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Enhancement of lateral flow immunoassay by alkaline phosphatase: a simple and highly sensitive test for potato virus X

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Abstract

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−1) 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.

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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Acknowledgments

The study was financially supported by the Russian Science Foundation (grant 16-16-04108).

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Authors and Affiliations

  1. A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, Moscow, 119071, Russia

    Vasily G. Panferov, Irina V. Safenkova, Anatoly V. Zherdev & Boris B. Dzantiev

  2. A.G. Lorch All-Russian Potato Research Institute, Kraskovo, Lorch street 23, Moscow region, 140051, Russia

    Yury A. Varitsev

Authors
  1. Vasily G. Panferov
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  2. Irina V. Safenkova
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  3. Yury A. Varitsev
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  4. Anatoly V. Zherdev
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Correspondence to Boris B. Dzantiev.

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Panferov, V.G., Safenkova, I.V., Varitsev, Y.A. et al. Enhancement of lateral flow immunoassay by alkaline phosphatase: a simple and highly sensitive test for potato virus X. Microchim Acta 185, 25 (2018). https://doi.org/10.1007/s00604-017-2595-3

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