Immobilization of DNA Aptamers on Polyester Cloth for Antigen Detection by Dot Blot Immunoenzymatic Assay (Aptablot)

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. 1. Introduction 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. One of the most widely used approaches for this purpose is the Enzyme Immunoassay (EIA) technique. The classic “sandwich” 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. The use of antibodies as assay reagents suffers from the high cost and time required for their production, as well as variability in stability, quality, and yield which may occur from one batch to another. Coating the solid phase with capture antibody consumes a high quantity of the available antibody, which must be applied in large excess to drive its immobilization on the solid phase. A variety of solid phases such as nonporous polystyrene microwells and microporous membranes lend themselves to the immobilization of antibodies for EIA procedures. Macroporous polyester cloth has also been used as a solid phase for the immobilization of antibodies in the development of simple dot blot assays [1]. Compared to nonporous and microporous solid phases, polyester cloth has the advantages of offering a readily available large surface for the immobilization of immunoreagents promoting rapid immunoreaction kinetics and ease of washing between reaction steps. Aptamers made of DNA or RNA oligonucleotides have emerged as a new class of synthetic receptors which in some applications may replace antibodies as reagents for the assay of macromolecule analytes, such as proteins