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Suspension Microarray Immunoassay Signal Amplification Using Multilayer Formation

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A method for rapid amplification of signals is demonstrated in multiplexed fluid microarrays using the 'super avidin–biotin system' (SABS). The Luminex xMAP system is a specialized flow cytometer that performs multiplexed microsphere based fluoroimmunoassays. The xMAP system's standard protocol utilizes antibodies immobilized to microspheres as the capture surface for target antigens and complementary biotinylated antibodies serve as the recognition "tracer" reagents. This anti-body:target:biotinylated antibody complex is fluorescently labeled by the addition of streptavidin–phycoerythrin (SA–PE). The present study shows that addition of two simple steps greatly amplifies the signal produced by the standard protocol, typically by a factor of ∼10. After addition of the SA–PE, the microspheres are subsequently exposed to biotinylated goat anti-streptavidin antibody; this is then followed by a second round of SA–PE, which further amplifies the initial signal. This amplification protocol was demonstrated for multiplexed detection of cholera toxin and botulinum toxoid A. In addition, several SA–PE conjugates having a variety of SA-to-PE stoichiometries were evaluated, as well as an alternative protocol, wherein biotinylated PE was substituted for the anti-streptavidin antibody.


Document Type: Research Article


Publication date: 2008-02-01

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