Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout |
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Authors: | Lasse Välimaa Johanna Ylikotila Hannu Kojola Tero Soukka Harri Takalo Kim Pettersson |
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Institution: | 1.Department of Biotechnology,University of Turku,Turku,Finland;2.Kojola Hannu (private enterprise),Riihikoski,Finland;3.Innotrac Diagnostics Oy,Turku,Finland |
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Abstract: | Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared
with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from
only a small part of the microtiter well’s bottom surface and it is inadequate for the optimal assay sensitivity when using
binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to
condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter
wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native
(SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu–biotin
binding densities of 0.080 and 0.47 pmol/mm2, respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in
the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared
with a reference assay.
Figure The condensation of the binding area into a spot (right) results in a denser collection of the labeled antibodies and more favorable signal-to-background ratios compared with a
regular approach using a large binding area (left) |
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Keywords: | Streptavidin Spot Immunoassay Solid phase Point-of-care |
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