Abstract
The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent. Immunoassays were then performed in a duplex format, demonstrating multiplex detection in a single well with limits of detection equivalent to the single assay format: 9.8 ng/mL chicken IgG and 7.8 ng/mL SEB.
Highlights
Labeled antibodies represent key reagents for numerous bioanalyses, including immunoassays and immunostaining, and are used in a myriad of applications that range from medical diagnosis through biological warfare agent monitoring
The plates were dried, and fluorescence intensities were measured at the emission maxima of the specific fluorescent label used (i.e., NC605 at 605 nm; NC650 at 650 nm; Cy5 at 670 nm)
NCs have several unique properties that make them ideal as fluorescent labels for a variety of applications. Their broad absorption and narrow emission profiles are excellently suited for multiplexing
Summary
Labeled antibodies represent key reagents for numerous bioanalyses, including immunoassays and immunostaining, and are used in a myriad of applications that range from medical diagnosis through biological warfare agent monitoring. The emission maxima of the NCs were centered at 605 ± 3 nm (NC605) and 650 ± 3 nm (NC650), and provided spectrally resolved multicolor detection with minimal crosstalk For this initial proof-of-concept study, SEB and chicken IgG immunoassays were selected as targets since they have been shown in previous fluorescent immunoassay studies and control experiments (data not shown) to be highly selective with no observable cross-reactivity, negating this potentially complicating issue [7,8,9,10]. The NC-based single sandwich immunoassays were readily adapted to a multiplex format for the simultaneous detection of two target antigens
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