This industrial-academic collaboration seeks to analytically validate a highly specific and sensitive multiplexed immunoassay for glial fibrillary acid protein (GFAP) and tau in serum and/or plasma to improve treatment decisions for neonates with hypoxic-ischemic encephalopathy (HIE). HIE, which affects approximately 1 in every 1,000 births, is a major cause of death and disability for children. Prompt whole body cooling of neonates improves outcomes in some cases; however, 24% of cooled neonates die from HIE, and 19% of survivors develop cerebral palsy despite best available treatment. Adjuvants to hypothermia promise to broaden the effectiveness of treatment. Clinical testing of these investigational adjuvants has been slow due to the difficulty in identifying neonates who are most likely to benefit from treatment. By prescreening neonates at the extremes of the injury spectrum, an objective simple blood test that stratifies neonates according to injury severity and monitors treatment responses would accelerate trials of investigational adjuvants. The multiplex employs a novel format with approximately 100-1,000x higher sensitivity than regular immunoassays, which improves quantitation in the clinically relevant ranges of these low abundance biomarkers. Our preliminary data demonstrate that our new assay format is sufficiently sensitive to measure GFAP and tau in serum and plasma. Collaborators at Johns Hopkins University (Drs. Everett and Northington) have demonstrated clinical utility of GFAP and tau as markers of injury severity and functional outcomes in HIE. This proposal seeks to fully develop and optimize the ultrasensitive multiplex using our phase-gated process, followed by extensive analytical validation. First-year tasks are extensive characterization of key critical reagents, assay fine-tuning, and establishment of analytical controls at key concentrations for stratifying neonates according to HIE severity. A risk analysis will be performed, and the effect of preanalytical factors and potential interferents will be assessed and mitigated. In year two, the design will be frozen and transferred to manufacturing. QC protocols for critical reagents will be established and executed, and components will be configured following MSD?s ISO 9001:2015-certified quality management system. Three kit lots at final design will be manufactured. Assay performance will be assessed throughout the project, and include testing of clinical samples. A formal analytical assay validation plan will be developed in collaboration with a clinical chemist (Dr. Sokoll from JHU), with input from the NIH Program Officers, and based on the 2018 FDA Bioanalytical Method Validation Guidance for Industry. The final analytical validation study will be performed at three sites: MSD, Johns Hopkins Hospital, and All Children?s Hospital. Multi-site testing will include testing of proficiency samples, a 20-day reproducibility study, and a lot-to lot comparison. Additionally, real-time and accelerated stability tests will be performed in year 4.
When caring for neonates with hypoxic-ischemic encephalopathy (HIE), neonatologists lack sufficient tools to assess severity of brain damage or monitor response to therapy. This proposal seeks to analytically validate a highly sensitive and specific research-use only blood immunoassay for objectively measuring the extent of brain injury caused by HIE. All critical components for the assay will be carefully characterized, three kit lots will be manufactured, and the immunoassay will be evaluated at three different sites. Long-term, the assay will help to stratify neonates with HIE for clinical trials of investigational adjuvants to therapeutic hypothermia and enable personalized treatments tailored to the severity of injury.