A novel approach for increasing sensitivity in lateral flow assays: Development of an enrichment module based on polyethylene sintered bodies

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Abstract

Lateral flow assays (LFAs, e.g. the well established pregnancy test) are frequently used, fast and easy-to-handle immunoassays with a broad application range. Nevertheless, the restriction to small sample volumes is one of the major drawbacks and can be the reason for lack of sensitivity. In order to detect even small amounts of analyte in big sample volumes without the need for time consuming sample preparation or sophisticated labelling or detection technologies, the aim of the here presented research was the development of an ‘enrichment module’, which can be integrated into the workflow of a LFA. The core element of this enrichment module was a cleavable biological interface structure. A fusion protein, which was expressed recombinant and consisted of a monomeric concanavalin A (Con A) domain and a monomeric streptavidin domain (SAv), was used as interface structure. For the construction of the entire module, the polysaccharide mannan was firstly covalently attached to a porous polyethylene (PE) sintered body in a four-stage procedure. This immobilisation procedure was monitored by a modified Bradford assay. In the next step, the Con A–SAv fusion protein was added. In order to assess the functionality of the enrichment module, a model assay was developed. The SAv domain of the attached fusion protein was specifically recognised by a primary and a secondary, horseradish peroxidase (HRP)-labelled, antibody. The HRP-reaction was used for photometric detection in order to characterise binding properties of the enrichment module and to prove binding characteristics of the fusion protein.

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