AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.
We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.