Scheme 1. All-in-one immunoassay with a fluorescence detection operated with enzyme-labeled antibodies and a substrate.
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Scheme 2. All-in-one immunoassay with a fluorescence detection operated with fluorescence dye-labeled antibodies.
The all-in-one immunoassay with a fluorescence detection can be also applied with a fluorescent dye-labeled antibody conjugated to antigen formed from the immunoreaction in a kit. As shown in Scheme 1, the fluorescent dye-labeled antibody conjugated to antigen emits visible fluorescence. The color of fluorescence emitted in the all-in-one immunoassay is dependent on the properties of a fluorescence dye as shown in Scheme 2. For example, D-dimer in human serum was quantified within 20 min with the all-in-one immunoassay with a fluorescence detection operated using fluorescein labeled with an antibodies. The detection limit (8.2 ng/ml) of the all-in-one immunoassay using fluorescein-labeled antibodies was about 16-fold higher than that (0.5 ng/ml) of the all-in-one immunoassay using HRP-labeled antibodies even though the same fluorescence detection was operated. This is because brightness of fluorescence emitted form a small fluorescence dye labeled with a large antibody is quenched due to the interference of the antibody. However, Scheme 1 indicates that the all-in-one immunoassay with a fluorescence detection operated with a fluorescent dye-labeled antibody conjugated to antigen can be applied as a multiplex assay method capable of simultaneously sensing different types of target antigens in a sample. In addition, an antibody conjugated with quantum dotsor fluorescent nanospheres is used in All-in-One immunoassays.