Josephine Ekomo's HRP Aptamer Project (2016)

Aptamer Selection: N71 Against Horseradish Peroxidase for ELISA

Introduction/Background

Horseradish Peroxidase(HRP) is a metalloenzyme that can be found within the root of a horseradish plant. It uses hydrogen peroxide to oxidize compounds and emits a blue color when exposed ultraviolet light. It has been said to be highly reactive

to human tumor cells and has a possible use in cancer therapy. Despite this its main use has been in diagnostics in one such procedure called an ELISA (Figure 1). An ELISA is an Enzyme-Linked ImmunoSorbent Assay with a plate-based technique that is designed to detect and quantify substances such as antibodies and proteins. It works through several methods; Direct ELISA(2.a) is where the target is immobilised onto a surface and blocked by the addition of an aptamer and conjugated HRP. This type of ELISA is used to estimate dissociation constants of aptamers against targets; determine affinity of any aptamer for a target.

In an Indirect ELISA(2.b), which will used in future work, the target is immobilized on a surface and the antibodies are allowed to bind to the target. The antibody capturing, enzyme labeled aptamer binds to that antibody and generates

signals. This is an unpopular method due to the uncertainty and unspecified impurities of antigen immobilization. However, this procedure allows an aversion to labelled antibodies to send signals when aptamers are more readily available.

A Sandwich ELISA isn’t dependent on target immobilization and instead falls back on the effectiveness of the aptamer being able to catch the target. The sandwich ELISA comes in many forms such as the Aptamer-target-antibody(2.c) where the aptamer is immobilized and it’s high affinity target will attach more so than an antibody(2.d). This is based around the idea of aptamers being easier to produce and being highly specific for a target.

In contrast to antibodies, aptamers have varied applications in assays and can be easily modified as required. Their small size also aids in transport to places antibodies are too large to fit through. Aptamers were selected in vitro starting with the incubation of an oligonucleotide library with target, separation of bound from unbound RNA sequences(Figure 3), and amplification which happens in several cycles to help generate an enriched pool of RNA that should have a high affinity for the target used during selection. Further methods can be seen in the methods and materials section below. Currently aptamer selection for HorseRadish Peroxidase is underway under conditions to be used in diagnostics.

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References