Development of a multiplexed antigen detection immunoassay for detection of viral agents

Abstract

Purpose: The spread of infectious disease continues to present a challenge for modern global public health initiatives, as was evidenced by the recent Ebola outbreak in West Africa. In disease outbreaks, diagnostics are the first line of defense in identifying the causative agent, treatment decisions, and control and prevention of future outbreaks. Methods & Materials: We designed a multiplexed immunoassay centered on detection of viral antigens in serum/plasma samples for EBOV, LASV, MARV, CCHFV, RVFV, alphaviruses, and flaviviruses. This flexible, immunoassay system, based on the MAGPIX® platform has faster sample-to-answer time over traditional methods and higher throughput, as assays can be multiplexed. Assays were developed and validated against live and inactivated viral supernatants and other antigen types. Inclusivity and exclusivity panels were run to ensure no crossreactivity between assays. Results: Each assay was able to detect the appropriate target, whether in inactivated, recombinant, and live agent form. Limits of detection were generated in plaque forming units (pfu) per ml of live agent. Limits of detection were also established for appropriate recombinant reagents when available, as these reagents serve as the positive controls for the multiplexed assays when deployed to overseas partners. Conclusion: Development of robust and sustainable immunoassays is important as they have great potential for use during outbreak events to provide a more complete picture of the presence of viral agents. Immunoassays are capable of providing further insight to shape outbreak response. The recent Ebola outbreak demonstrated the great sensitivity of RT-PCR, but often patients remained positive by PCR well after other symptoms subsided. An integrated diagnostic system that uses sensitive and specific RT-PCR in combination with higher throughput, multiplexed immunodiagnostic methods can provide the highest confidence in a diagnostic result.