title: A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA.
abstract: This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. sujathar@u.washington.edu.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: PATH, Seattle, WA 98121, USA. msinghal@path.org.
authors.forename: Mitra
authors.initials: M
authors.lastname: Singhal
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. k.g.mckenzie@gmail.com.
authors.forename: Katherine G
authors.initials: KG
authors.lastname: McKenzie
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. jen.l.osborn@gmail.com.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Osborn
authors.affiliation: Oxford University Clinical Research Unit-Patan Academy of Health Sciences, Kathmandu, Nepal. amitarjyal@yahoo.com.
authors.forename: Amit
authors.initials: A
authors.lastname: Arjyal
authors.affiliation: Oxford University Clinical Research Unit-Patan Academy of Health Sciences, Kathmandu, Nepal. dongolsabina@yahoo.com.
authors.forename: Sabina
authors.initials: S
authors.lastname: Dongol
authors.affiliation: The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City Q5, Vietnam. sbaker@oucru.org.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Baker
authors.affiliation: Oxford University Clinical Research Unit-Patan Academy of Health Sciences, Kathmandu, Nepal. rishibas@wlink.com.np.
authors.forename: Buddha
authors.initials: B
authors.lastname: Basnyat
authors.affiliation: The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City Q5, Vietnam. jfarrar@oucru.org.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Farrar
authors.affiliation: The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City Q5, Vietnam. cdolecek@oucru.org.
authors.forename: Christiane
authors.initials: C
authors.lastname: Dolecek
authors.affiliation: PATH, Seattle, WA 98121, USA. gdomingo@path.org.
authors.forename: Gonzalo J
authors.initials: GJ
authors.lastname: Domingo
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. yagerp@uw.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Yager
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. blutz@u.washington.edu.
authors.forename: Barry
authors.initials: B
authors.lastname: Lutz
keywords: Salmonella enterica serovar Typhi; enzyme-linked immunosorbent assay (ELISA); flow-through membrane immunoassay (FMIA); indirect IgM assay; low resource setting; multiplex; serodiagnosis; typhoid
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