New and resurgent viral and antibiotic-resistant bacterial diseases are being encountered worldwide. The US CDC now
ranks hospital acquired infections among the top 10 leading causes of death in the US, costing $20 billion annually. Such
nosocomial infections presently affect 5% - 10% of hospitalized patients leading to 2 million cases and 99,000 deaths
annually. Until now, assays available to mount comprehensive surveillance of infectious disease exposure by biosecurity
agencies and hospital infection control units have been too slow and too costly. In earlier clinical studies we have
reported proteomic microarrays combining 13 autoimmune and 26 viral and bacterial pathogens that revealed
correlations between autoimmune diseases and antecedent infections. In this work we have expanded the array to 40
viruses and bacteria and investigated a suspected role of human endogenous retroviruses in autoimmune neuropathies.
Using scanning laser imaging, and fluorescence color multiplexing, serum IgG and IgM responses are measured
concurrently on the same array, for 14 arrays (patient samples) per microscope slide in 15 minutes. Other advantages
include internal calibration, 10 μL sample size, increased laboratory efficiency, and potential factor of 100 cost
Bacterial and viral pathogens are implicated in many severe autoimmune diseases, acting through such
mechanisms as molecular mimicry, and superantigen activation of T-cells. For example, Helicobacter pylori, well
known cause of stomach ulcers and cancers, is also identified in ischaemic heart disease (mimicry of heat shock protein
65), autoimmune pancreatitis, systemic sclerosis, autoimmune thyroiditis (HLA DRB1*0301 allele susceptibility), and
Crohn's disease. Successful antibiotic eradication of H.pylori often accompanies their remission. Yet current diagnostic
devices, and test-limiting cost containment, impede recognition of the linkage, delaying both diagnosis and therapeutic
intervention until the chronic debilitating stage. We designed a 15 minute low cost 39 antigen microarray assay,
combining autoimmune, viral and bacterial antigens1. This enables point-of-care serodiagnosis and cost-effective
narrowly targeted concurrent antibiotic and monoclonal anti-T-cell and anti-cytokine immunotherapy.
Arrays of 26 pathogen and 13 autoimmune antigens with IgG and IgM dilution series were printed in
triplicate on epoxysilane covalent binding slides with Teflon well masks. Sera diluted 1:20 were incubated 10 minutes,
washed off, anti-IgG-Cy3 (green) and anti-IgM-Dy647 (red) were incubated for 5 minutes, washed off and the slide was
read in an ArrayWoRx(e) scanning CCD imager (Applied Precision, Issaquah, WA).
As a preliminary model for the combined infectious disease-autoimmune diagnostic microarray we surveyed
98 unidentified, outdated sera that were discarded after Hepatitis B antibody testing. In these, significant IgG or IgM
autoantibody levels were found: dsDNA 5, ssDNA 11, Ro 2, RNP 7, SSB 4, gliadin 2, thyroglobulin 13 cases.
Since control sera showed no autoantibodies, the high frequency of anti-DNA and anti-thyroglobulin
antibodies found in infected sera lend increased support for linkage of infection to subsequent autoimmune disease.
Expansion of the antigen set with synthetic peptide sequences should reveal the shared bacterial/human epitopes
Polylysine and aminopropylsilane treated glass comprised the majority of substrates employed in first generation genetic microarray substrates. Second generation single stranded long oligo libraries with amino termini provided for controlled terminal specific attachment, and rationally designed unique sequence libraries with normalized melting temperatures. These libraries benefit from active covalent coupling surfaces such as Epoxysilane. The latter's oxime ring shows versatile reactivity with amino-, thiol- and hydroxyl- groups thus encompassing small molecule, oligo and proteomic microarray applications. Batch-to-batch production uniformity supports entry of the Epoxysilane process into clinical diagnostics. We carried out multiple print runs of 21 clinically relevant bacterial and viral antigens at optimized concentrations, plus human IgG and IgM standards in triplicate on multiple batches of Epoxysilane substrates. A set of 45 patient sera were assayed in a 35 minute protocol using 10 microliters per array in a capillary-fill format (15 minute serum incubation, wash, 15 minute incubation with Cy3-labeled anti-hIgG plus Dy647-labeled anti-hIgM, final wash). The LOD (3 SD above background) was better than 1 microgram/ml for IgG, and standard curves were regular and monotonically increasing over the range 0 to 1000 micrograms/ml. Ninety-five percent of the CVs for the standards were under 10%, and 90% percent of CVs for antigen responses were under 10% across all batches of Epoxysilane and print runs. In addition, where SDs are larger than expected, microarray images may be readily reviewed for quality control purposes and pin misprints quickly identified. In order to determine the influence of stirring on sensitivity and speed of the microarray assay, we printed 10 common ToRCH antigens (H. pylori, T. gondii, Rubella, Rubeola, C. trachomatis, Herpes 1 and 2, CMV, C. jejuni, and EBV) in Epoxysilane-activated slide-wells. Anti-IgG-Cy3 direct binding to printed IgG calibration spots could be detected (3 x LOD) above background at 100 pg/ml (0.13 femtomoles sample content) in a 10 minute incubation. The LOD for detection of serum anti-H. pylori antibody level was 9 ng/ml in the same incubation time.