Antibody-Detection-by-Agglutination-PCR (ADAP): An Ultra-Sensitive, High-Throughput, Multiplexable Tool for T1D Diagnosis and Monitoring

PROJECT SUMMARY ABSTRACT Accurate and timely detection of circulating autoantibodies against pancreatic islet antigens is critical to both research and clinical care for patients with type diabetes T D However this measurement remains highly variable across commercially available assays and such assays also may not adequately detect particularly low but clinically relevant levels of circulating autoantibodies This deficit translates into missed opportunities for both the timely initiation of the most appropriate treatment regimens and to support much needed research into novel and improved disease modifying interventions Additionally large scale public health screening efforts for T D are hampered by the low throughput nature of current bioassays and or their requirement for expensive and specialized instrumentation We have developed a proprietary patent pending PCR based technology termed Antibody Detection by Agglutination PCR ADAP ADAP is a high throughput assay that can be used for simultaneous detection of multiple antibodies while requiring only a very small amount of patient serum L ADAP also detects autoantibodies with to times greater sensitivity than the currently used immunometric and radio immuno assays and can be readily integrated into common quantitative PCR qPCR workflows using pre existing instrumentation that is available at many hospitals clinics and public health screening sites Importantly ADAP represents a significant departure from less effective PCR driven platforms such as immuno PCR overcoming many of the deficits inherent to this class of assays to afford a high throughput ultrasensitive robust reliable and specific detection method In this Phase application we propose to develop an ADAP based assay kit for detection of the four autoantibodies that form the basis for T D diagnosis The corresponding GAD IA insulin and ZnT antigens will be barcoded by conjugation to unique single stranded DNA sequences Agglutination of an antigen upon incubation with its cognate antibody brings the DNA sequences near to each other An appropriate bridge oligo is then supplied which upon ligation affords an amplifiable DNA duplex This antigen autoantibody binding event provides a PCR amplicon that enables ultrasensitive detection with minimal background signal Next we will use these appropriately validated reagents for analysis of patient serum samples in the context of T D both before and after diagnosis Finally it is increasingly appreciated that high affinity autoantibodies are privileged indicators of disease severity In anticipation of the diagnostic value of this new finding we also propose to create an innovative variant of the ADAP assay which we term ADAP to enable high throughput quantification of high affinity autoantibodies Deployment of such an assay may allow more accurate determination of T D prognosis and enable improved choices of therapeutic interventions In summary we seek to establish the ADAP T D assay kit not only as an effective alternative to current T D diagnostic platforms but one with significantly expanded capabilities in terms of sensitivity speed reproducibility and multiplexability that can be cost effectively integrated into existing laboratory and clinical workflows PROJECT NARRATIVE of individuals newly diagnosed with type diabetes T D have no affected relatives and this disease remains the most common form of diabetes affecting children resulting in serious and often life threatening problems if the disease is not diagnosed and treated effectively in a timely manner The current tests for T D are costly and difficult to perform and are therefore not suitable for large scale public health screening of neonates and those with affected first degree relatives We propose to develop a new method of detection that we call ADAP which is inexpensive fast to times more sensitive than existing methods and be easily performed on site in hospitals clinics and public health screening sites using existing equipment