Detection of immunoglobulin E using an aptamer based dot-blot assay

Abstract

A novel aptamer based dot-blot assay for the detection of immunoglobulin E (IgE) was developed. A biotinylated aptamer was employed as the bio-recognition element to specifically interact with the target protein immobilized onto a nitrocellulose membrane substrate. Avidin conjugated horseradish peroxidase was introduced onto the membrane through the biotin-avidin system to catalyze the hydrogen peroxide mediated oxidation of 3,3′,5,5′-tetramethylbenzidine, thereby producing the blue-colored insoluble product. The intensity of the dots increased as the concentration of IgE increased. The spot intensity was quantified using a simple portable instrument. A linear response relationship between the spot intensity and the concentration of IgE over the range of 50 nmol/L to 1 μmol/L was obtained. The detection limit for IgE using the aptamer-based assay was 2.89 nmol/L. This assay was found to discriminate IgE from non-target proteins such as thrombin, bovine serum albumin and immunoglobulin G. immunoglobulin E, dot-blot assay, aptamer, biotin-avidin system Citation:Wang Y X, Ye Z Z, Ying Y B. Detection of immunoglobulin E using an aptamer based dot-blot assay.

Label-free colorimetric aptasensor for IgE using DNA pseudoknot probe

“…In comparison with previously reported methods, our approach avoids constructing complicated aptasensors and shows better or comparable sensitivity toward IgE molecules. [28][29][30][31] Of note, however, compared with the sticky-end pairing-based colorimetric aptasensor reported by Wu et al, 19 our approach is less sensitive. This existing assay, however, requires slow, multi-step preparation processes of nanoparticle-aptamer conjugates that take 2 days, thus our approach is more convenient, faster, and cheaper.…”

Petal-like CdS nanospheres-based electrochemiluminescence aptasensor for detection of IgE with gold nanoparticles amplification

“…5B). The limit of detection was experimentally found to be 8.0 Â 10 À14 M, which is lower than 37 pM for a label-free electrochemical aptasensor [19], 211 fM for a fluorescent method [20], 4.4 pM for an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay [21], 2.89 nM for an aptamer based dot-blot assay [25]. …”

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications

“…A fully microchip integrated version of the electrophoretic selection process by combining the bead-based aptamer selection (bead-based PCR amplification) with gel electrophoresis was improved [23], resulted in aptamers reaching a K D value of 18 nM in only 3 selection rounds [24]. [97]. However, the conventional HRP label can be replaced by DNAzymes exhibiting peroxidase activity that can be conveniently linked to the aptamer sequence, resulting in a bifunctional probe, e.g., IgE aptamer and peroxidase-active hemin aptamer.…”

“…Relevant examples indicate that such assays can be accomplished in ca. 40 min with LODs in the lower nanomolar range [97]. However, the conventional HRP label can be replaced by DNAzymes exhibiting peroxidase activity that can be conveniently linked to the aptamer sequence, resulting in a bifunctional probe, e.g., IgE aptamer and peroxidase-active hemin aptamer.…”