Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing β-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-β. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-β (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.

For many infectious agents, the detection of antibodies is critical for diagnosis, monitoring and understanding vaccine responses. To facilitate the highly quantitative and simultaneous analysis of antibodies against multiple proteins from infectious agents, we have developed Luciferase Immunoprecipitation Systems (LIPS) arrays. By configuring microtiter plates with multiple antigens and testing control and infected serum samples at one time in solution, LIPS arrays provided highly reproducible antibody titers to panels of antigens with a wide dynamic range of detection. While all serum samples showed similar positive and negative immunoreactivity with internal control antigens derived from Influenza and Renilla luciferase-alone protein, respectively, antibody titers to many HCV and HIV antigens were generally 10 to over 400-fold higher in the infected versus uninfected samples. Additional screening of 18 proteins from the EBV proteome with serum samples from healthy EBV-infected individuals showed statistically significant antibody titers to 50% of the proteins tested. Antibody titers for the different EBV antigens in the healthy EBV-infected individuals were markedly heterogeneous highlighting the complexity of host humoral responses. These results suggest that LIPS arrays offer a highly discriminating platform for simultaneously profiling a wide spectrum of antibodies associated with many infectious agents.

B-cell mediated humoral responses are triggered in many human diseases including autoimmune, cancer, neurologic, and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire, for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed Luciferase immunoprecipitation systems (LIPS) that harnesses light emitting proteins to generate high definition antibody profiles optimal for both diagnostics and biomarker discovery. Here we describe the results and implications from a range of LIPS antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis, and provide a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring.

Infection with Borrelia burgdorferi is common in horses and ponies from the New England and mid-Lyme disease caused by the spirochete Borrelia burgdorferi represents an important problem in veterinary medicine due to the wide-spread infection of dogs, cats, and horses in areas where the organism is endemic (3, 30). While most cases of equine B. burgdorferi infection remain asymptomatic, some horses show signs of illness, including sporadic lameness, weight loss, arthritis, and encephalitis (16). Unlike humans, where the first sign of B. burgdorferi infection is erythema migrans skin lesions, the diagnosis of Lyme disease in horses is complex, and diagnostic testing is only one parameter for confirmation (16).A variety of serological tests, including immunofluorescence assays (IFA), Western blotting, and enzyme-linked immunosorbent assays (ELISAs), have been employed to detect human antibodies to B. burgdorferi (29). Considerable progress has been made in employing defined antigenic targets, such as the C6 peptide (1, 20, 27) and other recombinant B. burgdorferi antigens, including BBK07 (18, 19) and decorin binding proteins (Dbp) DbpA and DbpB (21,22,24,28,31,32). Although extensive testing of different antigens and assay formats has been performed for human Lyme disease (29), less is known about the optimal serological diagnosis of equine B. burgdorferi infection. Recently, the C6 SNAP test has been used for the serological diagnosis of equine Lyme disease (23, 26). Unfortunately, Chang and colleagues found that the C6 SNAP test detected only 63% of known, experimentally B. burgdorferiinfected horses, suggesting that this test is suboptimal for the diagnosis of equine infection (26). In light of the poor sensitivity of the currently available C6 SNAP test, a better understanding of humoral responses in B. burgdorferi-infected horses is needed.Luciferase immunoprecipitation systems (LIPS) comprise a relatively new approach for the serological testing of antibodies associated with many different pathogens (5). LIPS is based on employing light-emitting Renilla luciferase-fusion proteins in a liquid-phase immunoprecipitation assay. Serologic testing by LIPS for a variety of human pathogens provides the full exploitation of antibody profiles because of its robustness, multiplex capacity, and high sensitivity and specificity (6,11,12,14,15). Recently, a LIPS test employing a synthetic VOVO antigen consisting of two variants of immunodominant epitopes of the C6 peptide and OspC achieved 98% sensitivity and 100% specificity for the diagnosis of human Lyme disease, but the findings were not statistically different from those of the C6 ELISA (10). LIPS antibody profiling of recombinant DbpA and DbpB also was highly informative (10). In this study, LIPS tests for VOVO, DbpA, and DbpB antigens were used for measuring B. burgdorferi antibodies in horses, and the results were compared to IFA results for the diagnosis of equine B. burgdorferi infection.