors and the University of Florida Pathology Laboratories, Endocrine
Autoantibody Laboratory were used to validate the LIPS assay for PAA. Antigenic specificity to Pdx1 was confirmed by using a Rluc-only control
compared to Rluc-Pdx1 fusion antigen and by competition assays using purified recombinant Pdx1 protein. We then used the LIPS assay to
assess the prevalence of triple autoantibodies (GADA, IA-2A, and IA-2βA), and PAA in non-T1D control sera, recent onset (RO)-T1D sera (mean
duration of T1D = 9.5 weeks), and long standing (LS)-T1D sera. Compared to clinical radioimmunoprecipitation assays (RIPA), the LIPS assay
showed comparable sensitivity and specificity for detection of GADA and IA-2A. PAA were detectable in human serum samples and higher in
triple-positive T1D autoantibodies (21% PAA positive in triple positive sera and 4% PAA positive in triple negative sera). Interestingly, PAA were
found to be highest in the non-T1D population, suggesting that PAA might have a clinical utility in screening high-risk population susceptible for
developing T1D. In conclusion, we have developed a liquid-phase, non-radioactive, sensitive and specific LIPS assay to detect PAA in human
sera, providing a useful tool for evaluating the clinical relevance of PAA. (IJCEP1302035).

Keywords: Pancreatic and duodenal homeobox 1 (Pdx1), Pdx1 autoantibodies (PAA), type I diabetes, luciferase immunoprecipitation systems
(LIPS) assay

Address correspondence to: Dr. Li-Jun Yang, Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of
Medicine, 1600 SW Archer Road, P.O. Box 100275, Gainesville, FL 32610, U.S.A. Phone: 352-392-0005; E-mail: yanglj@pathology.ufl.edu