Mary T. McBride

Toward a Multiplexed Serotyping Immunoassay for Foot-and-Mouth Disease Virus

Initial results demonstrating the feasibility of a multiplexed liquid array immunoassay for foot-and-mouth disease viral antigen detection and simultaneous serotype differentiation are presented. Serotype-specific antibodies from rabbit and guinea pig hyperimmunesera were isolated and prepared for use in a multiplexed, bead-based assay. The performance of all of the available antibodies as both capture and detector reagents was evaluated in the multiplexed system to establish a combination exhibiting the highest homotypic responses and lowest heterotypic reactions. The multiplexed assay was evaluated against inactivated cell culture supernatant samples of the same subtype as the virus used to raise the capture and detector antibodies. Distinct serotype differentiation was observed, except in the case of serotype SAT1. Subsequently, cell culture supernatant samples from a larger pool of viral subtypes were analyzed. Distinct serotype differentiation was obtained when analyzing cell culture supernatant samples from viral serotypes C, Asia, and SAT3, irrespective of the subtype. However, limitations of the current antibody pairs were realized in some inconclusive results obtained when analyzing samples from a broader range of O, A, and SAT2 subtypes. The results obtained in this initial study will be used to further optimize the assay using polyvalent or monoclonal antibodies and move toward the analysis of clinical samples.

A sample-in-answer-out instrument for the detection of multiple respiratory pathogens in unprepared nasopharyngeal swab samples

Multiplex RT-PCR suspension array assays provide a powerful tool for identifying the causative agent(s) of respiratory infections. These assays are time consuming and laborious on a time-per-sample basis if only a few samples require processing. To address this shortcoming and provide an automated solution for fast detection and identification of viral pathogens, we developed the first automated multiplex RT-PCR suspension array instrument capable of handling unprepared clinical samples. The instrument requires less than 3 minutes of hands-on time for a result generated in approximately 2.5 hours. In analytical studies, the instrument performed as well as manually performed assays. The performance of the instrument and loaded multiplex viral detection assay was then tested using unprepared nasopharyngeal samples. The instrument-performed assay detected 61 of 71 RSV positive samples, for a sensitivity of 85.9%. Adenovirus (n = 5) and influenza B (n = 3) were less prevalent in the sample set, but detected to similar levels, 80% and 75%, respectively. The same sample set was also tested using FDA approved immuno-assay rapid tests, and the instrument was found to be more sensitive than the rapid tests with the sole exception being influenza A (n = 16), which was poorly detected due to significant sequence mismatches between the influenza A primer/probe set included in the multiplex mixture and the circulating influenza A strains. Overall, these data demonstrate the developed prototype platform performs multiplex array assays as well as hand-performed assays, and that the instruments sensitivity and specificity are dictated by the quality of the loaded multiplex assay.

Apds, a Network-Ready, Broad Spectrum, Environmental Pathogen Detection System

The Autonomous Pathogen Detection System (APDS) 1 is a stand-alone pathogen detection system capable of rapid, continuous, low cost environmental monitoring of multiple airborne biological threat agents. Its basic design comprises aerosol sampling, in-line sample preparation, multiplex detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. Its primary application is to warn civilians and emergency preparedness personnel of a terrorist attack, the same system could also have a role in protecting military personnel from biological warfare attacks. APDS instruments can be used at high profile events such as the Olympics for short-term, intensive monitoring or more permanent installation in major public buildings or transportation nodes. All of these units can be networked to a single command center so that a small group of technical experts could maintain and respond to alarms at any of the sensors. The APDS has several key advantages over competing technologies: (1) the ability to measure up to 100 different agents and controls in a single sample, (2) the flexibility and ease with which new bead-based assays can be developed and integrated into the system, (3) the presence of an orthogonal, real-time detection module for highly sensitive and selective nucleic acid amplification and detection, (4) the ability to use the same basic system components for multiple deployment architectures, and (5) the relatively low cost per assay (<

Microbead immunoassay dipstick system (MIDS)

2 per 10-plex or

MiDAS (Micro-dot Array Sensor): toward a rapid, in-vivo, reproducible, multianalyte biosensor using inkjet printing technology

0.20 per assay) and minimal consumables.

Multiplexed Liquid Arrays for Simultaneous Detection of Simulants of Biological Warfare Agents

Summary form only given. We have developed a prototype system for reducing Luminex based immunoassay technology to a portable, customizable platform that can perform multiplexed assays on location - independent of clinical laboratory support. In principle, this device can be used to test for any biomolecular interaction in which strong binding occurs, including receptor-ligand coupling, enzyme-substrate binding, antibody-antigen interactions, and nucleic acid (DNA) hybridization reactions. Since an overwhelming majority of clinical laboratory tests are based on antibody-antigen interactions, however, we have focusing our initial efforts on multiplexed immunoassay detection.

Autonomous detection of aerosolized bacillus anthracis and Yersinia pestis

The Microdot Array Sensor (MiDAS) platform for making optical fiber-based sensors using inkjet printing technology is a paradigm shifting technology for delivering low cost, rapid, in-vivo, reproducible, multianalyte biosensors. A fast-response, reproducible, pH sensor is demonstrated. The MiDAS platform is very adaptable to new and existing indicator chemistries and can be used for detecting blood/gas and enzyme biomarkers.