title: Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus.
abstract: Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.
authors.affiliation: Sandia National Laboratories, Chemical and Radiation Detection Laboratories, P.O. Box 969, Livermore, CA 94551, USA. dreichm@sandia.gov
authors.forename: David S
authors.initials: DS
authors.lastname: Reichmuth
authors.forename: Serena K
authors.initials: SK
authors.lastname: Wang
authors.forename: Louise M
authors.initials: LM
authors.lastname: Barrett
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Throckmorton
authors.forename: Wayne
authors.initials: W
authors.lastname: Einfeld
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
mesh_terms: D000818:Animals; D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods*; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods*; D009980:Influenza A virus / Q000276:immunology* / Q000302:isolation & purification*; D013552:Swine / Q000821:virology*; D013997:Time Factors