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Alkaline phosphatase (ALP) was used as an amplification tool in lateral flow immunoassay (LFIA). Potato virus Х (PVX) was selected as a target analyte because of its high economic importance. Two conjugates of gold nanoparticles were applied, one with mouse monoclonal antibody against PVX and one with ALP-labeled antibody against mouse IgG. They were immobilized to two fiberglass membranes on the test strip for use in LFIA. After exposure to the sample, a substrate for ALP (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) was dropped on the test strip. The insoluble dark-violet diformazan produced by ALP precipitated on the membrane and significantly increased the color intensity of the control and test zones. The limit of detection (0.3 ng mL) was 27 times lower than that of conventional LFIA for both buffer and potato leaf extracts. The ALP-enhanced LFIA does not require additional preparation procedures or washing steps and may be used by nontrained persons in resource-limited conditions. The new method of enhancement is highly promising and may lead to application for routine LFIA in different areas. Graphical abstract Two gold nanoparticles (GNP) conjugates were used - the first with monoclonal antibodies (mAb) (GNP-mAb); the second - alkaline phosphatase-labeled antibody against mAb (GNP-anti-mAb-ALP). The immuno complexes are captured by the polyclonal antibodies (pAb) in the test zone. Addition of the substrate solution (BCIP/NBT) results in the accumulation of the insoluble colored product and in a significance increase in color intensity.

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Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves' extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.