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

摘要

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.