Development of a High Sensitivity, Multiplex Vertical Flow Immunoassay System for the Detection of Biothreat Agents

Abstract

Effective biodefense requires diagnostic technologies capable of rapidly screening potentially contaminant materials and individuals to safeguard public health. The historical reliance on centralized laboratory networks, with their delayed turnaround times, has motivated a shift toward developing decentralized systems that enable early detection and expedite appropriate countermeasures. However, a critical gap exists between field-deployable systems that are cost-effective and those that offer highly sensitive, multiplex capabilities for biothreat detection. The goal of this project was to advance a promising vertical flow immunoassay (VFI) technology from laboratory concept toward a field-deployable prototype through a systematic methodology. The foundation of this effort began with establishment of the manufacturing process for the VFI’s core antibody microarray, which successfully increased throughput by 130% and set a standard for quality production. Building upon this manufacturing base, the VFI subsystems were validated, first by demonstrating a 2.5-fold sensitivity enhancement over prior art LFI devices for detection of a viral select agent, and then by expanding the system into a portable, multiplexed format for detecting bacterial select agents (3 targets) with up to 8-fold sensitivity enhancement in a clinically-relevant matrix. This development culminated in a fully integrated sample-to-answer prototype that demonstrated up to 16-fold sensitivity enhancement for detecting a panel of select agents (4 targets) in diverse sample matrices including buffer, human serum and urine, and soil extracts. To prepare the prototype system for real-world use, its storage reliability was established through the use of an innovative study design, a statistical model, and trehalose excipient, to overcome challenges with signal variation to provide rigorous evidence for at least 6 months of stability with enhanced thermostability. This evidence was a critical requirement for the final phase of maturation, where 2 additional toxin assays were integrated, a quality management system was established to scale kit production, and the complete system was delivered to government agencies and industry partners for human factors and conformance testing. This final evaluation verified the system’s promising analytical specificity and competitive sensitivity for detecting viable Y. pestis pathogens, while identifying targeted paths for optimization and validation of the remaining panel members. While further work remains in the path toward final deployment, this dissertation delivers a prototype system validated for detecting a panel of high priority select agents (7 targets), as well as a comprehensive technology development methodology to enhance national security through advanced biodefense.