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A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA
PATH, Seattle, WA 98121, USA
Oxford University Clinical Research Unit-Patan Academy of Health Sciences, Kathmandu, Nepal
The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City Q5, Vietnam
* Author to whom correspondence should be addressed.
Received: 1 February 2013; in revised form: 13 March 2013 / Accepted: 21 March 2013 / Published: 2 April 2013
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.
Keywords: flow-through membrane immunoassay (FMIA); enzyme-linked immunosorbent assay (ELISA); multiplex; indirect IgM assay; Salmonella enterica serovar Typhi; typhoid; serodiagnosis; low resource setting
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Ramachandran, S.; Singhal, M.; McKenzie, K.G.; Osborn, J.L.; Arjyal, A.; Dongol, S.; Baker, S.G.; Basnyat, B.; Farrar, J.; Dolecek, C.; Domingo, G.J.; Yager, P.; Lutz, B. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA. Diagnostics 2013, 3, 244-260.
Ramachandran S, Singhal M, McKenzie KG, Osborn JL, Arjyal A, Dongol S, Baker SG, Basnyat B, Farrar J, Dolecek C, Domingo GJ, Yager P, Lutz B. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA. Diagnostics. 2013; 3(2):244-260.
Ramachandran, Sujatha; Singhal, Mitra; McKenzie, Katherine G.; Osborn, Jennifer L.; Arjyal, Amit; Dongol, Sabina; Baker, Stephen G.; Basnyat, Buddha; Farrar, Jeremy; Dolecek, Christiane; Domingo, Gonzalo J.; Yager, Paul; Lutz, Barry. 2013. "A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA." Diagnostics 3, no. 2: 244-260.