Abstract
A simple dot blot immunoenzymatic assay system was developed using polyester cloth coated with an oligo-DNA aptamer to provide a high-affinity macroporous surface for the efficient capture of a model protein analyte (thrombin) in complex sample matrices such as foods. Bound thrombin was detected immunoenzymatically using a peroxidase-linked antithrombin antibody and a chromogenic substrate. A unique feature of this approach, which we have termed “aptablot,” is the facile immobilization of DNA aptamers on the polyester surface by cross-linking with a brief exposure to ultraviolet light, and the simple assay format obviating the need for specialized instruments. The assay principle described herein should be broadly applicable to many situations where analytes must be detected in complex samples, with the main limiting factor being the availability of suitable DNA aptamers.
Highlights
The detection of specific macromolecules remains an important concern in the fields of human and animal health diagnostics, food safety testing, and basic research
The classic “sandwich” Enzyme Immunoassay (EIA) utilizes specific antibodies immobilized on a solid phase to create a high-affinity surface for the capture of antigens, which are subsequently detected by reaction with an enzyme-linked antibody
We have demonstrated the applicability of a simple aptablot system using a DNA aptamer as an inexpensive synthetic capture agent for the sensitive assay of a protein analyte in complex samples
Summary
The detection of specific macromolecules (e.g., protein antigens) remains an important concern in the fields of human and animal health diagnostics, food safety testing, and basic research. The immobilization of oligo-DNA probes on polyester cloth by brief exposure to ultraviolet light for use in the detection of polymerase chain reaction (PCR) products by hybridization has previously been shown [4], and it is surmised presently that this simple approach should permit the immobilization of DNA aptamers to create a high affinity capture surface for the assay of target analytes. In this “aptablot” approach, target molecules captured on the aptamer-coated cloth surface are detected by reaction with a dilute preparation of specific antibodies linked to a marker enzyme. The detection of thrombin is presently considered as a model system, since DNA aptamers for this protein have been well characterized [5, 6] and specific antithrombin antibodies are commercially available
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