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PubMed · 12741641

Solid-phase immunoassay detection of peptides from complex matrices without a separation.

Xiaoyan Zhao·Sumith Kottegoda·Scott A Shippy
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Abstract

A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps. Both direct and indirect immunoassay methods are demonstrated; the indirect is more sensitive and features a 1 pmol detection limit of neat dynorphin A solutions. To further increase the immunoassay sensitivity, a novel capillary blotting system with hydrophilic membranes was designed where optimized sample volumes of 167 nL were deposited for each spot. The area-reduced blotting method shows a 1000-fold improved, 1.3 fmol spot(-1) detection limit of a dynorphin A diluted in a buffered solution of 150 mg L(-1) of casein. Low-flow push-pull perfusates with volumes of 1 microL sampled from the striatum of the rat were assayed for dynorphin A by the method of standard addition. The detection limit was estimated to be 1.9 fmol in the low-flow push-pull perfusates. These data demonstrate a solid-phase near infrared immunofluorescence strategy for the study of peptides directly blotted from chemically complex biological fluid matrices.

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DynorphinsFluorescence Polarization ImmunoassaySpectroscopy, Near-InfraredJournal ArticleResearch Support, Non-U.S. Gov't

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Xiaoyan Zhao, Sumith Kottegoda, Scott A Shippy. 2003. Solid-phase immunoassay detection of peptides from complex matrices without a separation.. https://doi.org/10.1039/b210782a

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